Effects of two starch synthase IIa isoforms on grain components and other grain traits in barley

Effects of two starch synthase IIa isoforms on grain components and other grain traits in barley

Zhifen Pan1*_{, Xiaoqing Deng}1,2_{, Qiao Li}1_{, Rong Xie}1,2_{, Huisheng Zhai}1_{, Xingquan}

Zeng3,4_{, Zhaxi Luobu}3,4_{, Nyima Tashi}3,4_{, Zhongyi Li}5

1_{Chengdu Institute of Biology, Chinese Academy of Sciences, No. 9 Section 4,} Renmin South Road, Chengdu 610041, People’s Republic of China

2_{University of the Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing} 100039, People’s Republic of China

3_{Tibet Academy of Agriculture and Animal Sciences, No. 130 Jinzhu West Road,} Lhasa 850032, People’s Republic of China

4_{State Key Laboratory of Barley and Yak Germplasm Resources and Genetic} Improvement, Lhasa 850002, People’s Republic of China

5_{CSIRO Agriculture Flagship, GPO Box 1600, Canberra ACT 2601, Australia，retired}

Table S1. Summary of Nucleotide Variations in SSIIa Exons among Amplified Barley

Accessions

“-” indicates deletion; “N” indicates unsuccessful amplification.

Accession Position 1435 1544-1576 3776 3802 4216 4313 7834 Waxiro T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG G A A G N Torrens T _ G A A G N Sloop T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG G A G G N Morex T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG T A G G N Himalaya T _ G A G G N Golden premise T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG G A A G G Glacier T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG G A A G N Galleon T _ N N N N N Finnis T _ T A G G N Barque T _ G A G G N 012Z1594-1 G GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG T G G C A 012Z88 T _ G A A G G 012Z1663 G GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG N N N C N 012Z1631-3 T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG N N N G N 012Z1628-1 G GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG T A G G N 012Z1627-2 T _ N N N G N 012Z1614-3 T _ N N N G N 012Z1595-3 T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG N N N N N 012Z1594-3 G GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG T G G C N 012Z1594-2 T GCGCCGCCGTCGTCCGTTGT CCCGGCCAAGAAG G A G G N

Table S2. Different barley populations with different SSIIa isoforms

Barley accessions with different SSIIa isoforms from Australia

Accession SSIIa Type Accession SSIIa Type

Barque SSIIaL _{Riso1508 SSIIa}H

Finniss SSIIaL _{Golden premise SSIIa}H

Torrens SSIIaL _{Wanubet (naked) SSIIa}H

Him SSIIaL _{Washonvpana SSIIa}H

Galleon SSIIaL _{Waxiro SSIIa}H

Morex SSIIaH _{Wyalong SSIIa}H

Arapiles SSIIaH _{Weeah SSIIa}H

Blenheim SSIIaH _{Piccard SSIIa}H

Bomi SSIIaH _{Dash SSIIa}H

Bulokc SSIIaH _{Schooner SSIIa}H

Picola SSIIaH _{Glacier SSIIa}H

Barley cultivars with different SSIIa isoforms from China

Accession SSIIa Type Accession SSIIa Type Accession SSIIa Type Kunlun2 SSIIaL _{Zangqing1 SSIIa}L _{Beiqing14 SSIIa}H Kunlun3 SSIIaL _{Zangqing336 SSIIa}L _{Ganqing1 SSIIa}H Kunlun10 SSIIaL _{Zangqing690 SSIIa}L _{Kangqing3 SSIIa}H

Kunlun13 SSIIaL _{Fu8-4 SSIIa}L _{Kangqing6 SSIIa}H

Kunlun14 SSIIaL _{Gannongda7 SSIIa}L _{Ximala2 SSIIa}H

Kunlun164 SSIIaL _{Aqing6 SSIIa}L _{Ximala3 SSIIa}H

Beiqing4 SSIIaL _{Aqing7 SSIIa}L _{Ximala4 SSIIa}H

Beiqing5 SSIIaL _{Kangqing8 SSIIa}L _{Ximala14 SSIIa}H Beiqing7 SSIIaL _{Kangqing10-5 SSIIa}L _{Ximala15 SSIIa}H Beiqing8 SSIIaL _{Ganqing8 SSIIa}L _{Ximala17 SSIIa}H Beiqing9 SSIIaL _{Ganqing9 SSIIa}L _{Ximala19 SSIIa}H Gannan2 SSIIaL _{Diqing1 SSIIa}L _{Zangqing13 SSIIa}H Ganqing3 SSIIaL _{Diqing2 SSIIa}L _{Zangqing25 SSIIa}H Ganqing4 SSIIaL _{8908720135 SSIIa}L _{Zangqing85 SSIIa}H Ganqing5 SSIIaL _{8910720138 SSIIa}L _{Zangqing148 SSIIa}H Kangqing7 SSIIaL _{160505 SSIIa}L _{Zangqing311 SSIIa}H Ximala1 SSIIaL _{14575 SSIIa}L _{Zangqing320 SSIIa}H Ximala6 SSIIaL _{17438 SSIIa}L _{Zangqing2000 SSIIa}H Ximala7 SSIIaL _{Kangqing17 SSIIa}L _{Zangqing3179 SSIIa}H Ximala8 SSIIaL _{YF70 SSIIa}L _{Zangqing7239 SSIIa}H

Ximala9 SSIIaL _{YF127 SSIIa}L _{Aqing5 SSIIa}H

Ximala11 SSIIaL _{Kunlun1 SSIIa}H _{Kangqing9 SSIIa}H

Ximala16 SSIIaL _{Kunlun15 SSIIa}H _{C1 SSIIa}H

Ximala22 SSIIaL _{Beiqing1 SSIIa}H _{C2 SSIIa}H

Indigenous barley accessions with different SSIIa isoforms from China Accessions SSIIa Type Accessions SSIIa Type Accessions SSIIa Type

Z0202 SSIIaL _{Z0674 SSIIa}L _{Z0647 SSIIa}H

Z0236 SSIIaL _{Z0678 SSIIa}L _Z0650

SSIIaH

Z0242 SSIIaL _{Z0849 SSIIa}L _{Z0652 SSIIa}H

Z0247 SSIIaL _{Z0851 SSIIa}L _Z0681

SSIIaH

Z0262 SSIIaL _{Z0918 SSIIa}L _{Z0697 SSIIa}H

Z0263 SSIIaL _{Z0967 SSIIa}L _Z0920

SSIIaH

Z0295 SSIIaL _{Z0968 SSIIa}L _{Z0931 SSIIa}H

Z0296 SSIIaL _{Z0969 SSIIa}L _021SZ4-4

SSIIaH

Z0315 SSIIaL _{Z0115 SSIIa}H _{021SZ5-4 SSIIa}H

Z0328 SSIIaL _{ZDM4276 SSIIa}H _{021SZ7-4 SSIIa}H

Z0332 SSIIaL _{Z0123 SSIIa}H _{021SZ8-1 SSIIa}H

Z0339 SSIIaL _{ZDM4284 SSIIa}H _{021SZ9-2 SSIIa}H

Z0346 SSIIaL _{Z0214 SSIIa}H _{021SZ9-4 SSIIa}H

Z0352 SSIIaL _{Z0215 SSIIa}H _{021SZ11-1 SSIIa}H

Z0378 SSIIaL _{Z0219 SSIIa}H _{021SZ11-4 SSIIa}H

Z0379 SSIIaL _{Z0221 SSIIa}H _{021SZ12-1 SSIIa}H

Z0438 SSIIaL _{Z0235 SSIIa}H _{021SZ13-2 SSIIa}H

Z0441 SSIIaL _{ZDM4434 SSIIa}H _{021SZ13-4 SSIIa}H

Z0465 SSIIaL _{Z0282 SSIIa}H _{Heilaoya SSIIa}H

Z0479 SSIIaL _{Z0284 SSIIa}H _{Walan SSIIa}H

Z0481 SSIIaL _{Z0289 SSIIa}H _{Geer SSIIa}H

Z0543 SSIIaL _{Z0293 SSIIa}H _{Nimubai SSIIa}H

Z0551 SSIIaL _{Z0314 SSIIa}H _{021SZ241-2 SSIIa}H

Z0556 SSIIaL _{ZDM4500 SSIIa}H _{021SZ242-4 SSIIa}H

Z0561 SSIIaL _{Z0345 SSIIa}H _{021SZ245-4 SSIIa}H

Z0562 SSIIaL _{ZDM4506 SSIIa}H _{021SZ247-1 SSIIa}H

Z0567 SSIIaL _{Z0384 SSIIa}H _{021SZ248-3 SSIIa}H

Z0595 SSIIaL _Z0390 SSIIaH _{021SZ248-4 SSIIa}H Z0646 SSIIaL _Z0434 SSIIaH _{021SZ250-3 SSIIa}H Z0653 SSIIaL _Z0443 SSIIaH _{021SZ251-1 SSIIa}H

Z0654 SSIIaL _{Z0457 SSIIa}H _{021SZ251-4 SSIIa}H

Z0656 SSIIaL _Z0559

SSIIaH _{021SZ254-2 SSIIa}H

Barley progenitors with different SSIIa isoforms

Number SSIIa Type Number SSIIa Type

ZYM00025 SSIIaL

ZYM00012 SSIIaH

ZYM00044 SSIIaL _{ZYM00210-2 SSIIa}H

ZYM00151 SSIIaL

ZYM00221 SSIIaH

ZYM00210 SSIIaL

ZYM00293 SSIIaH

ZYM00212 SSIIaL _{ZYM00310 SSIIa}H

ZYM00213 SSIIaL

ZYM00315 SSIIaH

ZYM00284 SSIIaL

ZYM00316 SSIIaH

ZYM00407 SSIIaL _{ZYM00336 SSIIa}H

ZYM00529 SSIIaL

ZYM00469 SSIIaH

ZYM00590 SSIIaL

ZYM00553 SSIIaH

ZYM00606 SSIIaL _{ZYM00558 SSIIa}H

ZYM00654 SSIIaL ZYM00642 SSIIaH ZYM00839 SSIIaL ZYM00647 SSIIaH ZYM00845 SSIIaL ZYM00665 SSIIaH ZYM00006 SSIIaH ZYM00832 SSIIaH

Figure S1. Positions of PCR primers and amplified fragments of barley SSIIa. The

nucleotide positions of each primer refer to the SSIIa genomic DNA sequence Genbank No: AH011942.2.

Figure S2. Mass-spectrometric analysis on granule associated proteins in SSIIaH and SSIIaL isoform starch. (a) Band 1: barley starch branching enzyme IIa, SBEIIa

(AAC69753.1). (b) Band 1: barley starch branching enzyme IIb, SBEIIb (AAC69754.1). (c) Band 2: barley starch synthase II, SSII (AAN28307.1). (d) Band 2: barley predicted protein (BAK03716.1). (e) Band 3: barley starch synthase II, SSII (AAN28307.1). Protein bands were purified from the 1D-SDS-PAGE gel, separating proteins bound within starch granules of barley. The unipeptide residues were highlighted with green fronts and blue backgrounds. When two unipeptide residues had some amino acids overlapped, blue backgrounds were used. The upper number indicates that the number of times was detected the unipeptide residues with the blue background, and the lower numbers show that the number of times was detected for the unipeptide residues with green colour. Boxed unipepetide residue was the specific fragment encoded by the inserted 33 bp DNA fragment within the barley SSIIa gene.

10 20 30 40 50 60 70 80 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

rice Nipponbare MSSAVVASST-TFLVALASSASRGGP--RRGRVVGVAAPPALLYDGRAGRLALRAPPPPRPRPRRRDAGVVRRAD--- rice IR36--- MSSAVVASST-TFLVALASSASRGGP--RRGRVVGVAAPPALLYDGRAGRLALRAPPPPRPRPRRRDAGVVRRAD--- rice Kinmaze--- MSSAVVASST-TFLVALASSASRGGP--RRGRVVGVAAPPALLYDGRAGRLALRAPPPPRPRPRRRDAGVVRRAD--- sorghum --- MSSAAVSSASSTFFLALASSASPGGR--RRARVG--SPP----FHTGAGPNFAFWR--APPRVPRDAALVRAEAE--- maize --- MSSAAVSSSSSTFFLALAS-ASPGGR--RRARVG--SSP----FHTGASLSFAFWAPPSPPRAPRDAALVRAEAE--- barley Himalaya MSSAVASPAS---FLALAS-ASPGRSSRRRARVG--ASP----TRAGAGR--LQWRPSPLQRTARDGAVAARAAG--- barley JP--- MSSAVASPAS---FLALAS-ASPGRSSRRRARVG--ASP----TRAGAGR--LQWRPSPLQRTARDGAVAARAAG--- wheat JP--- MSSAVASAAS---FLALAS-ASPGRS-RRRTRVS--ASP----PHTGAGR--LHWPPSPPQRTARDGAVAARAAGKKDAG wheat Wyuna---- MSSAVASAAS---FLALAS-ASPGRS-RRRARVS--APP----PHAGAGR--LHWPPWPPQRTARDGGVAARAAGKKDAR wheat CS----fgh MSSAVASAAS---FLALAS-ASPGRS-RRRTRVS--ASP----PHTGAGR--LHWPPSPPQRTARDGAVAARAAGKKDAG

90 100 110 120 130 140 150 160 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

rice Nipponbare ---DGENEAAVERAGEDDDEEEEFSSGAWQPPRSRRGGVGKVLKRRGTVPPVGRYGSGGDAARVRGAAAPAPAPTQDA rice IR36--- ---DGENEAAVERAGEDDEEEEEFSSGAWQPPRSRRGGVGKVLKRRGTVPPVGRYGSGGDAARVRGAAAPAPAPTQDA rice Kinmaze--- ---DGENEAAVERAGEDDDEEEEFSSGAWQPPRSRRGGVGKVLKRRGTVPPVGRYGSGGDAARVRGAAAPAPAPTQDA sorghum --- ---GGGKDAPPERSGDAAAAA---ASRWPRAARRKTV---SKRRDPLQPVGRYGS--- maize --- ---AGGKDAPPERSGD---AARLPR-ARRNAV---SKRRDPLQPVGRYG--- barley Himalaya IDD-AAPGRQPRARRYGAATKVA---DPVKTLDRDAAEGGG---PSPPAPRQDAARLPSKNGTL---INGENKPTGGG barley JP--- IDD-AAPGRQPRARRYGAATKVA---DPVKTLDRDAAEGGG---PSPPAPRQDAARLPSKNGTL---INGENKPTGGG wheat JP--- IDD-AAPARQPRALRGGAATKVAERRDPVKTLDRDAAEGGA---PSPPAPRQEDARLPSMNGMP---VNGENKSTGGG wheat Wyuna---- VDDDAASARQPRARRGGAATKVAERRDPVKTLDRDAAEGGA---PAPPAPRQDAARPPSMNGTP---VNGENKSTGGG wheat CS----fgh IDD-AAPARQPRALRGGAATKVAERRDPVKTLDRDAAEGGA---PSPPAPRQEDARLPSMNGMP---VNGENKSTGGG

170 180 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare ASSKNGALLSGRDDDTPASRNGSVVTGADKPAAATPPVTITKLPAPDSPVILPSVDKPQPEFVIPDATAPAP--- rice IR36--- ASSKNGALLSGRDDDTPASRNGSVVTGADKPAAATPPVTITKLPAPDSPVILPSVDKPQPEFVIPDATAPAP--- rice Kinmaze--- ASSKNGALLSGRDDDTPASRNGSVVTGADKPAAATPPVTITKLPAPDSPVILPSVDKPQPEFVIPDATAPAP--- sorghum --- ---SGTGNTARSGAATASTQNAALAAVEIKSIVAAPPTSIVKFPAPGYRVILPSGDI-APETVVPAPKP--- maize --- ---SATGNTARTGAA--SCQNAALADVEIKSIVAAPPTSIVKFPAPGYRMILPSGDI-APETVLPAPK--- barley Himalaya GATKDSGLPTPARAPHLSIQNRVPVNGENKHKVASPPTSIVDVASPGSAANISISNK-VPPSVVPAKKTPPSSVFPAKK- barley JP--- GATKDSGLPTPARAPHLSIQNRVPVNGENKHKVASPPTSIVEVASPGSAANISISNK-VPPSVVPAKKTPPSSVFPAKKA wheat JP--- GATKDSGLPAPARAPQPSSQNRVPVNGENKANVASPPTSIAEVAAPDPAATISISDK-APESVVPAEKAPPS--- wheat Wyuna---- GATKDSGLPAPARAPHPSTQNRVPVNGENKANVASPPTSIAEVVAPDSAATISISDK-APESVVPAEKPPPS--- wheat CS----fgh GATKDSGLPAPARAPQPSSQNRVPVNGENKANVASPPTSIAEVAAPDPAATISISDK-APESVVPAEKAPPS---

250 260 270 280 290 300 310 320 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare ---PPPGSNPRSSAP-LPKPDNSEFAEDKSAKVVESAPKPKATRSSPIPAVEEETWDFKKYFDLNEPDA rice IR36--- ---PPPGSNPRSSAP-LPKPDNSEFAEDKSAKVVESAPKPKATRSSPIPAVEEETWDFKKYFDLNEPDA rice Kinmaze--- ---PPPGSNPRSSAP-LPKPDNSEFAEDKSAKVVESAPKPKATRSSPIPAVEEETWDFKKYFDLNEPDA

sorghum --- ---LPLHGSPGVDGDYSNGVAP---PPAEPVVQEATWDFKKYIGFEEPVE maize --- ---PLHESPAVDGD-SNGIAP---PTVEPLVQEATWDFKKYIGFDEPDE barley Himalaya ---TLPSSGSNFVSSASAPRLDTVSDVELAQKKDALIVKEAPKPKALSAPAAPAVQEDLWDFKKYIGFEEPVE barley JP--- PPSSVVPAKKTLPSSGSNFVSSASAPRLDTVSDVELAQKKDALIVKEAPKPKALSAPAAPAVQEDLWDFKKYIGFEEPVE wheat JP--- ---SGSNFVPSASAPGSDTVSDVELELKKGAVIVKEAPNPKALSPPAAPAVQQDLWDFKKYIGFEEPVE wheat Wyuna---- ---SGSNFVVSASAPRLDIDSDVEPELKKGAVIVEEAPNPKALSPPAAPAVQEDLWDFKKYIGFEEPVE wheat CS----fgh ---SGSNFVPSASAPGSDTVSDVELELKKGAVIVKEAPNPKALSPPAAPAVQQDLWDFKKYIGFEEPVE

330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare AEDGDDDDDWADSDASDSEIDQDDDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVAGALPKALARRGHRVMVVVPRYGD rice IR36--- AEDGDDDDDWADSDASDSEIDQDDDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVAGALPKALARRGHRVMVVVPRYGD rice Kinmaze--- AEDGDDDDDWADSDASDSEIDQDDDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVAGALPKALARRGHRVMVVVPRYGD sorghum --- AKD---DSGVAADGADSFEHYENNDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVVGALPKALARRGHRVMVVVPRYGD maize --- AKD---DSRVGADDAGSFEHYGDNDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVVGALPKALARRGHRVMVVVPRYGD barley Himalaya AKD---DGSAVADDAGSFEHHQNHDSGPLAGENVMNVVVVAAECSPWCKTGGLGDVAGALPKALAKRGHRVMVVVPRYGD barley JP--- AKD---DGSAVADDAGSFEHHQNHDSGPLAGENVMNVVVVAAECSPWCKTGGLGDVAGALPKALAKRGHRVMVVVPRYGD wheat JP--- AKD---DGRAVADDAGSFEHHQNHDSGPLAGENVMNVVVVAAECSPWCKTGGLGDVAGALPKALAKRGHRVMVVVPRYGD wheat Wyuna---- AKD---DGWAVADDAGSFEHHQNHDSGPLAGENVMNVVVVAAECSPWCKTGGLGDVAGALPKALAKRGHRVMVVVPRYGD wheat CS----fgh AKD---DGRAVADDAGSFEHHQNHDSGPLAGENVMNVVVVAAECSPWCKTGGLGDVAGALPKALAKRGHRVMVVVPRYGD

410 420 430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare YAEAQDVGIRKYYKAAGQDLEVKYFHAFIDGVDFVFIDAPLFRHRQDDIYGGNRQEIMKRMILFCKAAVEVPWHVPCGGV rice IR36--- YAEAQDVGIRKYYKAAGQDLEVKYFHAFIDGVDFVFIDAPLFRHRQDDIYGGNRQEIMKRMILFCKAAVEVPWHVPCGGV rice Kinmaze--- YAEAQDVGIRKYYKAAGQDLEVKYFHAFIDGVDFVFIDAPLFRHRQDDIYGGNRQEIMKRMILFCKAAVEVPWHVPCGGV sorghum --- YVEAFDMGIRKYYKAAGQDLEVNYFHAFIDGVDFVFIDAPLFRHRQNDIYGGSRQEILKRMILFCKVAVEVPWHVPCGGV maize --- YVEAFDMGIRKYYKAAGQDLEVNYFHAFIDGVDFVFIDAPLFRHRQDDIYGGSRQEIMKRMILFCKVAVEVPWHVPCGGV barley Himalaya YEEAYDVGVRKYYKAAGQDMEVNYFHAYIDGVDFVFIDAPLFRHRQQDIYGGSRQEIMKRMILFCKAAVEVPWHVPCGGV barley JP--- YEEAYDVGVRKYYKAAGQDMEVNYFHAYIDGVDFVFIDAPLFRHRQQDIYGGSRQEIMKRMILFCKAAVEVPWHVPCGGV wheat JP--- YEEAYDVGVRKYYKAAGQDMEVNYFHAYIDGVDFVFIDAPLFRHRQEDIYGGSRQEIMKRMILFCKAAVEVPWHVPCGGV wheat Wyuna---- YEEAYDVGVRKYYKAAGQDMEVNYFHAYIDGVDFVFIDAPLFRHRQEDIYGGSRQEIMKRMILFCKAAVEVPWHVPCGGV wheat CS----fgh YEEAYDVGVRKYYKAAGQDMEVNYFHAYIDGVDFVFIDAPLFRHRQEDIYGGSRQEIMKRMILFCKAAVEVPWHVPCGGV

490 500 510 520 530 540 550 560 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare PYGDGNLVFLANDWHTALLPVYLKAYYRDNGMMQYTRSVLVIHNIAYQGRGPVDEFPYMELPEHYLDHFKLYDPVGGEHA rice IR36--- PYGDGNLVFLANDWHTALLPVYLKAYYRDNGMMQYTRSVLVIHNIAYQGRGPVDEFPYMELPEHYLDHFKLYDPVGGEHA rice Kinmaze--- PYGDGNLVFLANDWHTALLPVYLKAYYRDNGMMQYTRSVLVIHNIAYQGRGPVDEFPYMELPEHYLDHFKLYDPVGGEHA sorghum --- CYGDGNLVFIANDWHTALLPVYLKAYYTDHGLMQYTRSILVIHNIAHQGRGPVAEFPYMDLPEHYLQHFELYDPVGGEHA maize --- CYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYTRSVLVIHNIAHQGRGPVDEFPYMDLPEHYLQHFELYDPVGGEHA barley Himalaya PYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYSRSVMVIHNIAHQGRGPVDEFPFTELPEHYLEHFRLYDPVGGEHA barley JP--- PYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYTRSVMVIHNIAHQGRGPVDEFPFTELPEHYLEHFRLYDPVGGEHA wheat JP--- PYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYTRSIMVIHNIAHQGRGPVDEFPFTELPEHYLEHFRLYDPVGGEHA

wheat Wyuna---- PYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYTRSIMVIHNIAHQGRGPVDEFPFTELPEHYLEHFRLYDPVGGEHA wheat CS----fgh PYGDGNLVFIANDWHTALLPVYLKAYYRDHGLMQYTRSIMVIHNIAHQGRGPVDEFPFTELPEHYLEHFRLYDPVGGEHA

570 580 590 600 610 620 630 640 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare NIFGAGLKMADRVVTVSPGYLWELKTTEGGWGLHDIIRENDWKMNGIVNGIDYREWNPEVDVHLQSDGYANYTVASLDSS rice IR36--- NIFGAGLKMADRVVTVSPGYLWELKTTEGGWGLHDIIRENDWKMNGIVNGIDYREWNPEVDVHLQSDGYANYTVASLDSG rice Kinmaze--- NIFGAGLKMADRVVTVSPGYLWELKTTEGGWGLHDIIRENDWKMNGIVNGIDYREWNPEVDVHLQSDGYANYTVASLDSS sorghum --- NIFAAGLKMADRVVTVSRGYLWELKTVEGGWGLHDIIRSNDWKINGIVNGIDHQEWNPKVDVHLRSDGYTNYSLETLDAG maize --- NIFAAGLKMADRVVTVSRGYLWELKTVEGGWGLHDIIRSNDWKINGIVNGIDHQEWNPKVDVHLRSDGYTNYSLETLDAG barley Himalaya NYFAAGLKMADQVVVVSPGYLWELKTVEGGWGLHDIIRQNDWKTRGIVNGIDNMEWNPEVDVHLKSDGYTNFSLKTLDSG barley JP--- NYFAAGLKMADQVVVVSPGYLWELKTVEGGWGLHDIIRQNDWKTRGIVNGIDNMEWNPEVDVHLKSDGYTNFSLKTLDSG wheat JP--- NYFAAGLKMADQVVVVSPGYLWELKTVEGGWGLHDIIRQNDWKTRGIVNGIDNMEWNPEVDVHLKSDGYTNFSLGTLDSG wheat Wyuna---- NYFAAGLKMADQVVVVSPGYLWELKTVEGGWGLHDIIRQNDWKTRGIVNGIDNMEWNPEVDVHLKSDGYTNFSLGTLDSG wheat CS----fgh NYFAAGLKMADQVVVVSPGYLWELKTVEGGWGLHDIIRQNDWKTRGIVNGIDNMEWNPEVDVHLKSDGYTNFSLGTLDSG

650 660 670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare KPRCKAALQRELGLEVRDDVPLIGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVLLGSGRRDLEVMLQRFEAQHNSKVRGW rice IR36--- KPRCKAALQRELGLEVRDDVPLIGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVLLGSGRRDLEVMLQRFEAQHNSKVRGW rice Kinmaze--- KPRCKAALQRELGLEVRDDVPLIGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVLLGSGRRDLEVMLQRFEAQHNSKVRGW sorghum --- KRQCKAALQRELGLEVRDDVPLLGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVMLGTGRADLERMLQHLEREHPSKVRGW maize --- KRQCKAALQRELGLEVRDDVPLLGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVMLGTGRADLERMLQHLEREHPNKVRGW barley Himalaya KRQCKEALQRELGLQVRGDVPLLGFIGRLDGQKGVEIIADAMPWIVSQDVQLVMLGTGRHDLESMLQHFEREHHDKVRGW barley JP--- KRQCKEALQRELGLQVRGDVPLLGFIGRLDGQKGVEIIADAMPWIVSQDVQLVMLGTGRHDLESMLQHFEREHHDKVRGW wheat JP--- KRQCKEALQRELGLQVRGDVPLLGFIGRLDGQKGVEIIADAMPWIVSQDVQLVMLGTGRHDLEGMLRHFEREHHDKVRGW wheat Wyuna---- KRQCKEALQRELGLQVRADVPLLGFIGRLDGQKGVEIIADAMPWIVSQDVQLVMLGTGRHDLESMLRHFEREHHDKVRGW wheat CS----fgh KRQCKEALQRELGLQVRGDVPLLGFIGRLDGQKGVEIIADAMPWIVSQDVQLVMLGTGRHDLEGMLRHFEREHHDKVRGW

730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| rice Nipponbare VGFSVKMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTMSAFDPFEDTGLGWTFDRAEPHKLIEAL rice IR36--- VGFSVKMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVSAFDPFEDTGLGWTFDRAEPHKLIEAL rice Kinmaze--- VGFSVKMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVSAFDPFEDTGLGWTFDRAEPHKLIEAL sorghum --- VGFSVPMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVAPFDPFGDAGLGWTFDRAEVNKLIEAL maize --- VGFSVPMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVAPFDPFSDAGLGWTFDRAEANKLIEAL barley Himalaya VGFSVRLAHRITAGADALLMPSRFEPCGLNQLYAMAYGTIPVVHAVGGLRDTVPPFDPFNHSGLGWTFDRAEAHKLIEAL barley JP--- VGFSVRLAHRITAGADALLMPSRFEPCGLNQLYAMAYGTIPVVHAVGGLRDTVPPFDPFNHSGLGWTFDRAEAHKLIEAL wheat JP--- VGFSVRLAHRITAGADALLMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVPPFDPFNHSGLGWTFDRAEAQKLIEAL wheat Wyuna---- VGFSVRLAHRITAGADALLMPSRFEPCGLNQLYAMAYGTVPVVHAVGGVRDTVPPFDPFNHSGLGWTFDRAEAHKLIEAL wheat CS----fgh VGFSVRLAHRITAGADALLMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVPPFDPFNHSGLGWTFDRAEAQKLIEAL

810 820 830 840 ....|....|....|....|....|....|....|....|....|.

rice Nipponbare GHCLETYRKYKESWRGLQVRGMSQDLSWDHAAELYEEVLVKAKYQW rice IR36--- GHCLETYRKYKESWRGLQVRGMSQDLSWDHAAELYEEVLVKAKYQW rice Kinmaze--- GHCLETYRKYKESWRGFQVRGMSQDLSWDHAAELYEEVLVKAKYQW sorghum --- RHCLDTYRNYGESWKSIQARGMAQDLSWDHAAELYEDVLVKAKYQW maize --- RHCLDTYRNYEESWKSLQARGMSQDLSWDHAAELYEDVLVKAKYQW barley Himalaya GHCLRTYRDHKESWRGLQERGMSQDFSWEHAAKLYEDVLVQAKYQW barley JP--- GHCLRTYRDHKESWRGLQERGMSQDFSWEHAAKLCEDVLVQAKYQW wheat JP--- GHCLRTYRDYKESWRGLQERGMSQDFSWEHAAKLYEDVLVKAKYQW wheat Wyuna---- GHCLRTYRDYKESWRGLQERGMSQDFSWEHAAKLYEDVLLKAKYQW wheat CS----fgh GHCLRTYRDYKESWRGLQERGMSQDFSWEHAAKLYEDVLVKAKYQW

Figure S3. Alignment of SSIIa peptide sequences of rice, sorghum, maize, barley, and wheat. The

11-amino acids InDel locates from 240 to 250 amino acids. The peptide sequences are encoded by the cDNA sequences of starch synthase IIa (SSIIa) as follows. Rice Nipponbare (GenBank No: AB115915), rice IR36(GenBank No: AB115917),rice Kinmaze(GenBank No: AB115916), sorghum (GenBank No: EU620718), maize (GenBank No: NM001301653), barley Himalaya (GenBank No: AY133249), barley JP (GenBank No: AK372518), wheat JP (GenBank No: AJ269504), wheat Wyuna (GenBank No: AF155217), wheat CS (GenBank No: AK455479).