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Supplementary Information

Supplementary Figures 1-21, Supplementary Tables 1-5 and supporting references

Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice

grain weight and increases yield

Ken Ishimaru1,4

, Naoki Hirotsu

1,4, Yuka Madoka

1, Naomi Murakami

1, Nao Hara

1,

Haruko Onodera1, Takayuki Kashiwagi

1, Kazuhiro Ujiie

1, Bun-ichi Shimizu

2, Atsuko

Onishi3, Hisashi Miyagawa

3 & Etsuko Katoh

1

1National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki

305-8602, Japan.

2Graduate School of Life Science, Toyo University, Itakura, Gunma 374-0193, Japan.

3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,

Kyoto 606–8502, Japan.

Nature Genetics: doi:10.1038/ng.2612

2

4These authors contributed equally to this work. Correspondence should be addressed

to: K. I. ([email protected]).


3

Supplementary Fig. 1. Amino acid sequence alignment of TGW6 from Nipponbare and

Kasalath. Conserved amino acids shown in red box.


4

Supplementary Fig. 2. Thousand grain weight in Nipponbare and Nipponbare

RNAi-transformed No.2 (T1). The same transformant was used in Fig. 1c. Data are means ±

s.d. (N = 5). Student’s t-test was used to generate P values.


5

Supplementary Fig. 3. Relationship between relative expression of TGW6 and grain lengths

of RNAi- transformed No.2, 4 and 8 (T1). Expression of TGW6 in leaves was analyzed using

real-time PCR. Actin was used as a control. Data are means ± s.d. (N = 3).


6

Supplementary Fig. 4. Grain length in RNAi-transformed NIL(TGW6) (T0) and control

plants, NIL(TGW6). Data presented are means ± s.d. (N = 10). Student’s t-test was used to

generate P values.


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Supplementary Fig. 5. Grain length (A) and ten grain weight (B) in transformants carrying

the Nipponbare tgw6 driven by the Kasalath promoter in NIL(TGW6) (T0) and control plants

(NIL(TGW6) and Nipponbare). Data presented are means ± s.d. (N = 10). Student’s t-test was

used to generate P values.


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Supplementary Fig. 6. Expression of TGW6 in Nipponbare and NIL(TGW6). Expression of

TGW6 in flag leaves was analyzed using real-time PCR. Actin was used as a control. Data are

means ± s.d. (N = 5). Student’s t-test was used to generate P values.


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Supplementary Fig. 7. Expression of core cell cycle genes in the endosperm of NIL(TGW6)

relative to expression levels in Nipponbare at 4 days after fertilization as determined by

real-time PCR. Data shown are means ± s.d. (N = 3). CycB1;1, Orysa;CyclinB1;1

(Os01t0805600-01); CycB2;2, Orysa;CyclinB2;2 (Os06g0726800); CycD2;2,

Orysa;CyclinD2;2 (Os07g0620800); Histon H2A, (Os08g0427700), E2F1, (Os02g0537500).


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Supplementary Fig. 8. Structure-based sequence alignment of TGW6 with strictosidine

synthase (STR1)1 and diisopropylfluorophosphatase (DFPase)

2. The sequence motifs of STR1

and DFPase that were homologous to TGW6 motifs, and that had known 3D structures within

the Protein Data Bank (PDB) were identified (Supplementary Table 1) using a BLASTP

search3. Structure-based sequence alignments of TGW6 with STR1 and DFPase were

performed both manually and with the ClustalX program4. As several signal peptide

prediction programs such as SignalP 3.05,6

and SOSUI7 indicated a possible cleavage site

between Ala30 and Ala31 in TGW6, we excluded the putative signal peptide from our further

studies of sequence and structure similarities between TGW6 and other proteins. Blue and green

background indicates identical and conservatively replaced amino acids, respectively. Blue and

red boxes show β-strands and α-helixes, respectively, in STR1 and DFPase. Active residues are

marked with magenta dots. The catalytic Ca2+

binding residues are highlighted with blue dots.

The putative active residues and catalytic Ca2+

binding residues of TGW6 (compare

Supplementary Fig. 6) are marked with pink and yellow dots, respectively. TGW6 possesses

36% sequence homology with STR1. There are 11 two helices (Lys86 to Glu90 and Ala95 to

TGW6 1:MRSTARQAATAAAFALIVFLVLLSPSPTAAATATTRMFKTIDARRSQHLDLGGSLVGPES 60

STR1 1:---------------MAKLSDSQTMALFTVFLLFLSSSLALSSPILKEILIEAPSYAPNS 45

DFPase 1:--------------------------------------MEIPVIEPLFTKVTEDIPGAEG 22

TGW6 61:VAFDGKGRGPYSGV--------SDGRIMRWNGEAAGWSTYTYSPSYT-KNKCAASTLPTV 111

STR1 46:FTFDSTNKGFYTSV--------QDGRVIKYEGPNSGFVDFAYASPYWNKAFCENSTD--A 95

DFPase 23:PVFDKNGD-FYIVAPQVEVNGKPAGEILRIDLKTGKKTVIC-----------------KP 64

TGW6 112:QTESKCGRPLGLRFHYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDI 171

STR1 96:EKRPLCGRTYDISYNLQNNQLYIVDCYYHLSVVGSEGGHATQLATSVDGVPFKWLYAVTV 155

DFPase 65:EVNGYGGIPAGCQCDRDANQLFVADMRLGLLVVQTDGTFEEIAKKDSEGRRMQGCNDCAF 124

TGW6 172:DQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMS 231

STR1 156:DQRTGIVYFTDVSTLYDDRGVQQIMDTSDKTGRLIKYDPSTKETTLLLKELHVPGGAEVS 215

DFPase 125:DYE-GNLWITAPAGEVAPADYTRSMQEKF--GSIYCFTTDGQMIQVDTAFQF-PNGIAVR 180

TGW6 232:----ADRTHLIVALTGPCKLMRHWIRGP-KTGKSEPFVDLP----GYPDNVRPDGKGGYW 282

STR1 216:----ADSSFVLVAEFLSHQIVKYWLEGP-KKGTAEVLVKIP-----NPGNIKRNADGHFW 265

DFPase 181:HMNDGRPYQLIVAETPTKKLWSYDIKGPAKIENKKVWGHIPGTHEGGADGMDFDEDNNLL 240

TGW6 283:IALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPT--EVMERKDG--KIYMGN 338

STR1 266:VSSSEELDGNMHGRVDPKGIKFD-EFGNILEVIPLPPPFAGEHFEQIQEHDG--LLYIGT 322

DFPase 241:VANW--------GSSHIEVFGPD--GGQPKMRIRCP----FEKPSNLHFKPQTKTIFVTE 286

TGW6 339:VELPYVGVVKSS 350

STR1 323:LFHGSVGILVYDKKGNSFVSSH 344

DFPase 287:HENNAVWKFEWQRNGKKQYCETLKFGIF 314


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Arg98) which are connected by a conserved disulfide bridge (between Cys89 and Cys101)

throughout the STR family. The two Cys residues (Cys103 and Cys117) of TGW6 are found at

the same position as in STR1. Therefore, the three dimensional structure of TGW6 appears to be

very similar to STR1, and the crystalline structure of STR1 might be a suitable template on

which to model the structure of TGW6 (related to Supplementary Fig. 9).


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Supplementary Fig. 9. Structural comparison of the modeled TGW6 structure (A)

with crystal structures of STR1 (B) and DFPase (C)2. The TGW6 model was built

using the Sybyl-ORCHESTRAR program based on the STR1 crystalline structure as a

template (B). The catalyticly active residues of STR1 (Glu309) and DFPase (Trp244

and His287) are shown as magenta sticks. The catalytic Ca2+ binding residues of

DFPase (Glu21, Asn112, Asn117 and Asp229) are highlighted as blue sticks. The

catalytic Ca2+ ion is shown as a green sphere. Cavities representing idealized substrate

binding sites called protomols were calculated by Surflex-Dock8 and are shown in

yellow. The catalytic active residues of STR1 and DFPase are located at the surface of

these pockets but at different sites. The catalytic Ca2+-binding residues of DFPase are

located at the bottom of the pocket. These four residues form a plane quadrangle with

approximately equal distances to the catalytic Ca2+. We compared the amino acid

residues located at the pocket surface of TGW6 with the active residues of STR1 and

DFPase. Four residues (Glu59, Asn167, Asn226 and Asp271) shown as cyan sticks

were located at the bottom of the TGW6 pocket at similar positions as the active

residues of DFPase. Furthermore, the side chains of His192 and Tyr224 which are

located at the surface of the TGW6 pocket (pink sticks) could possibly create a

catalytic dyad. Based on these results, TGW6 may be expected to possess hydrolytic activity. In fact, although β-propeller proteins have a surprising variety of functions,

roughly half of them are enzymes9. In particular, hydrolytic activity has been reported for numerous six-bladed β-propeller proteins2,10-13.


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Supplementary Fig. 10. Docking structure of TGW6 and IAA-glucose. IAA-glucose in the

lowest-energy docked location is shown in blue. Four residues (Glu59, Asn167, Asn226 and

Asp271) shown as yellow sticks and the side chains of His192 and Tyr224 (magenta) which are

located at the surface of the TGW6 pocket could possibly create a catalytic dyad. IAA-glucose

was modeled using Sybyl 8.1 (Tripos Inc.) and checked for allowable geometries. The initial

conformation of IAA-glucose, derived from IAA and glucose taken from the pdb database, was

energy-minimized using the Tripos force field. Formal charges were calculated automatically.

The docking program Surflex-Dock 2.38, a Sybyl 8.1.1 module, was employed to dock the

IAA-glucoses into the protomols. The protomols had been generated automatically using the

protomol mapping protocol within Surflex-Dock 2.3 as described previously8, which explored

the entire TGW6 surface. The resulting conformational energies were checked for steric clashes

and unrealistic geometries.


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Supplementary Fig. 11. Growth rates of endosperm length (A) and weight (B), calculated from

data shown in Fig. 2a, b.


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Supplementary Fig. 12. Starch contents of lower leaf sheaths of Nipponbare and NIL(TGW6)

just before heading. Data are means ± s.d. (N = 5). Student’s t-test was used to generate P

values.


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Supplementary Fig. 13. Relative expression levels of genes related to starch synthesis

following IAA treatment in leaf sheaths of NIL(TGW6) before heading, as determined

by real-time PCR using gene specific primers15. Data shown are means ± s.d. (N = 3).

Student’s t-test was used to generate P values.


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Supplementary Fig. 14. Expression of genes related to starch synthesis. Genes of the starch

synthesis pathway in endosperm cells and their expression levels at 15 days after heading in

NIL(TGW6) (A) and in plants in which the flag leaf had been removed (B) were analyzed by

real-time PCR using gene specific primers15. Red and blue boxes highlight genes whose

expression levels were significantly (P < 0.05) increased and decreased, respectively, compared

to Nipponbare. NIL(TGW6) increased the transcription levels of starch synthesis-related genes

in grains of intact plants, while the decreased source capacity in plants with removed flag leaves

correlated with a reduced expression of these genes. Removal of the flag leaf caused a reduction

of grain weight (20.60 mg ± 3.3) compared to the control (27.43 mg ± 0.47, P = 0.001), whereas

grain length remained unaffected (5.59 mm ± 0.06, compared to 5.65 mm ± 0.05, P = 0.078; all

data given are means ± s.d.).


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Supplementary Fig. 15. Proposed model for the role of TGW6 in regulating grain length and

weight. Loss of function in the Kasalath allele (TGW6) has desirable effects. Through

pleiotropic effects on sink and source organs, TGW6 enhances grain weight.


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Supplementary Fig. 16. Proposed model for the roles of TGW6, GW2, GS3, SW5, GS5, GW8

and GlF1 in regulating grain size and weight. TGW6 affects both source and sink tissues. In

contrast to TGW6, other genes do not function in the endosperm but control the size of the

spikelet hull, and thus indirectly regulate the width of the endosperm. Higher translocation rates

from a high-capacity source lead to greater accumulation in an expanded sink with an increased

number of endosperm cell layers, leading to increased grain length and consequently, a higher

final yield.


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Supplementary Fig. 17. Haplotype pattern of the TGW6 gene in the world rice collection

(WRC). (A) Comparison of the ORF sequences of 69 varieties revealed nine mutations

compared to the Nipponbare allele. Eight mutations are 1-bp substitutions, and one is a 1-bp

deletion (FNP). Four SNPs cause amino acid substitution (blue triangles). (B) Sequences of 69

WRC varieties arranged according to mutation patterns (left). Polymorphisms are indicated by

red boxes. The variety names shown in green, blue, and orange represent the japonica, indica1,

and Indica 2 (aus) groups, respectively. The letters in the dendrogram (left) stand for the same

haplogroups as in the haplotype network (C) Haplotype network of the TGW6 gene generated

by comparison of the ORF sequences of 69 varieties and 14 wild rice lines. (compare to Fig. 3a).

(D) Sequences of TGW6 alleles in various cultivars. (E) Model of the TGW6 protein. Mutated

and predicted active site residues are shown in magenta and blue, respectively. The mutated

positions are not located in the putative active site. (F) Effect of the IR64 tgw6 allele on grain

length. NIL(tgw6) contains IR64 tgw6 in the Koshihikari background. Data presented are means

± s.d. (N = 7). Student’s t-test was used to generate P values.


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Supplementary Fig. 18. Grain length (A) and thousand grain weight (B) in plants of the

Koshihikari background that were heterozygote (Nipponbare tgw6/Kasalath TGW6) or

homozygote (Kasalath TGW6); Koshihikari served as the control. Data are means ± s.d. (N = 8).

Means were separated using the least significant difference (LSD) test at probability of P = 0.05

only when the F-test showed significance at 0.05 probability level. Significant differences

according to LSD are indicated by different letters. These data show that the Kasalath TGW6

allele has semi-dominant effects.


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Supplementary Fig. 19. Husk length in the Koshihikari background NIL(TGW6) and control

plants (Koshihikari). Data are means ± s.d. (N = 3). Student’s t-test was used to generate P

values.


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Supplementary Fig. 20. Effect of low-source treatment on the proportion of perfect grains in

Koshihikari and NIL(TGW6). Data shown are means ± s.d. (N = 7). Student’s t-test was used to

generate P values.


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Supplementary Fig. 21. Ambient temperature at ripening stage of Koshihikari and NIL(TGW6)

in the last 5 years and 2010. Heading date of both lines was the same (1st August) in Tsukuba,

Japan 2010.


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Supplementary Table 1 Grain shape of Nipponbare and NIL(TGW6). Data are means ± s.d. (N

=10).

Length (mm) Width (mm) Thickness (mm)

Nipponbare 5.34±0.03 2.84±0.02 1.91±0.01

NIL(TGW6) 5.66±0.03 2.91±0.02 1.93±0.01

P value 0.0020 0.1356 0.3046


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Supplementary Table 2 Query results for the TGW6 primary structure according to a PDB

sequence identity search (BLASTP search).

PDB code Protein name Identity

%

Query

coverage

E-values

2FP8

2IAV

Strictosidine Synthase

Diisopropyl Fluorophosphatase 36(55)

25(42)

72

58

3 × e-43

0.002


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Supplementary Table 3 Relative gene expression levels of auxin-responsive genes

determined in young grains (3 days after flowering) by microarray analysis16. The log-ratio,

factors of change, and P values of expression levels in NIL(TGW6) compared to

Nipponbare are given. Blue and red fonts indicate gene expression levels that are

significantly (P < 0.05) increased and decreased, respectively.

Locus Accession Name Description Log ratio Fold change P

Os09g0545700. AK062898 Auxin induced protein -1.01 10.26 0.00

Os02g0445100 AK063247 OsSAUR8 Auxin-induced SAUR-like protein -0.59 3.87 0.00

Os01g0802700. AK059229 Auxin Efflux Carrier family protein -0.55 3.51 0.01

Os02g0445600 AK063150 Auxin-induced SAUR-like protein (Fragment) -0.46 2.91 0.01

Os08g0118500 CI261909 OsSAUR31 Auxin responsive SAUR protein family protein -0.45 2.82 0.00

Os09g0547100 AK058556 OsSAUR55 Auxin induced protein -0.43 2.70 0.00

Os09g0554300 AK070682 Auxin Efflux Carrier family protein -0.38 2.43 0.00

Os04g0664400 AB071292 OsARF11 Auxin response factor 1 -0.36 2.32 0.00

Os06g0691400 AK107208 IAA-amino acid conjugate hydrolase-like protein (Fragment) -0.35 2.26 0.00

Os06g0660200 AK101191 Auxin efflux carrier component 2 (AtPIN2) -0.35 2.23 0.00

Os03g0342900 AK060136 Dormancyauxin associated family protein -0.30 2.01 0.00


Os09g0546900 AK062365 OsSAUR53 Auxin induced protein -0.29 1.96 0.02


Os01g0785400 AK063368 OsGH3-1 GH3 auxin-responsive promoter family protein. -0.27 1.85 0.00



Os04g0664400 AK103452 OsARF11 Auxin response factor 1 -0.22 1.67 0.00



Os09g0546000 Auxin responsive SAUR protein family protein -0.21 1.61 0.00


Os10g0510500 AK068270 OsSAUR56 Auxin responsive SAUR protein family protein -0.20 1.59 0.00


Os07g0182400 AK103483 OsIAA24 AUX/IAA protein family protein -0.19 1.56 0.00

Os10g0510500 AK109491 OsSAUR56 Auxin responsive SAUR protein family -0.18 1.53 0.00


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Os05g0559400 AK104018 OsIAA19 IAA6 (Fragment) -0.17 1.48 0.01

Os01g0190300 AK100314 OsIAA2 AUX/IAA protein family protein. -0.15 1.40 0.02


Os05g0559400 AK109363 OsIAA19 IAA6 (Fragment) -0.13 1.36 0.03




Os05g0500900 AK101932 OsGH3-4 Auxin-responsive-like protein 0.16 1.45 0.01

Os06g0714300 AK107043 OsSAUR29 Auxin responsive SAUR protein family protein 0.21 1.63 0.00

Os05g0586200 AK071721 OsGH3-5 GH3 auxin-responsive promoter family protein 0.27 1.85 0.00

Os11g0221300 OsIAA29 AUX/IAA protein family protein 0.27 1.87 0.00


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Supplementary Table 4 List of rice and wild rice lines used for constructing the haplotype

network. The cultivars or accessions which have a Kasalath-type FNP are highlighted in blue.

1. World rice core collection

WRC No. Name Group* Origin FNP

WRC01 NIPPONBARE Japonica Japan Nipponbare

WRC02 KASALATH Indica 2 (Aus) India Kasalath

WRC03 BEI KHE Indica 1 Cambodia Nipponbare

WRC04 JENA 035 Indica 2 (Aus) Nepal Nipponbare

WRC05 NABA Indica 1 India Nipponbare

WRC06 PULUIK ARANG Indica 1 Indonesia Nipponbare

WRC07 DAVAO 1 Indica 1 Philipines Nipponbare

WRC09 RYOU SUISAN KOUMAI Indica 1 China Nipponbare

WRC10 SHUUSOUSHU Indica 1 China Nipponbare

WRC100 VANDARAN Indica 1 Sri Lanka Nipponbare

WRC11 JINGUOYIN Indica 1 China Nipponbare

WRC12 DAHONGGU Japonica China Kasalath

WRC13 ASU Indica 1 Bhutan Nipponbare

WRC14 IR 58 Indica 1 Philipines Nipponbare

WRC15 CO 13 Indica 1 India Nipponbare

WRC16 NARY FUTUI Indica 1 Madagascar Nipponbare

WRC17 KEIBOBA Indica 1 China Nipponbare

WRC18 QINGYU(SEIYU) Indica 1 China Nipponbare

WRC19 DENG PAO ZHAI Indica 1 China Nipponbare

WRC20 TADUKAN Indica 1 Philipines Nipponbare

WRC21 SHWE NANG GYI Indica 1 Myanmar Nipponbare

WRC22 CALOTOC Indica 1 Philipines Nipponbare

WRC23 LEBED Indica 1 Philipines Nipponbare

WRC24 PINULUPOT 1 Indica 1 Philipines Nipponbare

WRC25 MUHA Indica 2 (Aus) Indonesia Nipponbare

WRC26 JHONA 2 Indica 2 (Aus) India Nipponbare

WRC27 NEPAL 8 Indica 2 (Aus) Nepal Nipponbare

WRC28 JARJAN Indica 2 (Aus) Bhutan Nipponbare


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WRC29 KALO DHAN Indica 2 (Aus) Nepal Nipponbare

WRC30 ANJANA DHAN Indica 2 (Aus) Nepal Nipponbare

WRC31 SHONI Indica 2 (Aus) Bangladesh Nipponbare

WRC32 TUPA 121-3 Indica 2 (Aus) Bangladesh Nipponbare

WRC33 SURJAMUKHI Indica 2 (Aus) India Nipponbare

WRC34 ARC 7291 Indica 2 (Aus) India Nipponbare


WRC36 RATUL Indica 2 (Aus) India Nipponbare



WRC39 BADARI DHAN Indica 2 (Aus) Nepal Nipponbare

WRC40 NEPAL 555 Indica 2 (Aus) India Nipponbare

WRC41 KALUHEENATI Indica 2 (Aus) Sri Lanka Nipponbare

WRC42 LOCAL BASMATI Indica 2 (Aus) India Nipponbare

WRC43 DIANYU 1 Japonica China Nipponbare

WRC44 BASILANON Japonica Philipines Nipponbare

WRC45 MA SHO Japonica Myanmar Nipponbare

WRC46 KHAO NOK Japonica Laos Nipponbare

WRC47 JAGUARY Japonica Brazil Nipponbare

WRC48 KHAU MAC KHO Japonica Vietnam Nipponbare

WRC49 PADI PERAK Japonica Indonesia Nipponbare

WRC50 REXMONT Japonica USA Nipponbare

WRC51 URASAN 1 Japonica Japan Nipponbare

WRC52 KHAU TAN CHIEM Japonica Vietnam Nipponbare

WRC53 TIMA Japonica Bhutan Nipponbare

WRC55 TUPA 729 Japonica Bangladesh Nipponbare

WRC57 MILYANG 23 Indica 1 Korea Nipponbare

WRC58 NEANG MENH Indica 1 Cambodia Kasalath

WRC59 NEANG PHTONG Indica 1 Cambodia Nipponbare

WRC60 HAKPHAYNHAY Indica 1 Laos Nipponbare

WRC61 RADIN GOI SESAT Indica 1 Malaysia Kasalath

WRC62 KEMASHIN Indica 1 Malaysia Nipponbare

WRC63 BLEIYO Indica 1 Thailand Nipponbare


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WRC64 PADI KUNING Indica 1 Indonesia Nipponbare

WRC65 RAMBHOG Indica 1 India Nipponbare

WRC66 BINGALA Indica 1 Myanmar Nipponbare

WRC67 PHULBA Japonica India Nipponbare

WRC68 KHAO NAM JEN Japonica Laos Nipponbare

WRC97 CHIN GALAY Indica 1 Myanmar Nipponbare

WRC98 DEEJIAOHUALUO Indica 1 China Nipponbare

WRC99 HONG CHEUH ZAI Indica 1 China Nipponbare

2. Wild rice core collection (O. rufipogon)

Rank Name Old species Origin FNP

Rank1 W0106 O. spontanea India Nipponbare

Rank1 W0120 O. perennis India Nipponbare

Rank1 W1294 O. perennis Philippines Nipponbare

Rank1 W1866 O. perennis Thailand Nipponbare

Rank1 W1921 O. perennis Thailand Nipponbare

Rank1 W2003 O. perennis India Nipponbare

Rank2 W0630 O. perennis Myanmar Nipponbare

Rank2 W1236 O. perennis New Guinea Nipponbare

Rank2 W1807 O. nivara Sri Lanka Nipponbare

Rank2 W1945 O. perennis - Nipponbare

Rank2 W2051 - Bangladesh Nipponbare

Rank2 W2078 O. rufipogon Australia Nipponbare

Rank2 W2263 - Cambodia Nipponbare

Rank3 W1690 O. perennis Thailand Kasalath


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Supplementary Table 5 Primers used for the analysis of molecular markers and of TGW6. *thrombin-cleavage sites are underlined.

Name Type Forward primer (5'-3') Reverse primer (5'-3') SNP-specific primer (5'-3')

G001 Acycloprime FP method AGTGATGCTAATCTCAATGCTA CCAACAAGATTCACACTGCATG CTGATGAACCCAGGAATTTAG

G025 Acycloprime FP method GCCTATGTTTGCATAGCACTCC CCATGGGTGCTAGTTAGGTAG GGTACTAAACCACCTCCTACAAC

G040 Acycloprime FP method CCCCTCGGCGGCTTTGCTTAG GCTCGTAAGAGGTTTTAATACAG GCATTTCACCACACCACAATA

G203 PCR direct sequence GAGACCAACTGAAGTGATGGGA GGATTTGCGCTGAGAATTATGCC

G210 PCR direct sequence ACCTGGATCACGGACGGTGCCA GACATCGGCTGGCCAATTGCTG

G214 PCR direct sequence TTATCCACAGTACTTTAAGCAC GGACACACATCCAACAAGTCTT

G217 PCR direct sequence CCACGCTGCTCCTCCAATCTCT GCGTCGGCGATGTACAGGTTGC

G230 PCR direct sequence CAATCACGCAGCAGATCAAT CATCCTGCATCCTGGAATCT

G232 PCR direct sequence GGGAGCGATCGAGAAGTTTA TGTCGCAATTTACACGCAAT

G234 PCR direct sequence AAGAAGTGCAACAGCAGCAG CTGCCACGAGGCAAGTAAAT

G239 PCR direct sequence TTTGACGGAGGCAGTAGCTT TGCATAGCACATGCAGTCAA

TGW6 coding region for cloning GGTACCCATGAGAAGCACGGCGAGG AACCATCCTGCATCCTGGAATC

TGW6 promoter region for cloning AAGCTTTATGGGTAGTCACCAAATG GGTACCGCTGTAGCCTGTAGGAGCT

TGW6 for real-time PCR TTGAACTTGCAAAAGGCAGA CGGTTCCCCTAATGCAGAT

OsSUT1 for real-time PCR AGTTCCGGTCGGTCAGCAT ACCGAGGTGGCAACAAAG

Actin for real-time PCR GACTCTGGTGATGGTGTCAGC GGCTGGAAGAGGACCTCAGG

CycB1;1 for real-time PCR GGATTTACCGAGTCACAGCT CAAGCGTGTCAAGATTCCAA

Histone H2A for real-time PCR CGCGGGACAACAAGAAGAC CCTTCTTGGGGAGGAGCAG

E2F1 for real-time PCR GGTTGGGCAATGTAGCAACT AATGCTGACATCCCCTTCTG

TGW6 for cDNA CACCCTGGTGCCACGCGGTTCTGCCACAGCCACAACGAGAATGTTCA* TCACTAGCTGCTTTTGACGACTCCGACATACG


33

Supporting References

1. Stockigt, J., Barleben, L., Panjikar, S. & Loris, E.A. Plant Physiol. Biochem. 46, 340

(2008).

2. Scharff, E.I. et al. Structure 9, 493 (2001).

3. Berman, H.M. et al. Nat. Struct. Biol. 7, 957 (2000).

4. Thompson, J.D. et al. Nucl. Acids Res. 24, 4876 (1997).

5. Emanuelsson, O., Brunak, S., von Heijne, G. & Nielsen, H. Nat. Protoc. 2, 953 (2007).

6. Bendtsen, J.D., Nielsen, H., von Heijne, G. & Brunak, S. J. Mol. Biol. 340, 783

(2004).

7. Hirokawa, T., Boon-Chieng, S. & Mitaku, S. Bioinformatics 14, 378 (1998).

8. Jain, A.N. J. Comput. Aided Mol. Des. 21, 281 (2007).

9. Jawad, Z. & Paoli, M. Structure 10, 447 (2002).

10. Harel, M. et al. Nat. Struct. Mol. Biol. 11, 412 (2004).

11. Ha, N.C. et al. Nat. Struct. Mol. Biol. 7, 147 (2000).

12. Brandstetter, H., Kim, J.S., Groll, M. & Huber, R. Nature 414, 466 (2001).

13. Carapito, R. et al. J. Biol. Chem. 284, 12285 (2009).

14. Hirose, T. et al. Physiol. Plant. 425, 128 (2006).

15. Yamakawa, H., Hirose, T., Kuroda, M. & Yamaguchi, T. Plant Physiol. 144, 258

(2007).

16. Sato, Y. et al. BMC Plant Biol. 11:10 (2011).


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