Supplemental Figure S1. Ion Fragmentation Analysis of Anthocyanins Present in Wild Type

1

A.

B.

C.

D.

E.

Supplemental Figure S1.Ion fragmentation analysis of anthocyanins present in wild type and b mutant lines. A, Delphinidin-glucose-rhamnose (JI 2822 peak 1, see Fig. 2A). B, Petunidin-glucose-rhamnose (JI 2822 peak 2, see Fig 2A). C, Cyanidin-3-sambubioside-5-glucoside (FN 2271/3/pink, see Fig. 2C). D, Cyanidin-3-rhamnoside-5-glucoside (FN 2271/3/pink, see Fig. 2C). E, Peonidin-3-rhamnoside-5-glucoside peaks (2271/3/pink, see Fig. 2C). Full ion spectra (MS full) and secondary fragmentation spectra (MS2) are shown.

A.

>ADW66160 Pisum sativum

MILILYQTFLLKELSISMFIFFITHFILTFLSKKHHKKLPPGPNGYPFLGALPLMGAMPHLTLFKMSQKYGPIMYLKMGSNNMVVASSPSSAKAFLKTLDQNFSNRPLNAGATHLAYDSQDMVFADYGSRWKLLRKLSNLHMLGGKALEDWSKIREDEMGHMIHTMYDSSKKNESIVLPEMLTYAMANMIGQIILSRRVFETKGCESNEFKDMVVELMTTAGYFNIGDFIPILAWLDLQGIERGMKSLHKKFDALLTKMIEEHVASSHKNPRVKPDFLDTLIAQSKQDSDGGEKLTITNIKAVLLNLFTAGTDTSSSIIEWALAEMLKNPNIMKRAHEEMDRVIGKQRRLQDSDIQNLPYLQAICKETFRKHPSTPLNLPRVSSEACEVNGYYIPKNTRLSVNIWAIGRDPNVWENPLEFNPDRFLSGENAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSFDWKLPNGVVALDMDESFGLALQKKVPLAVVVTPRLSPSAYIP

>CU651565_9 MEDICAGO truncatula

mvitlyqtflykelsislfiflithlffrtfllkinhkklppgpkgypivgalplmgtmphltlfkmsqkygpvmylkmgshnmvvastpssakaflktldlnfsnrppnagathlaydsqdmvfadygsrwkllrklsnlhmlggkaledwskirgdemghmirtmydcskkdesivvsemltyamanmigqvilsrrvfetkgyesnefkdmvvelmttagyfnigdfipilawldlqgiergmkslhkkfdalltkmieehvasslkshrvkpdfldrliaqskedsdgeklsltnikalllnlftagtdtsssiiewalaemlknpkimkkvheemdqvigkqrrfqeadiqklpylqaicketyrkhpstplnlprvslepcqvngyyipkntrlsvniwaigrdpnvwenplefnperfltgknakidprgndfelipfgagrricagtrmgivlvqyilgtlvhsfdwklpngvvdlnmdesfglalqkkvplaaivsprlspsayil

>CU651565_21 MEDICAGO truncatula

mafdafllrdigiaififfishffiglilrntsknlppgprgwpilgvlphlgtmphvtlanmakkfgpimylkmgtcdtvvasssdaaraflktldhnflnrptiigatylgynsqdlvfakygpkwkllrklttlhmlggkalqnwanvrenevkhmvrsihesgkkgesievggllscaitnmvsqvvlskrmfaskgqeskefkemvvefmtisginigdfvpfigwidlegvvgkmkrlhkrfdefltkliedhvncaherkgnpdfldivlandddnfgerlslsnikalllyhfiiltqmylflyiknlftagtdtsssiiewalaemlkspniliraqkemnqvvgrermlvesdleklpylqaicketyrlhpstplsvprvsnkacqingyyipkntrfnvniwaigrdpniwanplefkperflsgkyaridpsgvdfelipfgagrrvcvgykmaivvieyilgtlvhsfdwklpngvelnmdeafgltlekavplsatvtprlvphayv

>LjT34E09_40 LOTUS Japonicus

MASTLDLMFLLREIFVSFFIFLVTRFSFRSLFKSHHQKLPPGPRGWPVLGALPLMGSMPHVTLSNMAKKYGPVMYLKMGTNSLVVASTPAAARAFLKTLDLNFSNRPPNAGATHLAYDSQDMVFAEYGSRWKLLRKLSNLHMLGGKALDDWAQFRDQEMGYMLRAMYDCSKKGEAVVVPEMLTYAMANMIGQVILSRRVFETKGSESNEFKDMVVELMTVAGYFNIGDFIPALGWFDLQGIEGGMKALHKKFDSLLTRMIEEHLASSHKRKLKPDFLDVVMAYQSENSGGEKLSLTNIKALLLNLFTAGTDTSSSIIEWSLAEMLNNPKIMRKAHEEMDRVIGKDRRLRESDLPNLPYFQAICKETYRKHPSTPLNLPRISTEPCEVNGYYIPKNTRLNVNIWAIGRDPDVWENPLEYNPERFLSGRNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSYDWKLPPGVKELDMEEAFGLALQKKVPLSVMVTPRLPPSAYIP

>BAJ14024 Glycine max CLARK

MDSLLLLKEIATSILIFLITRLSIQTFLKSYRQKLPPGPKGWPVVGALPLMGSMPHVTLAKMAKKYGPIMYLKMGTNNMVVASTPAAARAFLKTLDQNFSNRPSNAGATHLAYDARDMVFAHYGSRWKLLRKLSNLHMLGGKALDDWAQIRDEEMGHMLGAMYDCNKRDEAVVVAEMLTYSMANMIGQVILSRRVFETKGSESNEFKDMVVELMTVAGYFNIGDFIPFLAKLDLQGIERGMKKLHKKFDALLTSMIEEHVASSHKRKGKPDFLDMVMAHHSENSDGEELSLTNIKALLLNLFTAGTDTSSSIIEWSLAEMLKKPSIMKKAHEEMDQVIGRDRRLKESDIPKLPYFQAICKETYRKHPSTPLNLPRISSEPCQVNGYYIPENTRLNVNIWAIGRDPDVWNNPLEFMPERFLSGKNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVHYILGTLVHSFDWKLPNGERELDMEESFGLALQKKVPLAALVTPRLNPSAYIS

>BAF49293 Clitoria ternatea

MFLLREIGVSILIFMITHLVIRLVLKEKEQRKLPPGPKGWPIVGALPLMGSMPHVTLSEMAKKYGPVMYLKMGTNNMAVASTPSAARAFLKTLDLNFSNRPPNAGATHLAYDAQDMVFADYGSRWKLLRKLSNLHMLGGKALEEWSQVREIEMGHMLRAMYDCSGGGDGNNDNDGNKKKGTRHEPIVVAEMLTYAMANMIGQVILSRRVFETKGSESNEFKDMVVQLMTVAGYFNIGDFIPFLARFDLQGIERGMKTLHNKFDVLLTTMIHEHVASAHKRKGKPDFLDVLMAHHTNESHELSLTNIKALLLNLFTAGTDTSSSIIEWALAEMLINPKIMKKVHEEMDKVIGKDRRLKESDIENLPYLQAICKETYRKHPSTPLNLPRISSQACQVNGYYIPKNTRLSVNIWAIGRDPNVWENPLEFNPERFMGANKTIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSFDWKLPNGVVELNMEETFGLALQKKIPLSALITPRLPPTAYNVINS

>BAE86871 Gentiana scabra

MPIKMSPIYATLTLHLAAALFLFFHVQKLVHYLHGKATGHRLPPGPTGWPILGALPLLGNMPHVTFANMAKKYGPVMYLKVGSHGLAIASTPDAAKAFLKTLDLNFANRPPNAGATHLAYNAQDMVFAHYGPKWKLLRKLSNLHMLGGKALENWADVRKTELGYMLKAMFESSQNNEPVMISEMLTYAMANMLSQVILSRRVFNKKGAKSNEFKDMVVELMTSAGYFNIGDFIPSIGWMDLQGIEGGMKRLHKKFDVLLTRLLDDHERTSHERKQKPDFLDFVIANGDNSDGERLNTDNIKALLLNLFTAGTDTSSSIIEWALAELLKNRTLLTRAQDEMDWVIGRDRRLLESDIPNLPYLQAICKETFRKHPSTPLNLPRIASEPCEVNGYYIPKGTRLNVNIWAIGRDPSVWGDNPNEFDPERFLYGKNAKIDPRGNDFELIPFGAGRRICAGTRMGILLVEYILGTLVHSFDLKLGLPEEELNMDETFGLALQKAVPLAAMLIPRLPLHVYAP

>ABH06585 Vitis vinifera

MAIDTSLLLELAAATLLFFITRFFIRSLLLKSSRKLPPGPKGWPLVGALPLLGNMPHVALAKMAKRYGPVMFLKMGTNSMVVASTPGAARAFLKTLDINFSNRPPNAGATLLAYHAQDMVFADYGARWKLLRKLSNLHMLGGKALEDWSQVRAVELGHMLRAMLELSQRAEPVVVPEMLTFSMANMIGQVILSRRVFETKGSESNEFRDMVVELMTTAGYFNIGDFIPSIAWLDIQGIQRGMKHLHRKFDRLLTKMMEEHTASAHERKGNPDFLDVIMANQENSTGEKLTITNIKALLLNLFTAGTDTSSSVIEWSLAEMLKNPSILKRAHEEMDQVIGRSRRLVESDLPKLPYLQAICKESFRKHPSTPLNLPRVSTQACEVNGYYIPKNTRLSVNIWAIGRDPDVWESPEEFRPERFLSGRNEKIDPRGNDFELIPFGAGRRICAGTRMGIVLVEYILGTLVHSFDWKMPDGVEINMDEAFGLALQKAVSLSAMVTPRLHQSAYAV

>P48418 Petunia HYBrida

MMLLTELGAATSIFLIAHIIISTLISKTTGRHLPPGPRGWPVIGALPLLGAMPHVSLAKMAKKYGAIMYLKVGTCGMAVASTPDAAKAFLKTLDINFSNRPPNAGATHLAYNAQDMVFAHYGPRWKLLRKLSNLHMLGGKALENWANVRANELGHMLKSMSDMSREGQRVVVAEMLTFAMANMIGQVMLSKRVFVDKGVEVNEFKDMVVELMTIAGYFNIGDFIPCLAWMDLQGIEKRMKRLHKKFDALLTKMFDEHKATTYERKGKPDFLDVVMENGDNSEGERLSTTNIKALLLNLFTAGTDTSSSAIEWALAEMMKNPAILKKAQAEMDQVIGRNRRLLESDIPNLPYLRAICKETFRKHPSTPLNLPRISNEPCIVDGYYIPKNTRLSVNIWAIGRDPQVWENPLEFNPERFLSGRNSKIDPRGNDFELIPFGAGRRICAGTRMGIVMVEYILGTLVHSFDWKLPSEVIELNMEEAFGLALQKAVPLEAMVTPRLQLDVYVP

>NP196416 Arabidopsis thaliana tt7

MATLFLTILLATVLFLILRIFSHRRNRSHNNRLPPGPNPWPIIGNLPHMGTKPHRTLSAMVTTYGPILHLRLGFVDVVVAASKSVAEQFLKIHDANFASRPPNSGAKHMAYNYQDLVFAPYGHRWRLLRKISSVHLFSAKALEDFKHVRQEEVGTLTRELVRVGTKPVNLGQLVNMCVVNALGREMIGRRLFGADADHKADEFRSMVTEMMALAGVFNIGDFVPSLDWLDLQGVAGKMKRLHKRFDAFLSSILKEHEMNGQDQKHTDMLSTLISLKGTDLDGDGGSLTDTEIKALLLNMFTAGTDTSASTVDWAIAELIRHPDIMVKAQEELDIVVGRDRPVNESDIAQLPYLQAVIKENFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDPLAFKPERFLPGGEKSGVDVKGSDFELIPFGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAGGVTPEKLNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSG

>NP001064333 Oryza sativa

MEVAAMEISTSLLLTTVALSVIVCYALVFSRAGKARAPLPLPPGPRGWPVLGNLPQLGGKTHQTLHEMTKVYGPLIRLRFGSSDVVVAGSAPVAAQFLRTHDANFSSRPRNSGGEHMAYNGRDVVFGPYGPRWRAMRKICAVNLFSARALDDLRAFREREAVLMVRSLAEASAAPGSSSPAAVVLGKEVNVCTTNALSRAAVGRRVFAAGAGEGAREFKEIVLEVMEVGGVLNVGDFVPALRWLDPQGVVARMKKLHRRFDDMMNAIIAERRAGSLLKPTDSREEGKDLLGLLLAMVQEQEWLAAGEDDRITDTEIKALILNLFVAGTDTTSTIVEWTMAELIRHPDILKHAQEELDVVVGRDRLLSESDLSHLTFFHAIIKETFRLHPSTPLSLPRMASEECEIAGYRIPKGAELLVNVWGIARDPAIWPDPLEYKPSRFLPGGTHTDVDVKGNDFGLIPFGAGRRICAGLSWGLRMVTMTAATLVHAFDWQLPADQTPDKLNMDEAFTLLLQRAEPLVVHPVPRLLPSAYNIA

>ABH06586 Vitis vinifera

MNPLALFFCTALFCVLLYHFLTRRSVRLPPGPKPWPIVGNLPHLGPVPHHSIAALAKTYGPLMHLRMGFVDVVVAASASVAAQFLKTHDANFSNRPPNSGAKHIAYNYQDLVFAPYGPRWRMLRKICSVHLFSGKALDDFRHIRQEEVAVLTRALARAGQTPVNLGQLLNVCTTNALGRVMLGRRVFGDGSGGEDPKADEFKEMVVELMVLAGVFNIGDFVPALEWLDLQGVAAKMKKLHARFDAFLGAIVEEHKISGSAGSERHVDLLSTLISVRDNADGEGGKLTDVEIKALLLNLFTAGTDTSSSTVEWAIAELIRHPEMMAQAQQELDAVVGRGRLVTDLDLPQLTYLQAIIKETFRLHPSTPLSLPRMAAESCEINGYHIPKNATLLVNVWAIARDPEVWEKPLEFRPSRFLPGGERPNADVRGNDFEVIPFGAGRRICAGMSLGLRMVHLLTATLVHAFNWELPEGQVAEKLNMDEAYGLTLQRAPPLMVHPRPRLSPQVFGK

>BAB83261 Glycine max

MSPLIVALATIAAAILIYRIIKFITRPSLPLPPGPKPWPIVGNLPHMGPVPHHSLAALARIHGPLMHLRLGFVDVVVAASASVAEQFLKIHDSNFSSRPPNAGAKYIAYNYQDLVFAPYGPRWRLLRKLTSVHLFSGKAMNEFRHLRQEEVARLTCNLASSDTKAVNLGQLLNVCTTNALARAMIGRRVFNDGNGGCDPRADEFKAMVMEVMVLAGVFNIGDFIPSLEWLDLQGVQAKMKKLHKRFDAFLTSIIEEHNNSSSKNENHKNFLSILLSLKDVRDDHGNHLTDTEIKALLLNMFTAGTDTSSSTTEWAIAELIKNPQILAKLQQELDTVVGRDRSVKEEDLAHLPYLQAVIKETFRLHPSTPLSVPRAAAESCEIFGYHIPKGATLLVNIWAIARDPKEWNDPLEFRPERFLLGGEKADVDVRGNDFEVIPFGAGRRICAGLSLGLQMVQLLTAALAHSFDWELEDCMNPEKLNMDEAYGLTLQRAVPLSVHPRPRLAPHVYSMSS

>NP182079 cytochrome P450 76C4 Arabidopsis thaliana

MDIISGQALFLLFCFISSCFLISTTARSRRSSGRAATLPPGPPRLPIIGNIHQVGKNPHSSFADLAKIYGPIMSLKFGCLNSVVITSPEAAREVLRTHDQILSGRKSNDSIRCFGHEEVSVIWLPPSSARWRMLRKLSVTLMFSPQRTEATKALRMKKVQELVSFMNESSERKEAVDISRASYTTVLNIISNILFSVDLGSYDSKKSNEFQDTVIGAMEAAGKPDAANYFPFMGFLDLQGNRKAMRGLTERLFRVFRGFMDAKIAEKSLGNYSKDVSNRDFLDSLLILNEGDEAELDNNDIEHLLLDMFTAGTDTSSSTLEWAMAELLRNPKTMVKAQAEMDRVLGQNSVVQESDISGLPYLQAVVKETFRLHPAAPLLVPRKAESDVEVLGFMVPKDTQVLVNVWAIGRDPSVWENPSQFEPERFMGKDIDVKGRDYELTPFGGGRRICPGLPLAVKTVSLMLASLLYSFDWKLPNGVVSEDLDMDETFGITLHRTNTLYAIPVKKQTIN

>NP775426 cytochrome P450 2D10 Rattus norvegicus

MELLNGTGLWPMAIFTVIFILLVDLMHRHQRWTSRYPPGPVPWPVLGNLLQVDPSNMPYSMYKLQHRYGDVFSLQMGWKPMVIVNRLKAVQEVLVTHGEDTADRPPVPIFKCLGVKPRSQGVVFASYGPEWREQRRFSVSTLRTFGMGKKSLEEWVTKEAGHLCDAFTAQNGRSINPKAMLNKALCNVIASLIFARRFEYEDPYLIRMLTLVEESLIEVSGFIPEVLNTFPALLRIPGLADKVFQGQKTFMAFLDNLLAENRTTWDPAQPPRNLTDAFLAEVEKAKGNPESSFNDENLRMVVVDLFTAGMVTTATTLTWALLLMILYPDVQRRVQQEIDEVIGQVRCPEMTDQAHMPYTNAVIHEVQRFGDIAPLNLPRITSCDIEVQDFVIPKGTTLIINLSSVLKDETVWEKPLRFHPEHFLDAQGNFVKHEAFMPFSAGRRACLGEPLARMELFLFFTCLLQHFSFSVPAGQPRPSTLGNFAISVAPLPYQLCAAVREQGH

B.

ABH06586 ------MNPLALFFCTALFCVLLY---HFLTRRSVR--LPPGPKPWPIVGNLPHLG- 45

BAB83261 ------MSPLIVALATIAAAILIYRIIKFITRPSLP--LPPGPKPWPIVGNLPHMG- 48

NP196416 ------MATLFLTILLATVLFLILRIFSHRRNRSHNNRLPPGPNPWPIIGNLPHMG- 50

NP001064333 -MEVAAMEISTSLLLTTVALSVIVCYALVFSRAGKARAPLPLPPGPRGWPVLGNLPQLG- 58

ADW66160 --MILILYQTFLLKELSISMFIFFITHFIL-TFLSKKHHKKLPPGPNGYPFLGALPLMG- 56

CU651565_9 --MVITLYQTFLYKELSISLFIFLITHLFFRTFLLKINHKKLPPGPKGYPIVGALPLMG- 57

LJT34E09_40 --MASTLDLMFLLREIFVSFFIFLVTRFSFRSLFKSHHQ-KLPPGPRGWPVLGALPLMG- 56

BAJ14024 ------MDSLLLLKEIATSILIFLITRLSIQTFLKSYRQ-KLPPGPKGWPVVGALPLMG- 52

BAF49293 ------MFLLREIGVSILIFMITHLVIRLVLKEKEQRKLPPGPKGWPIVGALPLMG- 50

ABH06585 -----MAIDTSLLLELAAATLLFFITRFFIRSLLLKSSR-KLPPGPKGWPLVGALPLLG- 53

BAE86871 MPIKMSPIYATLTLHLAAALFLFFHVQKLVHYLHGKATGHRLPPGPTGWPILGALPLLG- 59

P48418 ------MMLLTELGAATSIFLIAHIIISTLISKTTGRHLPPGPRGWPVIGALPLLG- 50

CU651565_21 -----MAFDAFLLRDIGIAIFIFFISHFFIGLILRNTSK-NLPPGPRGWPILGVLPHLG- 53

NP182079 ----MDIISGQALFLLFCFISSCFLISTTARSRRSSGRAATLPPGPPRLPIIGNIHQVG- 55

NP775426 ----MELLNGTGLWPMAIFTVIFILLVDLMHRHQRWTSR--YPPGPVPWPVLGNLLQVDP 54

ABH06586 PVPHHSIAALAKTYGPLMHLRMGFVDVVVAASASVAAQFLKTHDANFSNRPPNSGAKHIA 105

BAB83261 PVPHHSLAALARIHGPLMHLRLGFVDVVVAASASVAEQFLKIHDSNFSSRPPNAGAKYIA 108

NP196416 TKPHRTLSAMVTTYGPILHLRLGFVDVVVAASKSVAEQFLKIHDANFASRPPNSGAKHMA 110

NP001064333 GKTHQTLHEMTKVYGPLIRLRFGSSDVVVAGSAPVAAQFLRTHDANFSSRPRNSGGEHMA 118

ADW66160 AMPHLTLFKMSQKYGPIMYLKMGSNNMVVASSPSSAKAFLKTLDQNFSNRPLNAGATHLA 116

CU651565_9 TMPHLTLFKMSQKYGPVMYLKMGSHNMVVASTPSSAKAFLKTLDLNFSNRPPNAGATHLA 117

LJT34E09_40 SMPHVTLSNMAKKYGPVMYLKMGTNSLVVASTPAAARAFLKTLDLNFSNRPPNAGATHLA 116

BAJ14024 SMPHVTLAKMAKKYGPIMYLKMGTNNMVVASTPAAARAFLKTLDQNFSNRPSNAGATHLA 112

BAF49293 SMPHVTLSEMAKKYGPVMYLKMGTNNMAVASTPSAARAFLKTLDLNFSNRPPNAGATHLA 110

ABH06585 NMPHVALAKMAKRYGPVMFLKMGTNSMVVASTPGAARAFLKTLDINFSNRPPNAGATLLA 113

BAE86871 NMPHVTFANMAKKYGPVMYLKVGSHGLAIASTPDAAKAFLKTLDLNFANRPPNAGATHLA 119

P48418 AMPHVSLAKMAKKYGAIMYLKVGTCGMAVASTPDAAKAFLKTLDINFSNRPPNAGATHLA 110

CU651565_21 TMPHVTLANMAKKFGPIMYLKMGTCDTVVASSSDAARAFLKTLDHNFLNRPTIIGATYLG 113

NP182079 KNPHSSFADLAKIYGPIMSLKFGCLNSVVITSPEAAREVLRTHDQILSGRKSNDSIRCFG 115

NP775426 SNMPYSMYKLQHRYGDVFSLQMGWKPMVIVNRLKAVQEVLVTHGEDTADRPPVPIFKCLG 114

ABH06586 YN--YQDLVFAPYG-PRWRMLRKICSVHLFS-GKALDDFRHIRQEEVAVLTRALARAGQT 161

BAB83261 YN--YQDLVFAPYG-PRWRLLRKLTSVHLFS-GKAMNEFRHLRQEEVARLTCNLASSDTK 164

NP196416 YN--YQDLVFAPYG-HRWRLLRKISSVHLFS-AKALEDFKHVRQEEVGTLTRELVRVGTK 166

NP001064333 YN--GRDVVFGPYG-PRWRAMRKICAVNLFS-ARALDDLRAFREREAVLMVRSLAEASAA 174

ADW66160 YD--SQDMVFADYG-SRWKLLRKLSNLHMLG-GKALEDWSKIREDEMGHMIHTMYDSS-- 170

CU651565_9 YD--SQDMVFADYG-SRWKLLRKLSNLHMLG-GKALEDWSKIRGDEMGHMIRTMYDCS-- 171

LJT34E09_40 YD--SQDMVFAEYG-SRWKLLRKLSNLHMLG-GKALDDWAQFRDQEMGYMLRAMYDCS-- 170

BAJ14024 YD--ARDMVFAHYG-SRWKLLRKLSNLHMLG-GKALDDWAQIRDEEMGHMLGAMYDCN-- 166

BAF49293 YD--AQDMVFADYG-SRWKLLRKLSNLHMLG-GKALEEWSQVREIEMGHMLRAMYDCSGG 166

ABH06585 YH--AQDMVFADYG-ARWKLLRKLSNLHMLG-GKALEDWSQVRAVELGHMLRAMLELS-- 167

BAE86871 YN--AQDMVFAHYG-PKWKLLRKLSNLHMLG-GKALENWADVRKTELGYMLKAMFESS-- 173

P48418 YN--AQDMVFAHYG-PRWKLLRKLSNLHMLG-GKALENWANVRANELGHMLKSMSDMS-- 164

CU651565_21 YN--SQDLVFAKYG-PKWKLLRKLTTLHMLG-GKALQNWANVRENEVKHMVRSIHESG-- 167

NP182079 HE--EVSVIWLPPSSARWRMLRKLSVTLMFS-PQRTEATKALRMKKVQELVSFMNESS-- 170

NP775426 VKPRSQGVVFASYG-PEWREQRRFSVSTLRTFGMGKKSLEEWVTKEAGHLCDAFTAQN-- 171

ABH06586 P------VNLGQLLNVCTTNALGRVMLGRRVFGDGSGGEDPKADEFKE 203

BAB83261 A------VNLGQLLNVCTTNALARAMIGRRVFNDGNGGCDPRADEFKA 206

NP196416 P------VNLGQLVNMCVVNALGREMIGRRLFG---ADADHKADEFRS 205

NP001064333 PGS------SSPAAVVLGKEVNVCTTNALSRAAVGRRVFA---AGAGEGAREFKE 220

ADW66160 ------KKNESIVLPEMLTYAMANMIGQIILSRRVFE----TKGCESNEFKD 212

CU651565_9 ------KKDESIVVSEMLTYAMANMIGQVILSRRVFE----TKGYESNEFKD 213

LJT34E09_40 ------KKGEAVVVPEMLTYAMANMIGQVILSRRVFE----TKGSESNEFKD 212

BAJ14024 ------KRDEAVVVAEMLTYSMANMIGQVILSRRVFE----TKGSESNEFKD 208

BAF49293 GDGNNDNDGNKKKGTRHEPIVVAEMLTYAMANMIGQVILSRRVFE----TKGSESNEFKD 222

ABH06585 ------QRAEPVVVPEMLTFSMANMIGQVILSRRVFE----TKGSESNEFRD 209

BAE86871 ------QNNEPVMISEMLTYAMANMLSQVILSRRVFN----KKGAKSNEFKD 215

P48418 ------REGQRVVVAEMLTFAMANMIGQVMLSKRVFV----DKGVEVNEFKD 206

CU651565_21 ------KKGESIEVGGLLSCAITNMVSQVVLSKRMFA----SKGQESKEFKE 209

NP182079 ------ERKEAVDISRASYTTVLNIISNILFSVDLGS----YDSKKSNEFQD 212

NP775426 ------GRSINPKAMLNKALCNVIASLIFARRFEY-----EDPYLIRMLT 210

ABH06586 MVVELMVLAGVFNIGDFVPALEWLDLQGVAAKMKKLHARFDAFLGAIVEEHKISG----S 259

BAB83261 MVMEVMVLAGVFNIGDFIPSLEWLDLQGVQAKMKKLHKRFDAFLTSIIEEHNNS-----S 261

NP196416 MVTEMMALAGVFNIGDFVPSLDWLDLQGVAGKMKRLHKRFDAFLSSILKEHEMN------259

NP001064333 IVLEVMEVGGVLNVGDFVPALRWLDPQGVVARMKKLHRRFDDMMNAIIAERRAGSLLKPT 280

ADW66160 MVVELMTTAGYFNIGDFIPILAWLDLQGIERGMKSLHKKFDALLTKMIEEHVAS----SH 268

CU651565_9 MVVELMTTAGYFNIGDFIPILAWLDLQGIERGMKSLHKKFDALLTKMIEEHVAS----SL 269

LJT34E09_40 MVVELMTVAGYFNIGDFIPALGWFDLQGIEGGMKALHKKFDSLLTRMIEEHLAS----SH 268

BAJ14024 MVVELMTVAGYFNIGDFIPFLAKLDLQGIERGMKKLHKKFDALLTSMIEEHVAS----SH 264

BAF49293 MVVQLMTVAGYFNIGDFIPFLARFDLQGIERGMKTLHNKFDVLLTTMIHEHVAS----AH 278

ABH06585 MVVELMTTAGYFNIGDFIPSIAWLDIQGIQRGMKHLHRKFDRLLTKMMEEHTAS----AH 265

BAE86871 MVVELMTSAGYFNIGDFIPSIGWMDLQGIEGGMKRLHKKFDVLLTRLLDDHERT----SH 271

P48418 MVVELMTIAGYFNIGDFIPCLAWMDLQGIEKRMKRLHKKFDALLTKMFDEHKAT----TY 262

CU651565_21 MVVEFMTISG-INIGDFVPFIGWIDLEGVVGKMKRLHKRFDEFLTKLIEDHVNC----AH 264

NP182079 TVIGAMEAAGKPDAANYFPFMGFLDLQGNRKAMRGLTERLFRVFRGFMDAKIAEKSLGNY 272

NP775426 LVEESLIEVSGFIPEVLNTFPALLRIPGLADKVFQGQKTFMAFLDNLLAENRTT-----W 265

ABH06586 AGSERHVDLLSTLISVR----DNADGEG-GKLTDVEIKALLL------NL 298

BAB83261 SKNENHKNFLSILLSLK----DVRDDHG-NHLTDTEIKALLL------NM 300

NP196416 GQDQKHTDMLSTLISLKG---TDLDGDG-GSLTDTEIKALLL------NM 299

NP001064333 DSREEGKDLLGLLLAMVQEQEWLAAGED-DRITDTEIKALIL------NL 323

ADW66160 KNPRVKPDFLDTLIAQS-----KQDSDGGEKLTITNIKAVLL------NL 307

CU651565_9 KSHRVKPDFLDRLIAQS-----KEDSDG-EKLSLTNIKALLL------NL 307

LJT34E09_40 KR-KLKPDFLDVVMAYQ-----SENSGG-EKLSLTNIKALLL------NL 305

BAJ14024 KR-KGKPDFLDMVMAHH-----SENSDG-EELSLTNIKALLL------NL 301

BAF49293 KR-KGKPDFLDVLMAHH-----TNES---HELSLTNIKALLL------NL 313

ABH06585 ER-KGNPDFLDVIMAN------QENSTG-EKLTITNIKALLL------NL 301

BAE86871 ER-KQKPDFLDFVIAN------GDNSDG-ERLNTDNIKALLL------NL 307

P48418 ER-KGKPDFLDVVMEN------GDNSEG-ERLSTTNIKALLL------NL 298

CU651565_21 ER-KGNPDFLDIVLAN------DDDNFG-ERLSLSNIKALLLYHFIILTQMYLFLYIKNL 316

NP182079 SKDVSNRDFLDSLLILN------EGDE-AELDNNDIEHLLL------DM 308

NP775426 DPAQPPRNLTDAFLAEV----EKAKGNPESSFNDENLRMVVV------DL 305

ABH06586 FTAGTDTSSSTVEWAIAELIRHPEMMAQAQQELDAVVGRGRLVTDLDLPQLTYLQAIIKE 358

BAB83261 FTAGTDTSSSTTEWAIAELIKNPQILAKLQQELDTVVGRDRSVKEEDLAHLPYLQAVIKE 360

NP196416 FTAGTDTSASTVDWAIAELIRHPDIMVKAQEELDIVVGRDRPVNESDIAQLPYLQAVIKE 359

NP001064333 FVAGTDTTSTIVEWTMAELIRHPDILKHAQEELDVVVGRDRLLSESDLSHLTFFHAIIKE 383

ADW66160 FTAGTDTSSSIIEWALAEMLKNPNIMKRAHEEMDRVIGKQRRLQDSDIQNLPYLQAICKE 367

CU651565_9 FTAGTDTSSSIIEWALAEMLKNPKIMKKVHEEMDQVIGKQRRFQEADIQKLPYLQAICKE 367

LJT34E09_40 FTAGTDTSSSIIEWSLAEMLNNPKIMRKAHEEMDRVIGKDRRLRESDLPNLPYFQAICKE 365

BAJ14024 FTAGTDTSSSIIEWSLAEMLKKPSIMKKAHEEMDQVIGRDRRLKESDIPKLPYFQAICKE 361

BAF49293 FTAGTDTSSSIIEWALAEMLINPKIMKKVHEEMDKVIGKDRRLKESDIENLPYLQAICKE 373

ABH06585 FTAGTDTSSSVIEWSLAEMLKNPSILKRAHEEMDQVIGRSRRLVESDLPKLPYLQAICKE 361

BAE86871 FTAGTDTSSSIIEWALAELLKNRTLLTRAQDEMDWVIGRDRRLLESDIPNLPYLQAICKE 367

P48418 FTAGTDTSSSAIEWALAEMMKNPAILKKAQAEMDQVIGRNRRLLESDIPNLPYLRAICKE 358

CU651565_21 FTAGTDTSSSIIEWALAEMLKSPNILIRAQKEMNQVVGRERMLVESDLEKLPYLQAICKE 376

NP182079 FTAGTDTSSSTLEWAMAELLRNPKTMVKAQAEMDRVLGQNSVVQESDISGLPYLQAVVKE 368

NP775426 FTAGMVTTATTLTWALLLMILYPDVQRRVQQEIDEVIGQVRCPEMTDQAHMPYTNAVIHE 365

ABH06586 TFRLHPSTPLSLPRMAAESCEINGYHIPKNATLLVNVWAIARDPEVW-EKPLEFRPSRFL 417

BAB83261 TFRLHPSTPLSVPRAAAESCEIFGYHIPKGATLLVNIWAIARDPKEW-NDPLEFRPERFL 419

NP196416 NFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQW-SDPLAFKPERFL 418

NP001064333 TFRLHPSTPLSLPRMASEECEIAGYRIPKGAELLVNVWGIARDPAIW-PDPLEYKPSRFL 442

ADW66160 TFRKHPSTPLNLPRVSSEACEVNGYYIPKNTRLSVNIWAIGRDPNVW-ENPLEFNPDRFL 426

CU651565_9 TYRKHPSTPLNLPRVSLEPCQVNGYYIPKNTRLSVNIWAIGRDPNVW-ENPLEFNPERFL 426

LJT34E09_40 TYRKHPSTPLNLPRISTEPCEVNGYYIPKNTRLNVNIWAIGRDPDVW-ENPLEYNPERFL 424

BAJ14024 TYRKHPSTPLNLPRISSEPCQVNGYYIPENTRLNVNIWAIGRDPDVW-NNPLEFMPERFL 420

BAF49293 TYRKHPSTPLNLPRISSQACQVNGYYIPKNTRLSVNIWAIGRDPNVW-ENPLEFNPERFM 432

ABH06585 SFRKHPSTPLNLPRVSTQACEVNGYYIPKNTRLSVNIWAIGRDPDVW-ESPEEFRPERFL 420

BAE86871 TFRKHPSTPLNLPRIASEPCEVNGYYIPKGTRLNVNIWAIGRDPSVWGDNPNEFDPERFL 427

P48418 TFRKHPSTPLNLPRISNEPCIVDGYYIPKNTRLSVNIWAIGRDPQVW-ENPLEFNPERFL 417

CU651565_21 TYRLHPSTPLSVPRVSNKACQINGYYIPKNTRFNVNIWAIGRDPNIW-ANPLEFKPERFL 435

NP182079 TFRLHPAAPLLVPRKAESDVEVLGFMVPKDTQVLVNVWAIGRDPSVW-ENPSQFEPERFM 427

NP775426 VQRFGDIAPLNLPRITSCDIEVQDFVIPKGTTLIINLSSVLKDETVW-EKPLRFHPEHFL 424

ABH06586 PGGERPNADVRGNDFEVIPFGAGRRICAGMSLGLRMVHLLTATLVHAFNWELPEGQVAEK 477

BAB83261 LGGEKADVDVRGNDFEVIPFGAGRRICAGLSLGLQMVQLLTAALAHSFDWELEDCMNPEK 479

NP196416 PGGEKSGVDVKGSDFELIPFGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAGGVTPEK 478

NP001064333 PGGTHTDVDVKGNDFGLIPFGAGRRICAGLSWGLRMVTMTAATLVHAFDWQLPADQTPDK 502

ADW66160 SG-ENAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSFDWKLPNGV--VA 483

CU651565_9 TG-KNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSFDWKLPNGV--VD 483

LJT34E09_40 SG-RNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSYDWKLPPGV--KE 481

BAJ14024 SG-KNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVHYILGTLVHSFDWKLPNGE--RE 477

BAF49293 G--ANKTIDPRGNDFELIPFGAGRRICAGTRMGIVLVQYILGTLVHSFDWKLPNGV--VE 488

ABH06585 SG-RNEKIDPRGNDFELIPFGAGRRICAGTRMGIVLVEYILGTLVHSFDWKMPDG---VE 476

BAE86871 YG-KNAKIDPRGNDFELIPFGAGRRICAGTRMGILLVEYILGTLVHSFDLKLGLPE--EE 484

P48418 SG-RNSKIDPRGNDFELIPFGAGRRICAGTRMGIVMVEYILGTLVHSFDWKLPSEV--IE 474

CU651565_21 SG-KYARIDPSGVDFELIPFGAGRRVCVGYKMAIVVIEYILGTLVHSFDWKLPNG---VE 491

NP182079 GK----DIDVKGRDYELTPFGGGRRICPGLPLAVKTVSLMLASLLYSFDWKLPNGVVSED 483

NP775426 DA-----QGNFVKHEAFMPFSAGRRACLGEPLARMELFLFFTCLLQHFSFSVPAGQ---- 475

ABH06586 LNMDEAYGLTLQRAPPLMVHPRPRLSPQVFGK--- 509

BAB83261 LNMDEAYGLTLQRAVPLSVHPRPRLAPHVYSMSS- 513

NP196416 LNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSG 513

NP001064333 LNMDEAFTLLLQRAEPLVVHPVPRLLPSAYNIA-- 535

ADW66160 LDMDESFGLALQKKVPLAVVVTPRLSPSAYIP--- 515

CU651565_9 LNMDESFGLALQKKVPLAAIVSPRLSPSAYIL--- 515

LJT34E09_40 LDMEEAFGLALQKKVPLSVMVTPRLPPSAYIP--- 513

BAJ14024 LDMEESFGLALQKKVPLAALVTPRLNPSAYIS--- 509

BAF49293 LNMEETFGLALQKKIPLSALITPRLPPTAYNVINS 523

ABH06585 INMDEAFGLALQKAVSLSAMVTPRLHQSAYAV--- 508

BAE86871 LNMDETFGLALQKAVPLAAMLIPRLPLHVYAP--- 516

P48418 LNMEEAFGLALQKAVPLEAMVTPRLQLDVYVP--- 506

CU651565_21 LNMDEAFGLTLEKAVPLSATVTPRLVPHAYV---- 522

NP182079 LDMDETFGITLHRTNTLYAIPVKKQTIN------511

NP775426 -PRPSTLGNFAISVAPLPYQLCAAVREQGH----- 504

Supplemental Figure S2.F3’5’H sequence analysis.

A, FASTA files of cytochrome P450 amino acid sequences from eudicot plants with NP775426 from Rattusnorvegicus included as an outgroup comparison. B, ClustalW 2.1 multiple sequence alignment. Absolutely conserved residues are highlighted in bold, cytochrome P450 motifs are underlined. The G305D polymorphism in soybean sequences ABQ96218 and BAJ14024 is highlighted.

1

|....|....|....|....|....|....|....|....|....|....|....|....|....|....

280 290 300 310 320 330 340

JI 2822 GCATTTCTCAAAACACTCGATCAAAATTTCTCAAACCGTCCGCTCAATGCGGGAGCGACACATCTAGCTT

A F L K T L D Q N F S N R P L N A G A T H L A Y

JI 73 GCATTTCTCAAA------CCGTCCGCTCAATGCGGGAGCGACACATCTAGCTT

A F L K P S A Q C G S D T S S L

JI 118 GCATTTCTCAAAACACTCGATCAAAATTTCTCAAACCGTCCGCTCAATGCGGAAGCGACACATCTAGCTT

A F L K T L D Q N F S N R P L N A E A T H L A Y

. | . . . . | . . . . | . . . . | . . . . | . .

95 100 105 110 115

|....|....|....|....|....|....|....|....|....|....|....|....|....|....

350 360 370 380 390 400 410

JI 2822 ACGATTCACAAGACATGGTTTTCGCGGATTACGGATCCAGGTGGAAATTACTTAGAAAACTAAGTAACTTA

D S Q D M V F A D Y G S R W K L L R K L S N L

JI 73 ACGATTCACAAGACATGGTTTTCGCGGATTACGGATCCAGGTGGAAATTACTTAGAAAACTAAGTAACTTA

R F T R H G F R G L R I Q V E I T *

JI 118 ACGATTCACAAGACATGGTTTTCGCGGATTACGGATCCAGGTGGAAATTACTTAGAAAACTAAGTAACTTA

D S Q D M V F A D Y G S R W K L L R K L S N L

. . | . . . . | . . . . | . . . . | . . . . |

120 125 130 135 140

Supplemental Figure S3.Sequence characterization of mutant b alleles.

Nucleotide and amino acid sequences of a wild type allele from line JI 2822, a spontaneous deletion allele from line JI 73 with the 23-bp deletion indicated by dashes and the b mutant type allele from line JI 118 with the single nucleotide polymorphism underlined. Predicted changes in the amino acid sequences of the mutants are italicized.Numbering is according to JI 2822, from the A of the ATG on genomic sequence and the start methionine on the amino acid sequence.

Supplemental Figure S4. Characterisation of mutant b alleles by PCR.

A, PCR products amplified from wild type line JI 2822 and b mutant line FN 1076/6. The B gene is shown as a horizontal line with exons as thick lines and with the ATG start codon labelled. The position of an Ogreretroelement insertion in line FN 1076/6 is indicated by a triangle. Positions of primers are indicated by black arrows with the name of the primer above (see Supplemental Table 1 for primer sequences). PCR amplification from genomic DNA is represented by a connecting line between primers and F3’5’H PCR products, indicated by red arrows, are shown in gel photographs above or below the connecting line. The gel photographs show a 100-bp marker lane in the left lane, PCR products from line JI 2822 in the middle lane and PCR products from line FN 1076/6 in the right lane. PCR products not indicated by a red arrow are controls amplified from Ago primers. B, Diagram of the b gene from line FN 1076/6. The b gene is shown as a horizontal line with exons as thick lines and with the ATG start codon labelled. The position of an Ogreretroelement insertion is indicated by open rectangles. Positions of primers are indicated by black arrows as in panel A. Insertion breakpoints are indicated with vertical curved lines and labeled with nucleotide positions in the b gene and an Ogre element. C, PCR products amplified from wild type line JI 2822 and b mutant lines. The B gene is shown as a horizontal line with exons as thick lines and with the ATG start codon labelled. The position of an Ogreretroelement insertion in line FN 1076/6 is indicated by a triangle. Positions of primers are indicated by black arrows and gel photographs of PCR products are as in panel A. PCR amplification between primers is represented by a colored horizontal line. A red line indicates that no F3’5’H PCR product was amplified from any of the FN lines shown. A green line indicates that a F3’5’H PCR product was amplified from line FN 1076/6, but none of the other FN lines.

Medtr3g169650

44305329

ATGGCTTTTG ATGCGTTTCT TCTAAGAGAT ATTGGTATTG CCATTTTCAT CTTCTTCATC TCACACTTCT TCATAGGCTT

GATTCTTAGA AACACTTCTA AAAATCTTCC ACCTGGCCCA AGAGGATGGC CAATTCTTGG TGTTCTTCCA CACTTGGGAA

CCATGCCTCA TGTCACACTC GCAAACATGG CGAAGAAATT TGGACCGATC ATGTATCTAA AAATGGGCAC TTGTGACACT

GTGGTTGCTT CAAGCTCAGA TGCAGCTAGA GCATTTCTCA AGACTTTAGA CCATAATTTC CTAAATAGGC CTACCATTAT

TGGTGCAACT TATTTAGGTT ACAATTCACA AGACCTTGTT TTTGCAAAAT ATGGACCTAA ATGGAAACTA CTTAGGAAAT

TAACAACCTT ACACATGCTT GGTGGGAAAG CACTTCAAAA TTGGGCTAAT GTGAGAGAAA ATGAGGTGAA ACACATGGTG

AGATCCATTC ATGAGTCTGG GAAAAAAGGC GAATCGATTG AGGTTGGTGG TTTGTTGAGT TGTGCAATAA CAAACATGGT

GAGCCAAGTT GTACTAAGCA AACGTATGTT TGCAAGTAAA GGGCAAGAGT CAAAGGAGTT TAAGGAAATG GTTGTGGAGT

TCATGACAAT ATCTGGAATC AACATTGGTG ATTTTGTACC TTTCATTGGT TGGATTGATT TGGAAGGTGT AGTGGGTAAG

ATGAAACGAT TGCATAAGAG GTTTGATGAG TTTTTGACCA AGTTGATTGA AGATCATGTG AATTGTGCTC ATGAACGTAA

AGGGAATCCA GATTTTTTGG ATATTGTTTT GGCAAATGAT GATGATAATT TTGGTGAGAG ATTGAGCTTG TCCAACATCA

AGGCTTTATT ATTGGTACTT ACTTCTCTCT TTCCCAACTA CTACTTTCTT TATTTTTTAT TTTTTGTGAC CGGAGTTTGA

ACCTCAGACT TTGCATATAT TATGCCTGTC TGTATCAACT AAGTTAAGCT CAGGAGAATA A

44306349

Intron size = 2976 nt (44306223 – 44309199)

Medtr3g169660

44309176

ATGTATCTTT TCCTATATAT CAAGAATTTG TTCACTGCAG GGACAGATAC ATCATCTAGC ATCATAGAGT GGGCACTTGC

TGAAATGCTA AAAAGTCCAA ACATCTTGAT AAGGGCACAA AAAGAGATGA ATCAAGTGGT AGGCAGGGAA AGAATGCTAG

TTGAATCTGA CCTAGAAAAA TTACCATACC TTCAAGCAAT ATGCAAGGAG ACATATCGTT TGCATCCATC TACACCCCTT

AGTGTTCCAA GGGTGTCAAA TAAAGCATGC CAAATTAATG GTTACTACAT CCCCAAAAAT ACAAGGTTCA ATGTTAACAT

ATGGGCAATA GGGAGAGACC CAAATATATG GGCTAACCCA TTAGAATTCA AACCTGAAAG ATTTTTAAGT GGAAAATATG

CAAGAATTGA TCCAAGTGGA GTTGATTTTG AATTGATTCC TTTTGGAGCT GGAAGAAGAG TTTGTGTTGG GTATAAAATG

GCTATTGTGG TGATTGAGTA CATTCTGGGC ACATTGGTAC ACTCTTTTGA CTGGAAATTA CCAAATGGTG TTGAGCTAAA

CATGGATGAG GCCTTTGGAC TTACATTGGA AAAAGCAGTG CCCTTGTCAG CTACGGTTAC CCCTCGCTTG GTCCCACATG

CTTATGTTTA A

44309826

Supplemental Figure S5.Proposed splicing model for medicago gene CU651565_21.

Proposed splicing model for the F3’5’H gene CU651565_21 joins ORFs Medtr3g169650 and Medtr3g169660, as annotated in version 3.0 of medicago genome pseudomolecules. Proposed splice donor and splice acceptor dinucleotides defining a 2976 nt intron are highlighted.

1