1
Supplementary Information
Supplementary Figure S1. Phenotypic comparison of the wild type and mutants.
Supplementary Figure S2. Transverse sections of anthers.
Supplementary Figure S3. DAPI staining and TUNEL assay of anthers.
Supplementary Figure S4. Analysis of the expression of tapetum-expressed genes.
Supplementary Figure S5. Sequence analysis of EAT1 and its homologs.
Supplementary Figure S6. Expression analyses of OsAP25 and OsAP37.
Supplementary Figure S7. Analysis of the EAT1 antibody and the ChIP-PCR control.
Supplementary Table S1. Close homologs of EAT1.
Supplementary Table S2. Peptide sequences used for constructing the NJ tree.
Supplementary Table S3. Synthesized coding sequences of EAT1 and OsAP25 and
OsAP37.
Supplementary Table S4. Primer sequences used in this study.
2
Supplementary Figures and Legends
Fig.S1
Supplementary Figure S1. Phenotypic comparison of the wild type and mutants.
(a) Whole plants. (b) Spikelets after the removal of the lemma. (c) Anthers. (d)
I2-KI-stained pollen grains. (e) Phenotypic segregation ratio of F1 plants from genetic
crosses among eat1-1, eat1-2 and eat1-3. Bars = 20 cm in (a), 2 mm in (b) and (c),
and 200 µm in (d). pa, palea; gl, glume; st, stamen.
3
Fig.S2
Supplementary Figure S2. Transverse sections of anthers.
The wild-type (a) and eat1-1 (b) anthers. S7, stage 7; S8a, stage 8a; S8b, stage 8b; S9,
stage 9; S11, stage 11; E, epidermis; En, endothecium; Ml, middle layer; T, tapetum;
MMC, microspore mother cell; Tds, tetrads; Msp, microspore; DMsp, degraded
microspore. Bars = 50 µm.
4
Fig.S3
Supplementary Figure S3. DAPI staining and TUNEL assay of anthers.
(a) DAPI (4’,6-diamidino-2-phenylindole) staining of microspore mother cells. (b)
TUNEL assays. Red signal is propidium iodide (PI) staining, and yellow
fluorescence is the merged signal from TUNEL (green) and PI. Arrows,
TUNEL-positive signal. Bars = 5 µm in (a) and 50 µm in (b). S8a, stage 8a; S8b,
stage 8b; S7 to 12, stage 7 to 12; T, tapetum; MMC, microspore mother cell; Tds,
tetrads; Msp, microspore; DMsp, degraded microspore.
5
Fig.S4
Supplementary Figure S4. Analysis of the expression of tapetum-expressed genes.
(a) to (g) Expression levels detected by quantitative RT-PCR (qRT-PCR) in the
wild-type and eat1-1 anthers. (h) Expression of EAT1 in gamyb-4, udt1, tdr and ptc1
mutants relative to the wild type. One-way ANOVA test was used. Results are presented as
mean ± standard error. S8a, stage 8a; S8b, stage 8b; S7 to 12, Stage 7 to 12.
6
Fig.S5
Supplementary Figure S5. Sequence analysis of EAT1 and its homologs.
Sequences from the HLH and DUF domains from EAT1 and homologous genes were
used for tree construction. At, Arabidopsis thaliana; Aral, Arabidopsis lyrata; Popt,
Populus trichocarpa; Medt, Medicago truncatula; Glym, Glycine max; Sb,
Sorghum bicolor; Zm, Zea mays; Os, Oryza Sativa; Brad, Brachypodium distachyon;
Selm, Selaginella moellendorffii; Phyp, Physcomitrella patens.
7
Fig.S6
Supplementary Figure S6. Expression analyses of OsAP25 and OsAP37.
(a) OsAP25 is highly expressed in undifferentiated callus (UDc) or differentiating
callus (DC). (b) Expression levels of OsAP25 and OsAP37 in the wild-type, eat1-1,
gamyb-4, udt1, tdr and ptc1 anthers at stage 9 and stage 10. (c) In situ hybridization
analysis of OsAP25 and OsAP37 in wild-type anther at stage 10. Bars = 50 µm.
One-way ANOVA test was used. Results are presented as mean ± standard error.
8
Fig.S7
Supplementary Figure S7. Analysis of the EAT1 antibody and the ChIP-PCR
control.
(a) Western blot analysis of EAT1 in the nuclear protein extracts of the wild-type and
eat1-1 flowers at stage 9. (b) qRT-PCR analysis of OsAP19 in wild-type and eat1-1
anther. (c) Presence of the E-box motifs in the promoters of OsAP37 and OsAP19
(left) and qChIP-PCR results (right) showing that the promoter fragments containing
the E-box motifs, E1 to E3, E5 to E8 on OsAP37 and E1 to E4 on OsAP19 can’t be
amplified by EAT1 antibody immunoprecipitation (IP), compared with no antibody
(No Ab). FRAG, fragment. One-way ANOVA test was used. Results are presented as
mean ± standard error.
9
Supplementary Table S1. Close homologs of EAT1.
Taxonomic
Group Species ID
Accession
Number
Protein Accession
Number Tissues expression
Bryophyta PhypXP_001784001 XP_001784001
Physcomitrella
patens PhypXP_001767478 XP_001767478
Pteridophyte SelmXP_002961225 XP_002961225
SelmXP_002969202 XP_002969202
Selaginella
moellendorffii SelmXP_002964440 XP_002964440
Angiospermae
Monocots BradXP_003580474 XP_003580474
Brachypodium
distich BradXP_003567568 XP_003567568
Oryza sativa EAT1 LOC_Os04g51070 NP_001053749 Anther, Inflorescence,
Root, Leaf, Seed
OsbHLH142 LOC_Os01g18870 NP_001042795
Anther, Inflorescence,
Pistil, Lemma, Palea,
Root, Leaf, Seed
Zea mays ZmLOC100282922 NP_001149299
ZmLOC100283549 NP_001149921
Sorghum
bicolor Sb04g030850 Sb04g030850 XP_002452697
Sb03g011940 Sb03g011940 XP_002457706
Eduicots Glycine max GlymXP_003532668 Glyma08g09420 XP_003532668 Pod, Flower, Nodule,
SAM, Root and Leaf
GlymXP_003524131 Glyma05g26490 XP_003524131 Nodule, Flower, Pod,
SAM, Root and Leaf
MedtXP_003638306 XP_003638306 Pod, Bud, Nodule,
Flower, Root and Leaf
Medicago
truncatula MedtXP_003638303 XP_003638303
Pod, Bud, Nodule,
Flower, Root and Leaf
Populus
trichocarpa PoptXP_002314221 XP_002314221
Male and Female
Catkin, Seedling, Root,
Leaf and Xylem
PoptXP_002323376 XP_002323376
Male and Female
Catkin, Seedling, Root,
Leaf and Xylem
PoptXP_002308327 XP_002308327
Male and Female
Catkin, Seedling, Root,
Leaf and Xylem
Arabidopsis
lyrata AralXP_002879311 XP_002879311
AralXP_002892324 XP_002892324
Arabidopsis
thaliana AtbHLH091 At2g31210 NP_180679
Flower and Floral
organ
10
AtbHLH089 At1g06170 NP_172107 Flower, Floral organ
and Apex
AtbHLH010 At2g31220 NP_180680 not present on array
11
Supplementary Table S2. Peptide sequences used for constructing the NJ tree.
ID Peptides Sequences (N - C)
AtbHLH010 RKGRGSRKSRTSPTERERRVHFNDRFFDLKNLIPNPTKIDRASIVGEAIDYIKELLRTIEEFKMLV
EKKRCGRFRSKKRARVGEGGGGEDQEEEEDTVNYKPQSEVDQSCFNKNNNNSLRCSWLKRKS
KVTEVDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGS
CVYASGIADTLMEVVEKQYMEAVPSNGY
AralXP_002879313 RKGRGSRKSRTFPTERERRVHFNDRFFDLKNLIPNPTKIGRASIVGEAIDYIKELLRTIEEFKMLV
EKKRCGRFRSKKRARVGEGGGEDQEEEEDTVNYKPQSEVDQSGFNKNNNTSLRCSWLKRKSK
VTEIDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGSC
VYASGIADTVMEVVEKQYMEAVPTNGY
AtbHLH089 RKGRGSKKRKIFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLLV
EKKRVKQRNREGDDVVDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTDVDVRIIDDEV
TIKIVQKKKINCLLFVSKVVDQLELDLHHVAGAQIGEHHSFLFNAKISEGSSVYASAIADRVMEV
LKKQYMEALSANNGYHCYSSD
AralXP_002892324 RKGRGSRKRKVFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLL
VEKKRTKQRNREGDDVIDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTEVDVRIIDDDV
TIKIVQKKKINCLVFVSKVVDQLQLDLHHVAGAQIGEHHSFLFNAKICEGSSVYASAIADRVME
VLEKQYMEALSTNNGYHCYSSD
AtbHLH091 RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPSPSKGDRASILQDGIDYINELRRRVSELKY
LVERKRCGGRHKNNEVDDNNNNKNLDDHGNEDDDDDDENMEKKPESDVIDQCSSNNSLRCS
WLQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLHHVAGGQIGEHYSFLF
NTKIYEGSTIYASAIANRVIEVVDKHYMASLPNSNY
AralXP_002879311 RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPNPSKGDRASILQDGIDYINELRRRVSELKY
LVERKRCGGRHKNNELDNNINNNNSNDHDNDEDDIDDENMEKKPESDVVDQCSSNNSLRCSW
LQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLYHVAGGQIGEHYSFLFN
TKIYEGSTIYASAIANRVIEVVDKHYMAALPINY
PoptXP_002308327 GKGIRKSGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGDAINYIKELLRTVEE
LKLLVEKKRNGRERIKRRKPEEDGGVDVLENSNTKVEQDQSTYNNGSLRSSWLQRKSKHTEV
DVRLIEDEVTIKLVQRKKVNCLLSVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA
SGIANKLLEVVDRQYASSTSVPAASC
PoptXP_002323376 GKGIKKTGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGEAIDYIKELLRTVQE
LKLLVEKKRCGRERSKWRKTEDDGGVEVLDNSDIKVEPDQSAYSNGSLRSSWLQRKSKDTEV
DVRLIEDEVTIKLVQRKRVNCLLYVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA
SAIANRLIEVVDRQYASSTTTVPAAGSCY
MedtXP_003638303 GKGKGKATEHLTTEKQRREQLKGRYKILRSLIPNSTKDDRASVVGDAIEYLRELIRTVNELKLL
VEKKRHEIEICKRHKTEDYAAESCHMKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLFQ
RKKVNCLLFVSKVLDELQLELNHVAGGHVGEYCSFLFNSKVIEGSSVHASAIANRVIDVLDTQY
AAVVPHNRM
MedtXP_003638306 GKGRGGKATKHFATEKQRREQLNGKYKILRDLIPSPTKTDRASVVGDAIEYIRELIRTVNELKLL
VEKKRHGREMCKRLKTEDDAAESCNIKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLF
QRKKVNCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKVNEGSSVYASAIANRVIDVMDT
QYAAGLPHISRL
12
GlymXP_003524131 GKGRGGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLIGWVWFNTDDRASVVGDAIDYIRE
LIRTVNELKLLVEKKRYAKERYKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVR
IIDDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSV
YASAIANRVIDVLDSQYTAAVPHTNSY
GlymXP_003532668 GKGRRGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLVGFVLTQTDRASVVGDAIDYIRELI
RTVNELKLLVEKKRYAKDRCKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVRII
DDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSVY
ASAIANRVIDVLDSQYAAAVPHTNSY
PoptXP_002314221 LIAFAIELQRRGQLNDNYKTLRDLIKNPSTKEDRATVIRDAIKYIIQLIRTVYELKQLVEKTRGKK
LDTIGGVDVFTKPVVRKSQYSHSYNDVSSGPSFSKKHSSVQRKSKDTEIDVRIIGDEVTIKVLRR
RKKNDYCLLFVSRVLDELHMDLHFVSSCYIGYETYYFQFKTKINGGPSSDNAHTIADKLIEVLD
SSCSI
Sb03g011940 RKRGRALGGGFHAGLANGVEKKEKQRRQRLTEKYTALMHLIPNVTKPDRATVISDAIEYIQEL
GRTVEELTLLVEKKRRRRELQGDVVDAAPTAVVVAAAATGGEAESSEGEVAPPPPPhypAVQR
QPIRSTYIQRRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC
HIYMFNTKIHKGSSVFASAVASRLMEVVDEY
ZmLOC100283549 RKRGRALGGGFHAVLANGVEKKEKQRRLRLTEKYTALMHLIPNVTKTDRATVISDAIEYIQEL
GRTVEELTLLVEKKRRRRELQGDVVDAAPAAVVAAAGEAESSEGEVAPPPhypVPRQPIRSTYIQ
RRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCQIYMFNTKI
HKGSSVFASAVAGRLMEVVDEY
OsbHLH142 RKRSRATAGFHGGGPANGVEKKEKQRRLRLTEKYNALMLLIPNRTKEDRATVISDAIEYIQELG
RTVEELTLLVEKKRRRREMQGDVVDAATSSVVAGMDQAAESSEGEVMAAAAMGAVAPPPRQ
APIRSTYIQRRSKETFVDVRIVEDDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC
HIYMFNTKIHSGSPVFASAVASRLIEVVDEY
BradXP_003567568 RKRNRGSRAAGGPAHGGVEKKEKQRRLRLTEKYTALMLLIPNRTKEDRATVISDAIEYIQELGR
TVEELTLLVGKKRRRNGAGEHHLHQGDVVDAAPAVGAAGELVLAAESSEGEVQAPLAALQPI
RSTYIQRKSKETFVDVRIVEDEVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCHIY
MFNTKIHQGSPVFASAVASKLIEVVDEY
Sb04g030850 RRQKGGFGKGKGKANFATERERREQLNVKYGALRSLFPNPTKNDRASIVGDAIDYINELNRTV
KELKILLEKKRNSTDRRKILKLDDEAADDGESSSMQPVSDDQNNQMNGAIRSSWVQRRSKECD
VDVRIVDDEINIKFTEKKRANSLLCAAKVLEEFRLELIHVVGGIIGDHHIFMFNTKIPKGSSVYAC
AVAKKLLEAVEIKKQALNIFN
ZmLOC100282922 RKQKGGFGKGKGKANFATERERRQFNVKYGALRSLFPNPTKNDRASIVGDAIEYINELNRTVK
ELKILLEKKRNSADRRKILKLDEEAADDGESSSMQPVSDDQNQMNGTIRSSWVQRRSKECDVD
VRIVDDEINIKFTEKKRANSLLCAAKVLEEFHLELIHVVGGIIGDHHIFMFNTKIPKGSSVYACAV
AKKLLEAVEIKKQAYNIFN
EAT1 (OsbHLH141) RRGKGEFGKGKGKANFATERERREQLNVKFRTLRMLFPNPTKNDRASIVGDAIEYIDELNRTV
KELKILVEQKRHGNNRRKVLKLDQEAAADGESSSMRPVRDDQDNQLHGAIRSSWVQRRSKEC
HVDVRIVDDEVNIKLTEKKKANSLLHAAKVLDEFQLELIHVVGGIIGDHHIFMFNTKVSEGSAV
YACAVAKKLLQAVDVQHQALDIFN
BradXP_003580474 RRGKGEFGKGKGKANFATERERREQLNVKYKTLKDLFPNPTKSDRASVVGDAIEYIDELNRTV
KELKILVEQKWHGNKRTKIIKLDEEVAADGESSSMKPMRDDQDNQFDGTIRSSWVQRRSKECH
IDVRIVENEVNIKLTEKKKVNSLLHAARVLDEFQLELIHAVGGIIGDHHIFMFNTKVSEGSSVYA
CAVAKRLLQAVDAQHQAINIFH
PhypXP_001767478 KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL
VQEKRRAAGDSSGAKRRRSLDATDTYPGACTPENASNGHLVMQKGNDTFSADGSQLRSSWLQ
RTSQNGTHVDVRIVHDEVTIKVNQRRGKTCLVFDVISVLQELQLDLLQASGATIGEHDVFLFNT
13
KASQMMILNVNTMKCYDLGTYSISQHRTLTSYYLSPKLICRVRK
PhypXP_001784001 KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL
VQEKRRAAGDSSGGKRRRSMDDADNYAGSCTTENASNGHLVMQKGNDTFSTDGSQLRSSWL
QRTSQNGTHVDVRIVHDEVTIKVNQRRGKNCLVFDVIAVLQELQLDLLQASGATIGEHDVFLF
NTKILEGSSTFAGYIAVKLLDALDRHLDITIDDSNR
SelmXP_002964440 KGAESRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIDYVKELKRTVQELQLL
VEEKRRGSNKRCKASPDDPSATDVESTTAMQQPGGTRVSKETTFLGDGSQLRSSWLQRTSQMG
THIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLDELRLDLLHANGASIGEHHIFMFNTKVVLAP
SSFLLSLFLFYFFLFSSALILPR
SelmXP_002969202 KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL
VEEKRRGSNKRRCKASPDNPSEGGGATDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS
QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM
EGTSTFAGQVATKLIDALGK
SelmXP_002961225 KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL
VEEKRRGSNKRRCKASPDNPSEGGGVTDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS
QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM
EGTSTFAGQVATKLIDAVDRHITLASSGL
UDT1 (OsbHLH164)1 QAAAAAMGKEFKSKNLEAERRRRGRLNGNIFALRAVVPKITKMSKEATLSDAIEHIKNLQNEV
LELQRQLGDSPGEAWEKQCSASCSESFVPTENAHYQGQVELISLGSCKYNLKIFWTKRAGLFTK
VLEALCSYKVQVLSLNTISFYGYAESFFTIEVKGEQDVVMVELRSLLSSIVEVPSI
HLH domains and DUFs were predicted by SALAD
(http://salad.dna.affrc.go.jp/CGViewer/SALADonARRAYs/cgv_search_pr.html)53
Homologs were retrieved by blastp searches using bHLH domains and DUF from NCBI.
14
Supplementary Table S3. Synthesized coding sequences of EAT1 and OsAP25 and OsAP37.
ID Sequences (5' - 3')
EAT1 ATGATTGTTGGGGCTGGTTACTTTGAGGATTCCCACGATCAAAGTCTCATGGCAGGATCTTTGATCCAT
GACTCAAATCAAGCTCCTGCAAGCAGTGAAAACACAAGCATTGATTTGCAGAAATTCAAAGTGCACCC
GTACTCAACAGAAGCTCTCTCGAATACGGCCAATCTAGCTGAAGCTGCAAGAGCAATTAACCACCTTCA
ACATCAACTAGAAATTGATTTGGAGCAAGAGGTTCCCCCAGTAGAAACTGCAAACTGGGATCCAGCTA
TCTGCACTATACCAGATCATATCATCAACCATCAGTTTAGCGAAGATCCACAAAACATATTGGTGGAGC
AACAGATCCAGCAGTATGATTCTGCACTTTATCCAAATGGTGTTTACACACCTGCACCAGATCTCCTTA
ATCTTATGCAGTGCACAATGGCTCCAGCATTCCCGGCAACGACATCCGTATTCGGTGACACAACACTGA
ATGGTACTAACTATTTGGATCTTAACGGTGAACTTACAGGAGTAGCAGCGGTTCCAGACAGTGGGAGT
GGGTTGATGTTTGCTAGTGATTCAGCTCTCCAGTTAGGGTACCATGGCACCCAGTCGCATCTGATTAAA
GATATCTGCCATTCGCTGCCGCAGAATTATGGCCTGTTTCCGAGCGAAGATGAACGCGATGTGATTATT
GGCGTGGGCAGCGGCGATCTGTTTCAGGAAATTGATGATCGCCAGTTTGATAGCGTGCTGGAATGCCGC
CGCGGCAAAGGCGAATTTGGCAAAGGCAAAGGCAAAGCGAATTTTGCGACCGAACGCGAACGCCGTG
AACAGCTGAATGTGAAATTTCGCACCCTGCGCATGCTGTTTCCGAATCCGACCAAAAATGATCGCGCGA
GCATTGTGGGCGATGCGATTGAATATATTGATGAACTGAATCGCACCGTGAAAGAACTGAAAATTCTG
GTGGAACAGAAACGCCATGGCAATAATCGCCGCAAAGTGCTGAAACTGGATCAGGAAGCGGCGGCGG
ATGGCGAAAGCTCAAGCATGCGCCCGGTGCGCGATGATCAGGATAATCAGCTGCATGGCGCGATTCGC
AGCAGCTGGGTGCAGCGCCGCAGCAAAGAATGCCATGTGGATGTGCGCATTGTGGATGATGAAGTGAA
TATTAAACTGACCGAAAAGAAAAAGGCGAATAGCCTGCTGCATGCGGCGAAAGTGCTGGATGAATTTC
AGCTGGAACTGATTCATGTGGTGGGCGGCATTATTGGCGATCATCATATTTTTATGTTTAATACCAAAG
TGAGCGAAGGCAGCGCGGTGTATGCGTGCGCGGTGGCGAAGAAACTGCTGCAGGCGGTGGATGTGCAG
CATCAGGCGCTGGATATTTTCAACTAA
OsAP25 CTCGAGGAATTCACCGGTTCCATGGCGGCGACCACCACCATCCCGCTGCTGCTGCTGCTGCTGGCGGCG
ACCGTGGCGGCGGCGGCGGCGGAACTGAGCGTGTATCATAATGTGCATCCGAGCAGCCCGAGCCCGCT
GGAAAGCATCATCGCGCTGGCGCGCGATGATGATGCGCGCCTGCTGTTTCTGAGCAGCAAAGCGGCGA
CCGCGGGCGTGAGCAGCGCGCCGGTGGCGAGCGGCCAGGCGCCGCCGAGCTATGTGGTGCGCGCGGGC
CTGGGCAGCCCGAGCCAGCAGCTGCTGCTGGCGCTGGATACCAGCGCGGATGCGACCTGGGCGCATTG
CAGCCCGTGCGGCACCTGCCCGAGCAGCAGCCTGTTTGCGCCGGCGAATAGCAGCAGCTATGCGAGCC
TGCCGTGCAGCAGCAGCTGGTGCCCGCTGTTTCAGGGCCAGGCGTGCCCGGCGCCGCAGGGCGGCGGC
GATGCGGCGCCGCCGCCGGCGACCCTGCCGACCTGCGCGTTTAGCAAACCGTTTGCGGATGCGAGCTTT
CAGGCGGCGCTGGCGAGCGATACCCTGCGCCTGGGCAAAGATGCGATCCCGAATTATACCTTTGGCTG
CGTGAGCAGCGTGACCGGCCCGACCACCAATATGCCGCGCCAGGGCCTGCTGGGCCTGGGCCGCGGCC
CGATGGCGCTGCTGAGCCAGGCGGGCAGCCTGTATAATGGCGTGTTTAGCTATTGCCTGCCGAGCTATC
GCAGCTATTATTTTAGCGGCAGCCTGCGCCTGGGCGCGGGCGGCGGCCAGCCGCGCAGCGTGCGCTAT
ACCCCGATGCTGCGCAATCCGCATCGCAGCAGCCTGTATTATGTGAATGTGACCGGCCTGAGCGTGGGC
CATGCGTGGGTGAAAGTGCCGGCGGGCAGCTTTGCGTTTGATGCGGCGACCGGCGCGGGCACCGTGGT
GGATAGCGGCACCGTGATCACCCGCTGGACCGCGCCGGTGTATGCGGCGCTGCGCGAAGAATTTCGCC
GCCAGGTGGCGGCGCCGAGCGGCTATACCAGCCTGGGCGCGTTTGATACCTGCTTTAATACCGATGAA
GTGGCGGCGGGCGGCGCGCCGGCCGTGACCGTGCATATGGATGGCGGCGTGGATCTGGCGCTGCCGAT
GGAAAATACCCTGATCCATAGCAGCGCGACCCCGCTGGCGTGCCTGGCGATGGCGGAAGCGCCGCAGA
ATGTGAATAGCGTGGTGAATGTGATCGCGAATCTGCAGCAGCAGAATATCCGCGTGGTGTTTGATGTGG
CGAATAGCCGCGTGGGCTTTGCGAAAGAAAGCTGCAATGCTAGCGAACAGAAGTTGATTTCCGAAGAA
GACCTCGCCGAGCAGAAACTGATCAGCGAAGAGGATCTGGCGGAACAGAAGCTGATTAGCGAGGAAG
ACCTGTAAACTAGTGTCGACCCCGGG
15
OsAP37 CCATGGTTCTCGAGGAATTCACCGGTATGAATGCGGCGGTGCTGCTGCTGCTGCTGGCGCTGGCGGCGC
TGCCGGCGAGCTGCGCGCCGCCGCGCAGCTTTCGCCTGGAACTGGCGAGCGTGGATGCGAGCGCGGCG
GATGCGGCGAATCTGACCGAACATGAACTGCTGCGCCGCGCGATCCAGCGCAGCCGCTATCGCCTGGC
GGGCATCGGCATGGCGCGCGGCGAAGCGGCGAGCGCGCGCAAAGCGGTGGTGGCGGAAACCCCGATC
ATGCCGGCGGGCGGCGAATATCTGGTGAAACTGGGCATCGGCACCCCGCCGTATAAATTTACCGCGGC
GATCGATACCGCGAGCGATCTGATCTGGACCCAGTGCCAGCCGTGCACCGGCTGCTATCATCAGGTGG
ATCCGATGTTTAATCCGCGCGTGAGCAGCACCTATGCGGCGCTGCCGTGCAGCAGCGATACCTGCGATG
AACTGGATGTGCATCGCTGCGGCCATGATGATGATGAAAGCTGCCAGTATACCTATACCTATAGCGGCA
ATGCGACCACCGAAGGCACCCTGGCGGTGGATAAACTGGTGATCGGCGAAGATGCGTTTCGCGGCGTG
GCGTTTGGCTGCAGCACCAGCAGCACCGGCGGCGCGCCGCCGCCGCAGGCGAGCGGCGTGGTGGGCCT
GGGCCGCGGCCCGCTGAGCCTGGTGAGCCAGCTGAGCGTGCGCCGCTTTGCGTATTGCCTGCCGCCGCC
GGCGAGCCGCATCCCGGGCAAACTGGTGCTGGGCGCGGATGCGGATGCGGCGCGCAATGCGACCAATC
GCATCGCGGTGCCGATGCGCCGCGATCCGCGCTATCCGAGCTATTATTATCTGAATCTGGATGGCCTGC
TGATCGGCGATCGCGCGATGAGCCTGCCGCCGACCACCACCACCACCGCGACCGCGACCGCGACCGCG
CCGGCGCCGGCGCCGACCCCGAGCCCGAATGCGACCGCGGTGGCGGTGGGCGATGCGAATCGCTATGG
CATGATCATCGATATCGCGAGCACCATCACCTTTCTGGAAGCGAGCCTGTATGATGAACTGGTGAATGA
TCTGGAAGTGGAAATCCGCCTGCCGCGCGGCACCGGCAGCAGCCTGGGCCTGGATCTGTGCTTTATCCT
GCCGGATGGCGTGGCGTTTGATCGCGTGTATGTGCCGGCGGTGGCGCTGGCGTTTGATGGCCGCTGGCT
GCGCCTGGATAAAGCGCGCCTGTTTGCGGAAGATCGCGAAAGCGGCATGATGTGCCTGATGGTGGGCC
GCGCGGAAGCGGGCAGCGTGAGCATCCTGGGCAATTTTCAGCAGCAGAATATGCAGGTGCTGTATAAT
CTGCGCCGCGGTCGCGTGACCTTTGTGCAGAGCCCGTGCGGCGCGCTGCGCGCTAGCGAACAGAAGTT
GATTTCCGAAGAAGACCTCTAAACTAGTGTCGACCCCGGG
16
Supplementary Table S4. Primer sequences used in this study.
Primer Name Sequence (5' - 3') Purpose
Os409-F 5'TACTTGAGCCAACCGAGCTA 3' map-based cloning
Os409-R 5'TGGAGATCGAGAGAGAATGG 3' map-based cloning
Os410-F 5'TATTGTCACCTCACCCGTCG 3’ map-based cloning
Os410-R 5'CGTCGATCTTGTATTTTCAC 3’ map-based cloning
XY409-1-F 5'TGACTTAATATCAGAGCACCT 3’ map-based cloning
XY409-1-R 5'ACGGCACTTCTTTTTCCTTC 3’ map-based cloning
XY409-2-F 5'TACGACAAATCTGAACCGAG 3’ map-based cloning
XY409-2-R 5'GCTCTTTCTCCACCTACCAAT 3’ map-based cloning
XY409-3-F 5'CACAAAGAACAAACATAGGTCA 3 map-based cloning
XY409-3-R 5'ATGGTGGAGATTGTTTGC 3’ map-based cloning
XY409-4-F 5'GCAATTGATTCCACTTCTT 3’ map-based cloning
XY409-4-R 5'ATAACCCGACCGCTACAT 3’ map-based cloning
XY409-5-F 5'TTTGGTGGATATGTATCTAGG 3’ map-based cloning
XY409-5-R 5'CCGTTCCACGTTGATACAG 3’ map-based cloning
eat1-2-F 5’ACGGCCAATCTAGCTGAAGC 3’ mutant identification
eat1-2-R 5’ATCTCGTTCGTCCTCACTGG 3’ mutant identification
eat1-3-F 5’TGGCTTGCCACCAAAGTTGG 3’ mutant identification
eat1-3-R 5'CCACTGCACATGCATAAACC 3’ mutant identification
EAT1-RT-F 5’CAGAGGAGGTCAAAGGAATG 3’ qRT-PCR
EAT1-RT-R 5’TCCAATCCTGGTCAAATAAG 3’ qRT-PCR
EAT1-pro-F 5’GGATCCTGCGAGCTCCCAGAAGTTAT 3’ GUS assay
EAT1-pro-R 5’CCATGGTTTGGCAAAACAGTGCTAGG 3’ GUS assay
EAT1-situ-F 5’CCACCTTCAACATCAACTAGA 3’ in situ analysis of EAT1
EAT1-situ-R 5’CAATAATCACATCTCGTTCGT 3’ in situ analysis of EAT1
MSP1-RT-F 5’CTCACAATGAAGTCCACCAC 3’ qRT-PCR
MSP1-RT-R 5’ACCAGAGGTCCACATTCCAT 3’ qRT-PCR
UDT1-RT-F 5’CCCACTGAGAACGCCCATTA 3’ qRT-PCR
UDT1-RT-R 5’CACCATCACAACATCCTGCT 3’ qRT-PCR
GAMYB-RT-F 5’CAGTGGCAATTCATTCACTGAATC 3’ qRT-PCR
GAMYB-RT-R 5’TCCAGATCCCATTGAAGTGCTTTG 3’ qRT-PCR
TDR-RT-F 5’GGAGGAGTAACAAGGACCCA 3’ qRT-PCR
TDR-RT-R 5’ACCTCCAGCAGCGAGTCCCT 3’ qRT-PCR
PTC1-RT-Probe 5’GCACGTAAGGCAAGGACGGTGAGC 3’ qRT-PCR
API5-RT-F 5’CCCCTTTATTTATTGGTGAC 3’ qRT-PCR
API5-RT-R 5’CCTCTACCACCACGGGATAA 3’ qRT-PCR
OsCP1-RT-F 5’CTACGGGATCAGGAACCACT 3’ qRT-PCR
OsCP1-RT-R 5’ CTCGGTAACAGTAACAAAGATA 3’ qRT-PCR
TDR-1F 5’CATATGGGAAGAGGAGACCACCT 3’ Y-2-H
TDR-mu-R 5’GGATCCTCAGTTGTCTTCCAGCTCGT 3’ Y-2-H
TDR-1R 5’TCAATCAAACGCGAGGTAAT 3' Y-2-H
EAT1-2F 5’GAATTCATTGTTGGGGCTGGTTA 3’ Y-2-H
EAT1-2R 5’GTCGACTTAGTTGAATATGTCGAGGGC 3’ Y-2-H
EAT1-muF 5’GGCCAATCCGGCCAAGGGTGAGTTCGGAAAGGG 3’ Y-2-H
EAT1-muR 5’GGCCTTAGAGGCCTTAGTTGAATATGTCGAGGGC 3’ Y-2-H
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EAT1-3F 5’ACTAGTGTTGAATATGTCGAGGGCCT 3’ BiFC
EAT1-3R 5’GTCGACGTTGAATATGTCGAGGGCCT 3’ BiFC
TDR-2F 5’ACTAGTATGGGAAGAGGAGACCACCT 3’ BiFC
TDR-2R 5’GTCGACATCAAACGCGAGGTAATGCA 3’ BiFC
EAT1-1F 5’GAATTCATGATTGTTGGGGCTGGTTA 3’ preparation of EAT1 protein
EAT1-1R 5’GTCGACTTAGTTGAATATGTCGAGGGC 3’ preparation of EAT1 protein
OsAP25-RT-F 5’AGCTTTCCGATCTGTGCAAT 3’ qRT-PCR
OsAP25-RT-R 5’GTCAAAGCTAACCATGAATACG 3’ qRT-PCR
OsAP37-RT-F 5’TACGACGAGCTGGTGAACGA 3’ qRT-PCR
OsAP37-RT-R 5’GTTGTAGAGCACCTGCATGTTC 3’ qRT-PCR
OsAP19-RT-F 5’ACAAACCAAGGATGGGAAAG 3’ qRT-PCR
OsAP19-RT-R 5’AAGGACTTGCGCCACTGAAC 3’ qRT-PCR
EAT1-anti-F 5’CCATGGATGATTGTTGGGGCTGGTTA 3’ preparation of EAT1 antibody
EAT1-anti-R 5’CTCGAGTTGTGGATCTTCGCTAAACTGA 3’ preparation of EAT1 antibody
OsAP25-ChIP-F 5’ACCTCGTGTTAGATTTCCTCCA 3’ qChIP PCR
OsAP25-ChIP-R 5’AACGTGCTCACGAAAAAGAGAG 3’ qChIP PCR
OsAP37-ChIP-F 5’ACAGCATTAACACCATTATTCTCC 3’ qChIP PCR
OsAP37-ChIP-R 5’TAGCTAAGAGAAATGAACTATGGC 3’ qChIP PCR
OsAP37-ChIP-SEG1-F 5’CATCCTCCATTTCTTGTATTTG 3’ qChIP PCR
OsAP37-ChIP- SEG1-R 5’TGCTACGTGATGAACTAGGA 3’ qChIP PCR
OsAP37-ChIP- SEG2-F 5’CATCCCACCATTTCCAAAGTA 3’ qChIP PCR
OsAP37-ChIP- SEG2-R 5’GAACTGGTTCAACTGCCGGC 3’ qChIP PCR
OsAP19-ChIP- SEG1-F 5’GGCAAACTAACCTGGTACTAAAC 3’ qChIP PCR
OsAP19-ChIP- SEG1-R 5’ATGGGGTCAAGGAGAATCAGTG 3’ qChIP PCR
OsAP19-ChIP- SEG2-F 5’ CCGTTGAGATCAACGCTCTT 3’ qChIP PCR
OsAP19-ChIP- SEG2-R 5’ATAGGTCGCGCGGCCTGAGG 3’ qChIP PCR
OsCP1-ChIP-1-F 5’TAAAGAAGCAGTTGCCATCC 3’ ChIP PCR
OsCP1-ChIP-1-R 5’GAAGCGTGATGCAGTAATAC 3’ ChIP PCR
OsCP1-ChIP-2-F 5’GTTCCGAGCCATTACGACAC 3’ ChIP PCR
OsCP1-ChIP-2-R 5’GATTGGGGATTGAGGTGTTA 3’ ChIP PCR
OsAP25-situ-F 5’GAATTCTCGCCAAGGAGTCCTGCAACTA 3’ in situ analysis of OsAP25
OsAP25-situ-R 5’AAGCTTAGCGATGCAAATGATGAAGAT 3’ in situ analysis of OsAP25
OsAP37-situ-F 5’GAATTCTGCGGAGGTTCGCCTACTGC 3’ in situ analysis of OsAP37
OsAP37-situ-R 5’AAGCTTCTCGTCGTACAGCGATGCCT 3’ in situ analysis of OsAP37
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Supplementary Reference
53 Mihara, M., Itoh, T. & Izawa, T. SALAD database: a motif-based database of protein annotations for
plant comparative genomics. Nucleic Acids Res 38, 835-842 (2011).