a novel single-nucleotide mutation in a gene homolog ... · brassica rapa . l. chuchuan fan....

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A Novel Single-Nucleotide Mutation in a CLV3 Gene Homolog Controls a Multilocular Silique Trait in Brassica rapa L. Chuchuan Fan National Key Lab of Crop Gentic Improvement Huazhong Agricultural University, Wuhan, China [email protected]

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Page 1: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

A Novel Single-Nucleotide Mutation in a CLV3 Gene Homolog Controls a Multilocular Silique Trait in Brassica rapa L

Chuchuan Fan

National Key Lab of Crop Gentic Improvement Huazhong Agricultural University Wuhan China

fanchuchuanmailhzaueducn

Bilocular silique

Background

uuml Brassica rapa is an important crop and a model plant for studying Brassica genome evolution

Brassica rapa AA 2n=2x=20 yellow sarson from India (Mohammad et al 1942) Sangribai from Tibet (He et al 2003)

B juncea AABB 2n=4x=36 such as Santong Silun Duoshi and Sileng (Liu 2000)

Multilocular silique

uuml Multilocular trait of Brassica is considered advantageous because the multilocular type potentially produces more seeds per silique than the bilocular type increasing the seed yield (Katiyar et al 1998 Lv et al 2012 Zhu et al 2012)

uuml Researchers made attempts to introgress the multilocular silique trait of B rapa to B juncea and B napus through interspecific hybridization and achieved success in developing stable and uniform multilocular lines with significantly improved seed numbers per silique and yields per plant (Katiyar et al 1998 Choudhary and Solanki 2007)

uuml Thus multilocular lines of Brassica as valuable germplasm resources can be used as materials for both practical breeding and for molecular mechanism studies of silique development

uuml Previous genetic analyses of multilocular traits had demonstrated that multilocular silique is controlled by a recessive gene in B rapa and is controlled by either one or two nuclear genes in B juncea

uuml Recently some studies reported the mapping of the multilocular genes in Brassica

u Paritosh et al (2013) mapped the multilocular gene to the A4 chromosome in B rapa

u Xu et al (2013) discovered that two recessive genes (mc1 and mc2) controlled the multilocular trait in B juncea and mc1 was mapped to the A7 chromosome

u Xiao et al (2013) reported the fine mapping of a multilocular gene Bjln1 in B juncea to a 208 kb region

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 2: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Bilocular silique

Background

uuml Brassica rapa is an important crop and a model plant for studying Brassica genome evolution

Brassica rapa AA 2n=2x=20 yellow sarson from India (Mohammad et al 1942) Sangribai from Tibet (He et al 2003)

B juncea AABB 2n=4x=36 such as Santong Silun Duoshi and Sileng (Liu 2000)

Multilocular silique

uuml Multilocular trait of Brassica is considered advantageous because the multilocular type potentially produces more seeds per silique than the bilocular type increasing the seed yield (Katiyar et al 1998 Lv et al 2012 Zhu et al 2012)

uuml Researchers made attempts to introgress the multilocular silique trait of B rapa to B juncea and B napus through interspecific hybridization and achieved success in developing stable and uniform multilocular lines with significantly improved seed numbers per silique and yields per plant (Katiyar et al 1998 Choudhary and Solanki 2007)

uuml Thus multilocular lines of Brassica as valuable germplasm resources can be used as materials for both practical breeding and for molecular mechanism studies of silique development

uuml Previous genetic analyses of multilocular traits had demonstrated that multilocular silique is controlled by a recessive gene in B rapa and is controlled by either one or two nuclear genes in B juncea

uuml Recently some studies reported the mapping of the multilocular genes in Brassica

u Paritosh et al (2013) mapped the multilocular gene to the A4 chromosome in B rapa

u Xu et al (2013) discovered that two recessive genes (mc1 and mc2) controlled the multilocular trait in B juncea and mc1 was mapped to the A7 chromosome

u Xiao et al (2013) reported the fine mapping of a multilocular gene Bjln1 in B juncea to a 208 kb region

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 3: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

uuml Multilocular trait of Brassica is considered advantageous because the multilocular type potentially produces more seeds per silique than the bilocular type increasing the seed yield (Katiyar et al 1998 Lv et al 2012 Zhu et al 2012)

uuml Researchers made attempts to introgress the multilocular silique trait of B rapa to B juncea and B napus through interspecific hybridization and achieved success in developing stable and uniform multilocular lines with significantly improved seed numbers per silique and yields per plant (Katiyar et al 1998 Choudhary and Solanki 2007)

uuml Thus multilocular lines of Brassica as valuable germplasm resources can be used as materials for both practical breeding and for molecular mechanism studies of silique development

uuml Previous genetic analyses of multilocular traits had demonstrated that multilocular silique is controlled by a recessive gene in B rapa and is controlled by either one or two nuclear genes in B juncea

uuml Recently some studies reported the mapping of the multilocular genes in Brassica

u Paritosh et al (2013) mapped the multilocular gene to the A4 chromosome in B rapa

u Xu et al (2013) discovered that two recessive genes (mc1 and mc2) controlled the multilocular trait in B juncea and mc1 was mapped to the A7 chromosome

u Xiao et al (2013) reported the fine mapping of a multilocular gene Bjln1 in B juncea to a 208 kb region

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 4: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

uuml Thus multilocular lines of Brassica as valuable germplasm resources can be used as materials for both practical breeding and for molecular mechanism studies of silique development

uuml Previous genetic analyses of multilocular traits had demonstrated that multilocular silique is controlled by a recessive gene in B rapa and is controlled by either one or two nuclear genes in B juncea

uuml Recently some studies reported the mapping of the multilocular genes in Brassica

u Paritosh et al (2013) mapped the multilocular gene to the A4 chromosome in B rapa

u Xu et al (2013) discovered that two recessive genes (mc1 and mc2) controlled the multilocular trait in B juncea and mc1 was mapped to the A7 chromosome

u Xiao et al (2013) reported the fine mapping of a multilocular gene Bjln1 in B juncea to a 208 kb region

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 5: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

uuml Recently some studies reported the mapping of the multilocular genes in Brassica

u Paritosh et al (2013) mapped the multilocular gene to the A4 chromosome in B rapa

u Xu et al (2013) discovered that two recessive genes (mc1 and mc2) controlled the multilocular trait in B juncea and mc1 was mapped to the A7 chromosome

u Xiao et al (2013) reported the fine mapping of a multilocular gene Bjln1 in B juncea to a 208 kb region

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 6: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

uumlBrassica rapa var yellow sarson

ml4 mutant line (silique with 2-4 locules) 98 of siliques are

multilocular (3-4 locules with a mean of 39 locules)

wt wild type (silique with two locules)

gynoecium 2-4 locules per silique

ml4

wt

Materials

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 7: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

A B C

D E F

G

H I

Fig A and D wt flowers carrying 6 stamens B C E and F mutant flowers carrying 6-7 stamens G Gynoecia of mutant (top) and wt (bottom) H and Istigma

1 The phenotype of ml4 mutant in B rapa

The mutation increases the number of the inner whorls of the floral organs

Results

uumlVariations of floral organs

0

1

2

3

4

5

6

7

Sepal Petal Stamen Carpel

wtml4

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 8: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

An extra gynoecium inside the fruit often called a fifth whorl was frequently observed (811) in the mutant (arrow)

The fifth whorl is forms when the FM fails to terminate due to prolonged WUS expression (Clark et al 1993 1997 Schoof et al 2000)

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 9: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

b

c d

e f

g h

i j

a Embryo (0d)

Seedling (15d)

The primary inflorescence meristems

SAM

area (μm2)

uumlThe multilocular plants exhibited abnormal enlargement of the shoot apical meristems (SAMs)

0

1000

2000

3000

4000

5000

6000

0 d 15 d

wtmutant

wt mutant

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 10: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

These observations suggest that multilocular mutation is important for the control of SAM size

A H B

uumlThe difference of SAM size in Near-Isogenic Lines

0

20000

40000

60000

80000

100000

120000

A B H

SAM

area (μm2)

ml4timeswt

ml4timesF1

ml4timesBC1F1

BC2F1

BC2F2

U

NIL development A homozygous of mutant B homozygous of wt H heterozygous

14d-old-seedling

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 11: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

It indicate that the extra locules in siliques were initiated from the extra carpels of early stages of the developing gynoecium in the mutant

uumlLocular numbers during different stages of the developing gynoecium

wt

ml4

early stage late stage middle stage

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 12: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Table 1 Descriptive statistics of the traits for parents and F1 hybrids

Fig Frequency distribution of siliques with mutiple carpels in the random F2 population

F2 population Bilocular multilocular=31 (χ2=248 Pgt005)

2 Genetic control of multilocular trait in B rapa

A recessive nuclear gene ml4 controls the multilocular trait in B rapa

Materials Silique Length(mm)

Seed Number per Silique

100-seed weight(g)

Siliques with multiple carpels

Range MeanplusmnSD ml4 mutant 344plusmn22A 233plusmn49A 036plusmn007 796-1000 979plusmn51 wt 385plusmn16B 166plusmn24B 035plusmn004 00 00plusmn00 ml4wt F1 381plusmn15B 174plusmn17B 036plusmn004 00 00plusmn00 wtml4 F1 370plusmn19B 171plusmn19B 035plusmn005 00 00plusmn00

020406080

100120140

No

of p

lant

s

percentage of multilocular siliques

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 13: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

3 Preliminary mapping of the ML4 gene

Fig Location of the ML4 locus on the molecular linkage map in B rapa

A random F2 subpopulation (183 individuals) was subsequently screened in the preliminary linkage analysis

A4_18

ML4 A4_38 A4_43

DXP21

116

00 19 12

31

A4_20 A4_26

A4

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 14: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

4 Fine-mapping and gene cloning of the ML4 gene

uuml1704 plants with the recessive phenotype from the F2 population of 6771 individuals were selected for recombinant screening

A4_38 DXP72 A4_18 DXP67 DXP63 DXP96 A4_20 31 17 6 2 2 4 17

DXP63 DXP96 DXP58 A4_26 DXP94 2 2 2 0 0

Scaffold000094

ML4 386 K

50 kb

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 15: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

B rapa gene Arabidopsis homologs E value Arabidopsis annotations

Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor

Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein

Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)

Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)

Bra034344 AT3G57020 0 strictosidine synthase family protein

Table 2 the information of candidate genes of ML4

p Bra034340 is the closest homologue to the CLV3 gene of Arabidopsis in B rapa (79 similarity) and thus this gene is named as BrCLV3

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 16: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

In Arabidopsis CLV3 gene plays a critical role in controlling the stem cells differentiation in the SAM and loos-of-function clv3 mutant shows enlargement of SAM and multicarpel siliques

BrCLV3 is most likely the candidate gene of ml4 in B rapa

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 17: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

(a)

ATG TAA

(b)

ml4

wt

100 bp

BrCLV3_ml4 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3_wt MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 BrCLV3 MDSRT-LVLLLLFCLMFLHDASDITHANANVHALPIRKMMVMKKDNEWGGANG-IEEEKE 58 AtCLV3 MDSKSFLLLLLLFCFLFLHDASDLTQAHAHVQGLSNRKMMMMKMESEWVGANGEAEKAKT 60 BrCLV3_ml4 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_wt KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

T

C

T

C ATAT

hellip T

C

secretion signal

CLE motif

uumlThe gene structure and comparative sequencing analysis of the candidate gene BrCLV3

2260 bp upstream + the entire gene + 2541 bp 3rsquo flanking

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 18: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Fig the relative contributions of individual residues in the core CLE motif the CLV3 function in SAMs

The alanine scan of MCLV3

In Arabidopsis Pro9 in the core CLE motif is critical for endogenous CLV3 function in SAM maintenance

Thus the C-to-T Nucleotide Substitution in BrCLV3 probably leads to the Multilocular phenotype in B rapa

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 19: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

J

Car

pel n

umbe

r

B C C C

C

B B B A

uumlOverexpression of BrCLV3Brclv3 in Arabidopsis

K

5 Genetic transformation of BrCLV3 gene in Arabidopsis and B rapa

BrCLV3 35S

BrCLV3 OE

Brclv3 35S

Brclv3 OE

NOS

NOS

clv3-2

Ler clv3-2 OE OE OE

Ler clv3-2 OE

clv3-2

no obvious changes

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 20: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Chart1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3-2 08193072487 08193072487
ler 0 0
Page 21: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

定位标记

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Table 3 Primer sequences of the molecular markers developed in the study
Marker Marker type Sequence (5-3) Production size (bp)
A4_18 SSR forward CTAGATGATTTTATGTGTCATGTGC 250
reverse AGAAAAGCAACACGGTTCGT
A4_20 SSR forward TGATTATGAAACACAATAAAAACATCA 230
reverse AGATGACGGCACTCACAGC
A4_26 SSR forward TCAATTCCATTTGCTCCTCC 238
reverse CATTCGCAAGGTTTGCTTCT
A4_38 SSR forward TCCATTTCCCTTTTGTTTCTTT 229
reverse TGAATGCCTTTATACGCGAG
A4_43 SSR forward AATCATACATTTTTCGTGTTGAAA 278
reverse TATCCGGCTTGGTGAAGAAG
DXP21 SSR forward AGTCATGGAAACAAACGGCG 196
reverse GGTTTACGCTATGGGCATGA
DXP58 SNP forward 1 TGGTAAGTCGCTTGGAAGCGC 949 (P17)
forward 2 GGTGGTAAGTCGCTTGGAAGATT 949 (P12)
reverse CACCTGCTACGGAAGGAGCA
DXP63 SNP forward 1 GCCGTCGTGGACGGTCTTA 655(P17)
forward 2 CCGTCGTGGACGGTCATG 655(P12)
reverse TCGCAGATGTATAGATCTCCCGTT
DXP67 SNP forward 1 ATTCTTCTTCAGGAGGGAGACAATG 460 (P17)
forward 2 TTCTTCTTCAGGAGGGAGACAACA 460 (P12)
reverse GTTTGGTAGGCTGAGAAGATGGG
DXP72 SNP forward 1 GATCGGTCACTTTCTCCATCGC 315(P17)
forward 2 AGGATCGGTCACTTTCTCCATACA 315 (P12)
reverse GATTCTCTTTGGTTGCTCCCTTTT
DXP94 SNP forward ATGGCATGTGGGAGTATGATCGCT 956 (TA)
reverse TACCAGCGGAGAATTGGAGAAGCA
DXP96 SNP forward ACACCCAGCCCTATCACACAGAAA 808 (GT)
reverse TGACTTGTCCCAAACTGATCTGGC
下划线为引入的错配碱基
SNP标记开发网址httpausubellabmghharvardedu 进入SNAP program
Page 22: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

候选基因

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
定位区段内的候选基因信息
B rapa gene Arabidopsis homologs E value Arabidopsis annotations
Bra034338 AT2G27230 0 LHW (LONESOME HIGHWAY) protein homodimerization transcription activator transcription factor
Bra034339 AT2G27240 0 Aluminium activated malate transporter family protein
Bra034340 AT2G27250 100E-22 CLV3 (CLAVATA3)
Bra034341 AT2G27260 500E-64 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034342 AT2G27270 100E-31 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family
Bra034343 AT2G27285 500E-72 Coiled-coil domain-containing protein 55 (DUF2040)
Bra034344 AT3G57020 0 strictosidine synthase family protein
Page 23: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

亲本表型

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
花瓣数 花萼数 雄蕊数 雌蕊数 Genotype No of plants Sepal Petal Stamen Carpelsflower
LXP 40plusmn00 40plusmn00 60plusmn01 10plusmn00 Wild type 19 40plusmn00 40plusmn00 60plusmn01 20plusmn00
DXP 40plusmn00 40plusmn00 62plusmn02 10plusmn00 DXP mutant 21 40plusmn00 40plusmn00 62plusmn02 35plusmn04
P18 P12
Sepal 4 4 0 0
Petal 4 4 0 0
Stamen 6 62 01 02
Carpel 2 35 0 04
Page 24: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

亲本表型

P18
P12

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
0 0 0 0
0 0 0 0
01 01 02 02
0 0 04 04
Page 25: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

35S OE T1

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
20135 35sclv3 OE材料T1植株考种结果
carpel number silique length seed number per silique
clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C seed number per silique
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
silique length
carpel number
Page 26: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

35S OE T1

33
22222222222
32142857143
30333333333
21
22222222222
3
45333333333
2

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
OE64 07022132499 07022132499
OE13 05483188806 05483188806
OE4 07867957925 07867957925
OE72 08502873078 08502873078
OE74 03051285766 03051285766
OE47 06666666667 06666666667
OE71 1 1
clv3 08193072487 08193072487
ler 0 0
Page 27: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

35S OE T2

59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
Page 28: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

35S OE T3

328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
Page 29: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
角果粒数 角果粒数
1 OE64 18 21 23 39 69 49 36 42 28 18 19 46 29 42 29 32 41 7 13 70 19 44 32 28 43 57 16 12 38 25 1 OE64 328 158 carpel number silique length seed number per silique
11 OE13 35 18 37 16 46 26 13 13 6 8 5 13 29 5 12 9 9 53 11 OE13 196 147 clv3-2 45plusmn08 A 42plusmn06 E 206plusmn89 CD
13 OE4 20 15 41 15 29 5 5 9 34 51 18 34 15 7 23 42 33 23 18 58 21 17 15 15 16 16 14 21 13 OE4 225 133 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
17 OE72 22 48 17 50 39 29 39 50 47 39 45 31 11 8 20 41 43 36 24 16 13 28 30 17 50 20 19 66 26 20 17 OE72 315 144 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
18 OE74 51 44 47 48 54 31 32 52 51 54 42 47 27 31 35 48 44 66 23 28 56 54 51 56 47 50 42 58 52 38 18 OE74 453 105 OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
22 OE47 6 12 22 9 15 27 9 11 12 22 OE47 137 67 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
24 OE71 17 21 35 29 15 6 19 30 39 24 OE71 234 106 OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
clv3 clv3 14 19 21 13 22 27 12 17 15 26 14 18 32 16 25 22 30 37 10 17 28 23 34 9 14 6 38 6 33 19 clv3-2 clv3 206 89 OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
ler ler 34 31 36 33 30 22 34 32 13 30 35 27 19 24 24 22 38 21 20 24 32 24 31 21 24 25 33 33 20 25 ler ler 272 62 OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
角果长 角果长 ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
1 OE64 413 593 624 871 714 62 548 771 564 595 536 743 579 615 465 582 688 367 451 787 488 764 734 591 639 555 425 384 585 525 1 OE64 59 13
11 OE13 607 466 59 478 765 599 466 45 409 476 4 471 523 441 429 395 381 68 11 OE13 50 11
13 OE4 625 444 577 51 542 411 387 34 466 579 386 475 423 365 442 577 508 382 443 601 446 381 417 418 438 299 361 398 13 OE4 45 08
17 OE72 498 546 462 75 808 644 76 826 758 709 628 533 416 49 436 587 539 624 567 435 362 521 513 481 576 432 462 736 501 525 17 OE72 57 13
18 OE74 957 878 934 748 902 748 697 96 947 895 913 907 789 923 738 788 791 771 758 722 856 922 992 897 84 825 784 678 753 652 18 OE74 83 09
22 OE47 504 389 428 422 336 639 427 383 525 22 OE47 45 09
24 OE71 432 5 492 482 424 366 393 49 485 24 OE71 45 05
clv3 clv3 429 451 375 314 459 413 392 42 421 463 398 462 535 346 441 526 567 465 355 377 441 393 456 361 33 302 476 425 477 404 clv3-2 clv3 42 06
ler ler 647 693 694 697 628 619 712 671 562 577 765 619 562 601 554 625 737 53 494 552 779 531 623 567 621 702 813 719 682 638 ler ler 64 08
心皮数 心皮数
1 OE64 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 1 OE64 33 07
11 OE13 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 11 OE13 22 05
13 OE4 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 13 OE4 32 08
17 OE72 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 17 OE72 30 09
18 OE74 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 18 OE74 21 03
22 OE47 2 2 2 2 2 2 2 2 4 22 OE47 22 07
24 OE71 2 2 2 2 3 4 4 4 4 24 OE71 30 10
clv3 clv3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 clv3-2 clv3-2 45 08
ler ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ler ler 20 00
方差分析单因素方差分析
SUMMARY
观测数 求和 平均 方差
OE64 30 985 328333333333 2490402298851 233796728972 900
OE13 18 353 196111111111 2150751633987 35108366251 324
OE4 28 630 225 1765555555556 784
OE72 30 944 314666666667 2086022988506 900
OE74 30 1359 453 1096655172414 57753262483 900
OE47 9 123 136666666667 455 816620599 81
OE71 9 211 234444444444 1115277777778 81
clv3 30 617 205666666667 794264367816 900
ler 30 817 272333333333 380471264368 900
方差分析 carpel number silique length seed number per silique
差异源 SS df MS F P-value F crit clv3 45plusmn08 A 42plusmn06 E 206plusmn89 CD
组间 158545109034268 8 19818138629284 137541319502 639109212884869E-16 19837794548 OE64 33plusmn07 B 59plusmn13 B 328plusmn158 B
组内 295381666666667 205 1440886178862 OE4 32plusmn08 B 45plusmn08 DE 225plusmn133 C
OE72 30plusmn09 B 57plusmn13 C 315plusmn144 B
总计 453926775700935 213 OE71 30plusmn10 B 45plusmn05 DE 234plusmn106 C
OE13 22plusmn05 C 50plusmn11 D 196plusmn147 CD
OE47 22plusmn07 C 45plusmn09 DE 137plusmn67 D
OE74 21plusmn03 C 83plusmn09 A 453plusmn105 A
ler 20plusmn00 C 64plusmn08 B 272plusmn62 BC
Page 30: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
59386666667
50144444444
45146428571
57083333333
83216666667
45033333333
45155555556
42246666667
64046666667
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
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32
2
47
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343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
OE64 12617522937 12617522937
OE13 10604339967 10604339967
OE4 08464456498 08464456498
OE72 12667500729 12667500729
OE74 09437638634 09437638634
OE47 09169514709 09169514709
OE71 04923949409 04923949409
clv3 06305319775 06305319775
ler 08022027147 08022027147
Page 31: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
328333333333
196111111111
225
314666666667
453
136666666667
234444444444
205666666667
272333333333
82
11
88
75
83
51
3
2
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32
2
47
461
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343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
OE64 157810085193 157810085193
OE13 146654411253 146654411253
OE4 13287420952 13287420952
OE72 144430709633 144430709633
OE74 104721305015 104721305015
OE47 67453687816 67453687816
OE71 10560671275 10560671275
clv3 89121510749 89121510749
ler 61682352774 61682352774
Page 32: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
07022132499 07022132499
05483188806 05483188806
07867957925 07867957925
08502873078 08502873078
03051285766 03051285766
06666666667 06666666667
1 1
08193072487 08193072487
0 0
Page 33: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
82
11
88
75
83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
角果长度 心皮数 每角果粒数 角果长度 心皮数 每角果粒数
OE13-20 82plusmn10 30plusmn12 461plusmn177 OE13-20 82 1 3 12 461 177
OE74-18 110plusmn13 20plusmn00 520plusmn132 OE74-18 11 13 2 0 52 132
OE47-22 88plusmn15 24plusmn07 406plusmn247 OE47-22 88 15 24 07 406 247
OE13-11 75plusmn18 32plusmn10 453plusmn174 OE13-11 75 18 32 1 453 174
ler 83plusmn17 20plusmn00 251plusmn146 ler 83 17 2 0 251 146
clv3 51plusmn11 47plusmn09 343plusmn151 clv3 51 11 47 09 343 151
角果长度 OE13-20-5 793 991 68 692 822 858 874 827
角果长度 OE74-18-3 115 1121 1004 109 1124 775 1185 1172 1048 1212 1260
角果长度 OE47-22-7 814 796 635 783 819 901 1088 105 1042
角果长度 OE13-11-14 93 739 569 911 679 986 761 63 482 95 602 608 421 556 657 402 893 859 987 992 586 999 88 555 571 64 773 93 651 774 947 720 731 1109 584 779 963 864
角果长度 ler 744 698 71 655 77 778 701 745 827 846 869 881 533 863 787 713 718 81 792 876 845 677 1132 1057 797 775 832 738 731 779 786 802 659 879 767 825 647 81 742 1008 8 783 741 694 681 776 569 935 612 482 572 879 755 746 85 866 934 1222 1075 1095 1104 1026 1264 735 747 892 1031 115 1117 737 1161 1026 1049
角果长度 clv3 571 855 379 391 42 662 582 461 534 481 525 497 293 663 603 379 519 538 437 560 540 454 601 509 455 482 489
每角果粒数 OE13-20-5 50 47 54 42 8 51 71 46
每角果粒数 OE74-18-3 39 56 74 53 28 47 59 58 55 65 38
每角果粒数 OE47-22-7 36 68 7 32 85 39 40 11 47
每角果粒数 OE13-11-14 38 77 31 33 59 50 59 37 56 77 30 44 30 29 58 71 45 62 28 47 22 68 50 18 28 28 65 85 16 53 37 36 51 57 41 34 32 38
每角果粒数 ler 20 17 16 12 18 14 21 23 27 20 21 24 21 13 10 24 21 30 22 15 61 51 19 14 14 22 11 20 15 19 31 22 13 13 18 15 15 21 12 11 10 23 22 13 26 10 13 18 16 13 23 27 35 53 50 46 60 45 47 19 20 44 17 49 35 16 62 57 58
每角果粒数 clv3 39 34 28 26 14 60 28 39 18 31 25 29 27 46 47 13 37 42 56 41 58 27 72 14 24 35 17
心皮数 OE13-20-5 2 2 5 4 2 2 4 3
心皮数 OE74-18-3 2 2 2 2 2 2 2 2 2 2 2
心皮数 OE47-22-7 3 3 2 2 4 2 2 2 2
心皮数 OE13-11-14 2 4 4 3 4 2 2 4 4 4 4 4 4 4 4 5 2 2 2 2 4 2 2 4 4 4 4 3 4 2 2 4 4 2 4 2 2 2
心皮数 ler 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
心皮数 clv3 4 2 5 5 4 5 4 7 5 6 4 5 5 5 5 5 5 4 5 5 4 4 5 5 4 5 5
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 47037037037 2 平均 47037037037 31842105263 平均 47037037037 24444444444 平均 47037037037 2 平均 47037037037 3
方差 0754985755 0 方差 0754985755 10192034139 方差 0754985755 05277777778 方差 0754985755 0 方差 0754985755 14285714286
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 02003023432 合并方差 09101612055 合并方差 07015250545 合并方差 05452674897 合并方差 08978675645
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat 268200382705 t Stat 63278561277 t Stat 70080366466 t Stat 102362461311 t Stat 44666382373
P(Tlt=t) 单尾 312135071098724E-47 P(Tlt=t) 单尾 00000000146 P(Tlt=t) 单尾 00000000218 P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 00000439814
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 624270142197448E-47 P(Tlt=t) 双尾 00000000292 P(Tlt=t) 双尾 00000000437 P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 00000879629
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 343333333333 25115942029 平均 343333333333 452631578947 平均 343333333333 405555555556 平均 343333333333 52 平均 343333333333 46125
方差 2268461538462 2133393009378 方差 2268461538462 301226173542 方差 2268461538462 6082777777778 方差 2268461538462 173 方差 2268461538462 3129821428571
观测值 27 69 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2170752389763 合并方差 270529657477 合并方差 3165947712418 合并方差 2118888888889 合并方差 2451174242424
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 94 df 63 df 34 df 36 df 33
t Stat 27559649625 t Stat -26401144355 t Stat -09085431012 t Stat -33930241395 t Stat -18710308791
P(Tlt=t) 单尾 00035144325 P(Tlt=t) 单尾 00052174808 P(Tlt=t) 单尾 01849933467 P(Tlt=t) 单尾 00008470438 P(Tlt=t) 单尾 00351149838
t 单尾临界 16612258553 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0007028865 P(Tlt=t) 双尾 00104349615 P(Tlt=t) 双尾 03699866935 P(Tlt=t) 双尾 00016940877 P(Tlt=t) 双尾 00702299676
t 双尾临界 19855234419 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设 t-检验 双样本等方差假设
clv3 ler clv3 OE13-11-14 clv3 OE47-22-7 clv3 OE74-18-3 clv3 OE13-20-5
平均 51407407407 83164383562 平均 51407407407 75447368421 平均 51407407407 88088888889 平均 51407407407 110372727273 平均 51407407407 817125
方差 12145763533 2722806583 方差 12145763533 33223445235 方差 12145763533 22926111111 方差 12145763533 17156218182 方差 12145763533 10042982143
观测值 27 73 观测值 27 38 观测值 27 9 观测值 27 11 观测值 27 8
合并方差 2322663869 合并方差 24524719453 合并方差 14682315904 合并方差 13537556491 合并方差 11699718996
假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0 假设平均差 0
df 98 df 63 df 34 df 36 df 33
t Stat -92510126111 t Stat -60988610235 t Stat -78650497103 t Stat -141681434111 t Stat -69601845189
P(Tlt=t) 单尾 0 P(Tlt=t) 单尾 0000000036 P(Tlt=t) 单尾 00000000019 P(Tlt=t) 单尾 134962586222509E-16 P(Tlt=t) 单尾 00000000295
t 单尾临界 16605512171 t 单尾临界 16694022217 t 单尾临界 16909242552 t 单尾临界 16882977141 t 单尾临界 1692360309
P(Tlt=t) 双尾 0 P(Tlt=t) 双尾 0000000072 P(Tlt=t) 双尾 00000000037 P(Tlt=t) 双尾 269925172445019E-16 P(Tlt=t) 双尾 00000000589
t 双尾临界 19844674545 t 双尾临界 19983405425 t 双尾临界 20322445093 t 双尾临界 2028094001 t 双尾临界 20345152974
Page 34: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
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88
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83
51
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
OE13-20 1 1
OE74-18 13 13
OE47-22 15 15
OE13-11 18 18
ler 17 17
clv3 11 11
Page 35: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
3
2
24
32
2
47
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
OE13-20 12 12
OE74-18 0 0
OE47-22 07 07
OE13-11 1 1
ler 0 0
clv3 09 09
Page 36: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,
461
52
406
453
251
343

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
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  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
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OE13-20 177 177
OE74-18 132 132
OE47-22 247 247
OE13-11 174 174
ler 146 146
clv3 151 151
Page 37: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

1 9 10 2 3 4 5 6 7 8 M

Detection of positive transgenic plants in B rapa M 3kb marker 1~9 transgenic plants 10 positive control 11 negative control

Control Transgenic plants

uumlTransgenic complementation in B rapa

BrCLV3 COM construct

ml4 mutant

11

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
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Page 38: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uumlIn vitro peptide assay

BrCLV3 RTVPSGPDPLHH BrCLV3Leu9 RTVPSGPDLLHH

The endogenously functional CLV3 signal is a 12-aa peptide derived from the CLE motif in Arabidopsis (Kondo et al 2006)

Synthesize peptides

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
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  • Slide Number 11
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  • Slide Number 24
  • Slide Number 25
  • Slide Number 26
  • Slide Number 27
  • Slide Number 28
  • Slide Number 29
  • Slide Number 30
  • Slide Number 31
  • Slide Number 32
Page 39: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

ml4 wt

A B

Ler clv3-2 Ler clv3-2 Ler clv3-2

00

100

200

300

400

500

600

700

800

7d 10d 13d 7d 10d 13d

Ler clv3-2

12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1microM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1microM BrCLV3

12 MS BrCLV3 10nM

BrCLV3Leu9 10nM 12 MS BrCLV3

1μM BrCLV3Leu9 1μM

Main root length (mm) Main root length (mm)

Root length inhibition assay of the synthetic peptides

ml4 wt ml4 wt

Arabidopsis B rapa

05

101520253035404550

wt ml4

12 MS

1μM BrCLV3Leu9

1μM BrCLV3

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
  • Slide Number 10
  • Slide Number 11
  • Slide Number 12
  • Slide Number 13
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  • Slide Number 24
  • Slide Number 25
  • Slide Number 26
  • Slide Number 27
  • Slide Number 28
  • Slide Number 29
  • Slide Number 30
  • Slide Number 31
  • Slide Number 32
Page 40: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

a b c d e f g

h i j k l m n

00

1000

2000

3000

4000

5000

Ler clv3-2

RA

M le

ngth

of m

ain

root

(μm

) 12 MS

10nM BrCLV3Leu9

100nM BrCLV3Leu9

1μM BrCLV3Leu9

10nM BrCLV3

100nM BrCLV3

1μM BrCLV3

12MS

clv3-2

Ler

BrCLV3Leu9 10nM 100nM 1μM 10nM 100nM 1μM

BrCLV3 Root apical meristem inhibition assay of the

synthetic peptides

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
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  • Slide Number 12
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Page 41: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

0

1000

2000

3000

4000

Ler clv3-2

SAM

size

(μm

2 ) 12 MS

1 μm 1 μm

A B C

D E F

G H I

J K L

M N

clv3-2

Ler

ml4

wt

12MS 1μM BrCLV3Leu9 1μM BrCLV3

SAM inhibition assay of the synthetic peptides

Arabidopsis

B rapa

BrCLV3Leu9

0

1000

2000

3000

4000

5000

6000

wt ml4

SAM

size

(μm

2 )

BrCLV3

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
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  • Slide Number 29
  • Slide Number 30
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  • Slide Number 32
Page 42: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

The results of peptide assay indicate that

BrCLV3_P12 KVFGLNEELRTVPSGPDLLHHHVNPPRKPRTDSHIP 94 BrCLV3_P18 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 BrCLV3 KVFGLNEELRTVPSGPDPLHHHVNPPRKPRTDSHIP 94 AtCLV3 KGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP 96

CLE motif

uuml The BrCLV3 peptide can function in both Arabidopsis and B rapa uuml Pro9rarrLeu9 mutation could disrupts the function of CLV3 peptide

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
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Page 43: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

00000000020000400006000080001000012000140001600018

Lev

el o

f gen

e ex

pres

sion

A

5 Expression analysis of BrCLV3 gene

How does the abnormal activation of BrCLV3 in the SAM of ml4 plants happen

Lev

el o

f gen

e ex

pres

sion

0

0001

0002

0003

0004

0005

wt ml4

B

SAM BrCLV3

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
  • Slide Number 10
  • Slide Number 11
  • Slide Number 12
  • Slide Number 13
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  • Slide Number 32
Page 44: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

00

05

10

15

P17 P12

CLV1

00

01

02

03

04

P17 P12

CLV2

000001002003004005006

P17 P12

POL

0000000100020003000400050006

P17 P12

CRN

0000

0001

0002

0003

P17 P12

WUS

000002004006008010012

P17 P120000

0004

0008

0012

0016

P17 P12

PLL1

Lev

el o

f gen

e ex

pres

sion

RPK2

uumlThe expression of the Arabidopsis homologues in the CLV3WUS pathway increased by three to nine fold in the SAM of ml4

Perales and Reddy et al Plant Biology 2012

wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4 wt ml4

a positive-negative feedback loop involved in the CLV3WUS maintains the SAM size in Arabidopsis

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
  • Slide Number 10
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  • Slide Number 32
Page 45: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Receptor complex

POLPLL1

WUS

ml4 mutant

times

BrCLV3Leu9

Membrane Receptor complex

POLPLL1

WUS

BrCLV3

wt

uumlThe possible mechanism of multilocular formation in B rapa

Extracellular

Intracellular

Fan et al Mol Plant 2014

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
  • Slide Number 10
  • Slide Number 11
  • Slide Number 12
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  • Slide Number 28
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  • Slide Number 32
Page 46: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Summary uumlThe multilocular mutant ml4 of B rapa shows enlarged SAM increased numbers of inner whorls of floral organs and seed number per silique without reduction of seed weight

uumlThe multilocular trait of B rapa is controlled by a recessive gene ml4 on the A4 chromosome

uumlThe multilocular gene ml4 was identified by map-based cloning approach it encodes a small putative secreted peptide that is the putative orthologue of the Arabidopsis CLAVATA3 gene

uumlIn the ml4 mutat the Pro-to-Leu substitution in the core CLE motif disrupts the function of BrCLV3

uumlExpression analyses indicated that the putative negative pathway in the feedback loop between CLV3 and WUS was disrupted in ml4

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

  • Slide Number 1
  • Slide Number 2
  • Slide Number 3
  • Slide Number 4
  • Slide Number 5
  • Slide Number 6
  • Slide Number 7
  • Slide Number 8
  • Slide Number 9
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  • Slide Number 28
  • Slide Number 29
  • Slide Number 30
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  • Slide Number 32
Page 47: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Acknowledgement u Prof Yongming Zhou

u Dr Qingyong yang

Yudi Wu Keqiao Zhang

Qingwei Meng Yang Yang

u Prof Zaiyun Li

u Students participated in the work

Thanks for your attention

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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Page 48: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

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Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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Page 49: A Novel Single-Nucleotide Mutation in a Gene Homolog ... · Brassica rapa . L. Chuchuan Fan. National Key Lab of Crop Gentic Improvement . Huazhong Agricultural University, Wuhan,

Seed number per silique

Silique length

Seed weight

Seed number per silique 1 Silique length 050 1 Seed weight 002 -014 1 Mutilocular silique 020 -054 018

Seed number per silique

Silique length (mm)

Seed weight (g)

Mutilocular type 198plusmn43 314plusmn36 039plusmn007 Bilocular type 153plusmn42 345plusmn46 037plusmn006

Table 2 Coefficients of pairwise correlations of the silique related traits in F2 population

Table 3 Statistics of the traits for bilocularmultilocular silique in F2 population

ml4 mutant holds great potential for the future high-yield breeding

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