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Genetic characterization and fine mapping of a chlorophyll-deficient

mutant (BnaC.ygl) in Brassica napus

Lixia Zhu, Xinhua Zeng, Yanli Chen, Zonghui Yang, Liping Qi,

Yuanyuan Pu, Bin Yi, Jing Wen, Chaozhi Ma, Jinxiong Shen, Jinxing Tu,

Tingdong Fu (*)

National Key Laboratory of Crop Genetic Improvement, National Sub-

center of Rapeseed Improvement in Wuhan, Huazhong Agricultural

University, Wuhan 430070, China

* Corresponding author: Tingdong Fu

E-mail: [email protected]

Tel: 86-27-87281900

Fax: 86-27-87280009

Abstract

A chlorophyll-deficient mutant with yellow-green leaves of Brassica

napus was obtained by treatment with the chemical mutagen ethyl

methanesulfonate (EMS). Compared with the wild type at seedling stage,

the mutant displayed decreased total chlorophyll content, less granal

stacks and granal membranes. Genetic analysis confirmed that the mutant

phenotype was controlled by a recessive gene, which was designated as

BnaC.ygl. Mapping of the gene was subsequently conducted in two

populations with yellow-green leaves (populationⅠBC8 and ⅡBC4, which

comprised 3472 and 5288 individuals respectively) . Analysis on the

public simple sequence repeat markers (SSR) showed that four SSR

markers linked to BnaC.YGL gene displayed polymorphism. Based on the

information of these SSR markers, the BnaC.YGL gene was mapped to

the linkage group N17. From a survey of amplified fragment length

polymorphism (AFLP), 15 of 47 AFLP markers were successfully

converted into sequence characterized amplified region (SCAR) markers.

BnY5 and CB10534, the closest flanking markers, were 0.32 cM and 0.03

cM away from the BnaC.YGL gene, respectively. And in the two

populations, 18 makers cosegregated with BnaC.YGL. BLAST analysis

revealed that the sequences of the makers displayed highly conserved

homology with C06 of B. oleracea. The collinearity of makers to makers

on N17 and on C06 showed that there might be an inversion occurring on

the N17 group. These results are expected to accelerate the process of

cloning the BnaC.YGL gene and facilitate the understanding of the

biological processes of chloroplast development in Brassica napus.

Keywordschlorophyll-deficient mutant

Brassica napus

Fine mapping

Chloroplast development

Introduction

Chlorophyll (Chl) plays several important roles in photosynthetic

light-harvesting and energy transduction both directly and indirectly.

Meanwhile, its biosynthesis, accumulation and degradation are also

associated with chloroplast development, photo-morphogenesis and

chloroplast-nuclear signalling (Eckhardt et al. 2004; Vothknecht and

Westhoff 2001). Extensive studies have been focused on the Chl

metabolism in various organisms by biochemical and genetic approaches

(CG and SP 1978; Gaubier et al. 1995; Oster et al. 2000). Biochemical

researches on the enzymatic steps have promoted the identification of

their encoding genes (Eckhardt et al. 2004). So far, almost all of Chl

biosynthetic genes have been identified in higher plants (Beale 2005;

Nagata et al. 2005; Tripathy and Pattanayak ， GK. 2012). The whole

pathway of Chl biosynthesis can be subdivided into four parts: (1)

formation of 5-aminolevulinic acid (ALA), the committed step for all

tetrapyrroles, (2) formation of a pyrrole ring porphobilinogen and the

synthesis of the first closed tetrapyrrole having inversion of ring D, (3)

formation of protoporphyrin IX (Proto IX), which is a common precursor

for Chl and heme/bilin biosynthesis, and (4) formation of Chl from Proto

IX (Tanaka et al. 2011; Tripathy and Pattanayak，GK. 2012). The ATP-

dependent insertion of Mg2+ into Proto IX is the branch point for

chlorophyll and heme biosynthesis (Tripathy and Pattanayak ， GK.

2012). In this metabolic pathway, plants need to prevent excessive

accumulation of the intermediate molecules because most of Chl

biosynthetic intermediates are strong photosensitizers that would lead to

oxidative damage or cell death under illumination (Tanaka et al. 2011).

Mutant lines are important resources for forward genetic studies of

gene functions as well as for a better understanding of the regulatory

mechanisms of biological metabolism because of the abnormal

expression of some metabolic intermediates. Mutants can be obtained by

chemical and irradiation mutagenesis such as EMS, γ-ray, or through

biological methods such as tissue culture and T-DNA/ transposon

insertions. The various methods used by different researchers have

resulted in a large number of Arabidopsis, rice, barley, maize, soybean

and rapeseed mutants which have been extensively used to study gene

functions (Falbel and Staehelin 1999; Hirochika et al. 2004; Krishnan et

al. 2009; Lunde et al. 2003; Zhao et al. 2000).

The mutants with chlorophyll-deficient or chlorophyll-modified

leaves are very common and widely used to study the molecular

mechanisms that regulate Chl biosynthesis and chloroplast development

in many plants such as Arabidopsis (Eckhardt et al. 2004), rice (Kurata et

al. 2005; Sakuraba et al. 2013; Wu et al. 2007), soybean (Palmer et al.

2000), barley (Falbel and Staehelin 1994; Falbel and Staehelin 1996;

Falbel and Staehelin 1999; Mueller et al. 2012; Yaronskaya et al. 2003),

maize (Lunde et al. 2003) and so on. Currently, at least 27 genes that

encode 15 enzymes from glutamyl-tRNA to Chl b in Chl biosynthesis

have been identified in Arabidopsis (Nagata et al. 2005; Nagata et al.

2007). A correlation analysis between the gene expression and the level

of Mg-Proto IX in a range of mutants (gun, chlm, crd1) showed that the

steady-state level of Mg-Proto IX was not a determinant of plastid-to-

nucleus signalling in Arabidopsis, which revised the previous model on

plastid signalling (Mochizuki et al. 2008). In rice, more than 70

chlorophyll mutants that exhibit albino, chlorina, stripe, virescent,

yellow-green and zebra leaves have been reported (Kurata et al. 2005).

However, only a few genes involved in the Chl biosynthesis have been

cloned and characterized, such as OsChlD, OsChlH, OsChlI, OsDVR,

OsCAO, OsGluRS, and OsPOR (Jung et al. 2003; Liu et al. 2007;

Sakuraba et al. 2013; Wang et al. 2010; Wu et al. 2007; Zhang et al.

2006). In Brassica napus, only one chlorophyll- deficient mutant (Cr) has

been reported. The yellow-green leaf phenotype in Cr is controlled by a

recessive gene and has been successfully turned into CMS lines (Zhao et

al. 2000). Moreover, it has a decreased amount of light-harvesting

complexes but an increased amount of some core polypeptides of PSII

(Guo et al. 2007). However, the mapping of the recessive gene which is

responsible for the mutational phenotype in Cr was not reported so far.

Currently, a combination of map-based cloning strategy and

comparative genome analysis is usually used for gene mapping, which is

an effective approach for gene mapping in cultivated Brassicas described

in “triangle of U” (Kowalski et al. 1994; Parkin et al. 2005; Wang et al.

2011; Xia et al. 2012). However, only several genes have been cloned in

cultivated Brassicas by this way, such as the dwarf gene dwf2，seed coat

color genes BrTT8 and TTG1 homologue in B. rapa (Li et al. 2012;

Muangprom and Osborn 2004; Zhang et al. 2009), the high-β-carotene

gene Or in B. oleracea (Lu et al. 2006), the RGMS genes (BnMs1,

BnMs2, BnMs3), the dwarf gene BnRGA and the cleistogamy gene Bn-

CLG1A in B. napus (Dun et al. 2011; Lei et al. 2007; Liu et al. 2010; Lu

et al. 2012; Yi et al. 2010). Since a large amount of B. rapa, B. oleracea

and B. napus sequence information has been released to the public

databases, and especially with the completion of B. rapa genome

sequencing, cloning genes with this approach would be more effective in

cultivated Brassicas.

In this study, we isolated a rapeseed Chl-deficient mutant, which

exhibited a yellow-green leaf phenotype, reduced Chl level, and affected

chloroplast development. The objectives of our study were to: (1) analyse

the inheritance model of the mutant, (2) develop the molecular markers

linked to the BnaC.YGL gene, and (3) fine map the BnaC.YGL gene

through a classic map-based cloning strategy in combination with

comparative mapping among B. napus and other Brassica species. The

results will promote the map-based cloning of the BnaC.YGL gene as well

as the understanding of the biological processes of chloroplast

development in Brassica napus

Materials and Methods

Plant materials

The chlorophyll-deficient mutant (BnaC.ygl) was obtained from the B.

napus inbred line T6 (+/+) whose seeds were treated with 1% EMS for

8h. The mutant with a yellow- green leaf phenotype at seedling stage

(Fig.1a) was identified at the M2 generation and was selfed to generate an

inbred line. Two breeding lines (B409 and 1161) with normal green

leaves were used to construct the populations.

Chlorophyll content determination and transmission electron microscopy

analysis

Total Chl was determined with UVmini-1240 (Shimadzu) according

to the method of Arnon (Arnon 1949). The leaf samples of the wild-type

(T6) and chlorophyll-deficient mutant (BnaC.ygl) were harvested from

the plants at different stages in normal conditions. Leaves of the wild-

type and the mutant (approximately of 30 mg fresh weight respectively)

were cut and homogenized in 5 mL 80% (V/V) aqueous acetone for 48h,

and then centrifuged at 3,000 g for 10 min. The combined supernatants

were diluted with acetone for spectrophotometric analysis.

For transmission electron microscopy analysis, leaves were cut into

1×1 cm sections and fixed in 2.5% (w/v) glutaraldehyde in 0.1 M

phosphate buffer (pH 7.4) and further fixed in 1% OsO4 in the same

buffer. The subsequent steps were carried out as described by Yi et al

(2010).

Genetic analysis

The cross between the mutant (BnaC.ygl) line and B409 was carried

out (hereafter referred to as populationⅠ). The F2 population was derived

from the self-pollination of F1 plants. The BC1 was derived by the

backcrosses of F1 to the mutant line. The phenotype of the reciprocal

hybrid F1 and the segregation ratio of ⅠF2 andⅠBC1 population were used

to detect the genetic pattern of the chlorophyll-deficient mutant. The

other similar hybrid population (F1) was obtained by crossing 1161 with

the mutant line (hereafter referred to as population Ⅱ). The ⅡF2 and ⅡBC1

were also obtained. Data from the above experiments were analysed

using the SAS system (SAS 8.1). All the measurements in the

experiments were analysed as completely random design.

DNA extraction and marker analysis

Total DNA was extracted individually from the leaves at seedling

stage using the CTAB method with some modifications (Doyle and Doyle

1990). Based on the measurement of DNA concentration by a Beckman

spectrophotometer (Beckman, Fullerton, USA), final DNA concentration

was set as 50 ng/μl in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

The SSR markers available in public were used to identify the

polymorphisms between the parents (the mutant and B409). SSR analysis

was performed as described previously by Piquemal et al (2005).

Equivalent amounts of DNA (12 chlorophyll-deficient leaf plants and 12

normal green leaf plants) from populationⅠwere randomly selected to

construct three chlorophyll-deficient bulks (CB) and three normal green

leaf bulks (GB). All the SSR markers were used to firstly analyse the two

parents of populationⅠand then the two bulks. The amplification products

were separated on a 6% polyacrylamide denaturing sequencing gel and

visualised using a silver staining system (Lu et al. 2004) with some

modifications.

For AFLP technique, the CB and GB DNA samples were digested

with the enzyme combinations (EcoRI, MseI and SacI) to produce 12.5 μl

of digested solution, respectively. Subsequently, the digested restriction

fragment ends were ligated to specific double-stranded adapters by T4

DNA ligased. The adapter-ligated DNA was diluted five-fold and then

pre-amplified with AFLP primers (EA/MC, EA/MG, EC/MC, EC/MG,

SA/MG) into 25 μl solution. The pre-amplified products were analysed in

a 1.0% agarose gel electrophoresis, and diluted 10 to 30 folds for

selective amplification (Negi et al. 2000). The products of the selective

amplification were separated and silver stained as described for AFLP

markers.

The polymorphic AFLP fragments were cloned and sequenced as

described previously by Ke (Ke et al. 2004) and Yi (Yi et al. 2006).

These sequences of markers linked to the BnaC.YGL gene were extended

using BLAST searches in http://brassica.bbsrc.ac.uk/IMSORB/. Based on

these sequences, the specific primers were designed by the software

Primer3 (Rozen and Skaletsky 2000). These primers were used to detect

the polymorphisms in the CB and GB bulks.

Genetic map of the BnaC.YGL gene

To construct a rough flanking map, we used the BC3 population with

500 individuals which showed the BnaC.ygl mutant phenotype

(populationⅠ). For a fine map of the region around the BnaC.YGL gene,

we enlarged the backcross population I BC8 ((ygl×B409)×ygl) to 3472

plants showing the BnaC.ygl mutant phenotype and constructed the

backcross Population ⅡBC4 ((1161×ygl)×ygl) with 5288 individuals

showing the BnaC.ygl mutant phenotype. The data of these markers and

individual phenotypes were analysed with the MAPMAKER/EXP 3.0

program (Lander et al. 1987; Lincoln et al. 1992). The map order was

estimated by maximum-likelihood.

Development of markers and comparative mapping with Arabidopsis

thaliana and B. oleracea

The genome sequence from B. oleracea became available for blast

since April in 2011 (http://brassicadb.org/brad/). All the polymorphic

fragments linked to the BnaC.ygl gene were sequenced to determine their

putative homology using BLAST search against the B. oleracea genome

database (Brassica database, BRAD; http://brassicadb.org/brad/). To

identify the putative syntenic region around the BnaC.YGL gene in

Arabidopsis genome, the scaffolds in B. oleracea genome with which the

markers had homology were used to identify their homologous regions in

the Arabidopsis genome database [http://www.arabidopsis.org/]. IP

markers were developed according to the information on the

corresponding Arabidopsis loci. The detailed procedures of developing IP

makers were performed as previously described by Xia et al (2012).

Results

Reduced Chl accumulation and affected chloroplast development in

BnaC.ygl mutant

The BnaC.ygl mutant exhibited a yellow-green leaf phenotype at

seeding stage and became green at the later stage (Fig. 1a, d). Compared

with the wild type, it showed deficiency in chlorophyll and exhibited

higher level of Chl a/b (Table 1) at seedling stage. The leaves of the

BnaC.ygl mutant had 7.02% to 35.19% reduction of total Chl compared

with those in the wild type at different stages (Table 1). The Chl a and

Chl b levels in BnaC.ygl mutant and the wild type were significant

differences at 6-week-old and 14-week-old plants, but not at 20-week-old

plants. In BnaC.ygl mutant, the Chl a/b ratio appeared highest at 6-week-

old plants, which was possibly due to that the synthesis of Chl b was

more severely declined than that of Chl a. However, at the later stages,

the Chl a /b ratio declined to almost the level of the wild-type (Table 1).

The above results suggested that the BnaC.ygl mutant showed delayed

greening during photomorphogenesis due to the slow rate of Chl

accumulation. Thus, it can be speculated that when the mutant plants

accumulated a sufficient amount of Chl, which almost reached the level

of the wild type, the leaves looked almost the same as the wild-type in the

later development.

To investigate how the BnaC.ygl mutation affects chloroplast

development, we compared the ultrastructures of the plastids in the

BnaC.ygl mutant and wild-type leaves at different stages using

transmission electron microscopy. The shapes of the chloroplasts in the

BnaC.ygl mutant and wild-type plants were normal but the membrane

organizations differed substantially (Fig. 1b, c, e, f). Chloroplasts in the

developing leaves of the one-month-old wild type had abundant and well-

ordered granal stacks. In BnaC.ygl mutant, the chloroplasts displayed less

dense granal stacks and fewer granal membranes compared with those of

the wild type. However, in twenty-week-old plants, Chloroplasts in the

BnaC.ygl mutant and wild-type had well-ordered granal stacks and

normal granal membranes (Fig. 1e, f). There was no apparent difference

in the structures of chloroplasts in the BnaC.ygl mutant and wild-type.

Furthermore, the number of plastoglobules in the chloroplasts of the

BnaC.ygl mutant increased significantly (Fig. 1b, c). It has been reported

that the size and number of plastoglobules would strongly increase during

light stress, senescence and in mutants with thylakoid formation being

blocked (Brehelin et al. 2007).

Genetic analysis of the chlorophyll-deficient mutant (BnaC.ygl)

The phenotype of the filial generations obtained by the crosses of the

BnaC.ygl mutant and the normal parents (B409 and 1161) was thoroughly

investigated. The reciprocal F1 plants exhibited green leaves as B409 and

1161, which indicated that the phenotype of the BnaC.ygl mutant was

controlled by nuclear genes. The F2 populations ( FⅠ 2 and FⅡ 2) from both

crosses showed an expected Mendelian inheritance ratio of 3:1 (green:

yellow plants, χ2<χ20.05=3.84; P>0.05; Table 2). In addition, the ratio of

green plants to yellow plants in the BC1 ( BCⅠ 1and BCⅡ 1) progenies was

approximately 1:1 (χ2< χ20.05=3.84; P>0.05; Table 2). These data indicated

that the phenotype of the BnaC.ygl mutants was controlled by one single

recessive gene.

Screening of the markers for the yellow-green leaves

In our studies, 338 SSR markers available in public were used to

detect the polymorphisms between chlorophyll-deficient bulks (CB) and

normal green leaf bulks (GB) in populationⅠ. Two SSR makers (CB10299

and CB10534) in the reference map (Piquemal et al. 2005) showed

polymorphisms in this population and were located in the same linkage

group. In population Ⅱ, four of the twenty-two SSR makers from the

same linkage group were identified to be polymorphic between CB and

GB bulks. In addition to the above two markers (CB10299 and

CB10534), Ol10-F06 and BRAS019 in the reference map (Cheng et al.

2009; Piquemal et al. 2005) were identified. Fig.S1 shows the

amplification profile of the SSR maker CB10534 for 24 yellow-leaf and

24 green-leaf individuals in populationⅠ.

In the BSA analysis, the E ＋ /M ＋ , SA/MG combinations (1280

primers) were used to identify the polymorphisms between CB and GB

bulks . As a result, 47 polymorphic markers linked to the BnaC.ygl gene

were identified, 15 of which were successfully converted into the SCAR

markers (Table S1).

The sequences of all the AFLP and SSR makers were BLASTed

against the B. oleracea genome (http://brassicadb.org/brad). Most of the

makers showed sequence homology with the scaffolds of B. oleracea

(Table S2). The 240 SSR makers were designed based on the information

of the scaffolds in B. oleracea (Liu, personal communication), among

which 18 SSR makers showed polymorphisms between the CB and GB

bulks. Additionally, the SSR makers BnY3,Y4,Y5,Y6 were identified to

be polymorphic only in population while BnY2, Y7,Y8 were identified toⅠ

be polymorphic in population (Ⅱ Fig.2c, d),and the others showed

polymorphism in both populations (some data not shown in Table S2).

Based on the comparative mapping of the scaffolds in B. oleracea with

their homologous regions in Arabidopsis, 36 IP makers were designed

according to the sequences of the genes in Arabidopsis. IP4 (At3g24560)

and IP5 (At3g24590) displayed polymorphisms just in populationⅡ while

IP20(At1g20620) exhibited polymorphism in both populations.

Genetic mapping of the BnaC.ygl gene

The SSR markers (CB10299 and CB10534) were analyzed in

population BCⅠ 8 with 3472 plants. There was no recombinant between

CB10299 and BnaC.YGL gene while one recombinant was detected

between CB10534 and BnaC.YGL gene, corresponding to a distance of

0.03 cM (Fig. 2c). In a larger populationⅡBC4 which comprised 5288

yellow-green leaf individuals, CB10534 showed five recombinants with

BnaC.YGL gene, corresponding to a distance of 0.09 cM (Fig. 2d). And

BRAS019 and Ol10-F06 were on the same side with CB10534 and

located at a distance of 3.54cM and 3.37cM to the BnaC.YGL gene

respectively (Fig. 2d). The other maker CB10299 had no recombinant

with the BnaC.YGL gene in populationⅡas well as in populationⅠ(Fig.2c,

d), which suggested that the recombination between CB10299 markers

and BnaC.YGL was severely suppressed. From the above results, it could

be concluded that the BnaC.YGL gene was located on the linkage group

N17 of B. napus.

For AFLP analysis, only the 15 SCAR markers converted from AFLP

makers were used to analyze the two populations. The maker SC6 was

identified to be on the opposite side of CB10534. 60 and 82

recombination events were detected between the BnaC.YGL gene and

SC6 in population BCⅠ 8 andⅡBC4, respectively (Fig.2c, d). There was no

recombination event occurring between BnaC.YGL and other SCAR

makers in the two populations (Table S1 and Fig.2c, d). The results

indicated that the SCAR makers (except SC6) cosegregated with

BnaC.YGL, which further suggested that the recombination between the

SCAR markers and BnaC.YGL might be severely suppressed.

The developed makers in our maps were designed from the scaffold

sequences of B. oleracea to analyse the two populations. The SSR maker

BnY1, which was on the same side with SC6, was 1.70cM (population )Ⅰ

and 1.55cM (populationⅡ) away from BnaC.YGL. BnY10 and IP20 also

cosegregated with BnaC.YGL in the two populations. Additionally, the

recombinants between BnY11 and BnaC.YGL were the same as CB10534

in the two populations (Fig. 2c, d). In population , the identifiedⅠ

recombinants between BnY5, Y4, Y3, Y2 and BnaC.YGL were included

in the recombinants between SC6 and BnaC.YGL (Fig.2c, d), and BnY5

was the nearest marker at the SC6 side, which was 0.32 cM away from

BnaC.YGL. In summary, integration of the two mapping populations

enabled the BnaC.YGL locus to the interval between the markers BnY5

and CB10534, corresponding to a genetic distance of 0.35 cM (Fig. 2c,

d).

Identification of synteny in the B. oleracea genome

The sequences of the SCAR and SSR markers were submitted to the

BRAD database (http://www.brassicadb.org/brad/) for identification of

putative orthologues. Eight SCAR and ten SSR markers showed sequence

homology to the C06 of B. oleracea (Table S2). Based on the extent of

the homology between the A genome and the C genome, Panjabi et al

(2008) proposed that N16 and N17 should be designated as C07 and C06

respectively. The makers on the linkage group N17 in this study also

displayed sequence homology to C06 linkage group of B. oleracea, but

were in different orders in the maps (Fig.3). The BnY3, Y4, Y5 and

BnY1, Y2, SC6 showed sequence homology to Scaffold000280 and

Scaffold000281 respectively, corresponding to the rough region of 27000

kb -28190 kb on C06 linkage group, while BRAS019, BnY8, BnY9 and

IP4, which were at the opposite side of SC6, had sequence homology to

Scaffold000181, corresponding to the rough region of 2945 kb -3724 kb

on C06 linkage group (Table S2).However, eight makers cosegregating

with the BnaC.YGL were located on the Scaffold000078 and

Scaffold000200, respectively corresponding to the rough region of 636 kb

-1757 kb, 23612 kb -23745 kb on C06 linkage group (Table S2). These

results indicated that an inversion occurred on the N17 linkage group of

B. napus compared to the homologous region on the C06 linkage group

of B. oleracea. Moreover, three makers (CB 10299, IP20 and SC37)

cosegregating with the BnaC.YGL were homologous to the

Scaffold000087 of B. oleracea while the other two (SC20, SC39) showed

homology to the Scaffold000370 of B. oleracea (Table S2). But there was

no information about the linkage groups of Scaffold000087 and

Scaffold000370.

Discussion

In the present study, we identified a chlorophyll-deficient mutant

(BnaC.ygl), which showed yellow-green leaf phenotype at seedling stage

and became green almost like the wild type at the later stage. The

BnaC.ygl gene affected Chl biosynthesis dramatically and possibly

delayed the chloroplast development at the early developmental stage

(Fig.1 and Table 1). Previously, there were studies suggesting that Chl

synthase is more stable in chloroplasts than in etioplasts (Soll et al. 1983)

and is bound to the thylakoid membranes (Block et al. 1980; Rudiger et

al. 1980), which indicates that Chl biosynthesis is associated with

chloroplast development (Biswal et al. 2003; El-Saht 2000). Therefore,

the delayed formation of thylakoid membranes of stunted chloroplasts

might lead to the decease of Chl accumulation in the BnaC.ygl mutant at

seedling stage in the present study. In rice, the ygl1 and ylc1 mutants

display the similar yellow-green phenotype at seedling stage and also

become green with increased leaf Chl accumulation at the mature stage.

However, the reasons for the yellow-green phenotype at seedling stage

and the gradual change to normal greenness as the plant ages have not yet

been completely understood. One hypothesis is that there might be other

paralogs/ homologs with redundant functional activities at later growth

stages which eventually offset the Chl loss caused by mutations (Wu et

al. 2007; Zhou et al. 2013), but this hypothesis remains to be confirmed.

Further researches should be carried out to figure out the reasons why Chl

biosynthesis is dramatically reduced by the BnaC.ygl mutation at the

early developmental stage but restored gradually at the later stages.

As a young allopolyploid species, B. napus originated from a

spontaneous hybridisation of B. rapa (AA, 2n=20) and B. oleracea (CC,

2n=18). According to the previous comparative mappings between the

Arabidopsis and Brassica genomes, B. rapa and B. oleracea has

undergone an evolutionary process of a whole-genome triplication

(WGT) (Cheung et al. 2009; Parkin et al. 2005; Rana et al. 2004;

Snowdon et al. 2002; Town et al. 2006; Wang et al. 2011). Although the

comparative mapping of B. oleracea and B. napus has revealed perfect

collinearity, extensive chromosomal rearrangements also occur in the

subsequent polyploidization process of B. napus (Panjabi et al. 2008;

Parkin et al. 2005; Snowdon et al. 2002). In this study, we focused on the

fine mapping of the BnaC.YGL gene which was related to the yellow-

green leaf phenotype through a map-based cloning strategy. The

BnaC.YGL gene was mapped into a genetic region of 0.35cM on the

linkage group N17 of B. napus (Fig.2c). And there were eighteen makers

cosegregating with BnaC.YGL gene in the two populations, which

showed sequence homology to the scaffolds and the C06 linkage group of

B. oleracea (Table S2). The makers cosegregating with the BnaC.YGL

gene at least covered a region of 1.2 Mb in C06 linkage group of B.

oleracea. Therefore, the recombination rate is significantly lower than the

average level of the genome (approximately 600 kb per cM) in B. napus

identified in the previous studies(Raman et al. 2013). Moreover, the SSR

maker CB10299 was in the vicinity of Athila-like S-SAP marker

A100E36b (Fig.3a) designed from the Athila-like retroelement Bn21G50-

04 which was preferentially inserted in the pericentromeric regions of B.

oleracea chromosomes(Pouilly et al. 2008). Additionally, the makers

around BnaC.YGL gene in our maps were in different orders as those on

the C06 linkage group of B. oleracea (Fig.3). According to the previous

studies, conserved synteny is likely to be broken down in the vicinity of

centromeres (Pouilly et al. 2008). Parkin (1997) has found that N17

linkage group has one translocation end point coincident with the

centromere position, which provides further circumstantial evidence for

the centric fission and (or) fusion in the evolution of Brassica

chromosomes (Parkin and Lydiate 1997). These observations support our

conclusion that BnaC.ygl gene may be located in the centromeric or

pericentromeric region of the N17 linkage group, and the region around

BnaC.ygl gene was identified to be inverted on the N17 linkage group of

B. napus compared with the homologous region on the C06 linkage group

of B. oleracea.

In the present study, the region around BnaC.ygl gene was extremely

complex, which greatly hampered the identification of genes by a map-

based cloning strategy. Thus, future efforts should be focused on the

following aspects. Firstly, to construct a new population using the

materials with significant background differences to the mutant as parents

would be helpful. It has been reported that the population used for

mapping will determine the recombination frequency around a particular

locus (Bentolila and Hanson 2001; Casselman et al. 2000; Imai et al.

2003). The study of Li (Li et al. 2012) has showed that the suppression of

the recombination is significantly reduced in two new mapping

populations. Secondly, the candidate gene approach, which has been

successfully used in human and animal genetics and plant genetics, would

be an alternative strategy when the positional cloning is limited by the

lack of genetic recombination around centromeric and pericentromeric

regions (Bout and Vermerris 2003; Pflieger et al. 2001; Ramalingam et

al. 2003; Rothschild and and Soller 1997; Wayne and McIntyre 2002;

Xue et al. 2008). Currently, RNA-seq has been recognized as an efficient

method for gene discovery and remains the gold standard for annotation

of both coding and non-coding genes (Haas and Zody 2010). RNA-Seq

also provides a far more precise measurement of the levels of transcripts

and their isoforms than other methods (Wang et al. 2009). Thus, a

combination of the candidate gene and the RNA-Seq approach would be

an effective way to identify BnaC.ygl gene.

Acknowledgments: We sincerely thank Dr. Shengyi Liu (Oilcrops

Research Institute, Chinese Academy of Agricultural Sciences) for

providing the scaffold sequences of B. oleracea. This research was

financed by funds from the Program for Modern Agricultural Industrial

Technology System (nycytx-00501) ， the Nature High-tech R & D

Program “863’’ (2012AA101107, 2011AA10A104), the National Science

& Technology Support Program （2012BAD49G00, 2011BAD35B04）.

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Fig 1Phenotypic characterization of the BnaC.ygl mutant. a one-month-

old plants, BnaC.ygl (left) and wild type T6(right). d twenty-week-old

plants. b Chloroplasts of BnaC.ygl mutant with one-month-old blades

have abnormally stacked grana structures and indistinct thylakoid

membrane layers. c Chloroplasts of wild type T6 with one-month-old

blades show abundant, well-ordered stacks. e, f Chloroplasts of BnaC.ygl

mutant and wild type T6 with twenty-week-old blades have abundant,

well-ordered stacks. cp chloroplast, st starch, tm thylakoid membrane, pg

plastoglobule. Scale bar = 0.5μm (c) and 1μm (b, e, f).

Fig 2The partial linkage maps of B. napus indicate the relative location of

the BnaC.ygl gene on linkage group N17 of the reference maps. a The

linkage group N17 (Cheng et al. 2009) showing the location of OI10-F06;

b The linkage group N17 (Piquemal et al. 2005) indicating the position of

BRAS019, CB10534 and CB10299; c The genetic linkage map of the

BnaC.ygl gene and associated molecular markers in population I; d The

genetic linkage map of the BnaC.ygl gene and associated molecular

markers in populationⅡ. Dotted lines indicate the common makers.

Fig 3a The linkage group N17 (Pouilly et al. 2008) indicating that the

SSR maker CB10299 was in the vicinity of Athila-like S-SAP markers

A100E36b. b The genetic linkage map of the BnaC.ygl gene and

associated molecular markers in populationⅡ.c The scaffolds from top to

bottom are scaffold000181 ， scaffold000078 ， scaffold000200 and

scaffold000281, respectively in B. oleracea. d The part physical map of

the linkage group C06 in B. oleracea. Dotted lines between a and b

indicate the common makers, and those between b, c, d show the

locations of homologous sequences that the makers correspond to in B.

oleracea.

Table 1Pigment contents in the leaves of wild-type and BnaC.ygl mutant,

in mg/g fresh weight

Growt

h stage

Genotyp

e

Chl a Chl b Total Chl Chl a/b

Ratio

6

weeks

old

Wild

type

0.85±0.02 0.22±0.02 1.08±0.04 3.79±0.10

BnaC.yg

l

0.60±0.04*

*

0.10±0.0

1**0.70±0.0

4**5.72±0.3

2**14 Wild 0.87±0.05 0.25±0.02 1.11±0.06 3.56±0.22

weeks

old

type

BnaC.yg

l

0.63±0.0

2**0.14±0.0

1∗∗0.77±0.03*

*

4.43±0.03*

*

20

weeks

old

Wild

type

0.92±0.03 0.22±0.01 1.14±0.04 4.20±0.22

BnaC.yg

l

0.85±0.03 0.21±0.01 1.06±0.04 4.12±0.11

Chl were measured in acetone extracted from the third leaf at different

growth stages. Values shown are the mean SD (±SD) from four

independent determinations. Asterisks indicate significant differences

between wild-type and BnaC.ygl mutant at the given time (t-test, n= 4,

** P < 0.01).

Table 2Segregation of F2 and BC1from two population ( and )Ⅰ Ⅱ

Population FⅠ 2 BⅠ

C1

FⅡ 2 BⅡ

C1

Numbers of green plantsa 293 487 306 295

Numbers of yellow

plants

92 452 110 313

Total numbers 385 939 416 608

Expected 3:1 1:1 3:1 1:1

χ2b 0.19 1.23 0.3

9

0.48

aGreen plants and yellow plants were determined at seedling stage by

visual inspection. bχ2>χ20.05=3.84 is considered as significant.

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