Supplementary Table 1. Primers used in this study.
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1 Supplementary Tale 1. Primers used in this study. Name Primer sequence (5'-3') Primers of PCR-ased molecular markers developed in this study M1 F M1 R M2 F M2 R M3 F M3 R M4 F M4 R M5 F M5 R M6 F M6 R M7 F M7 R M8 F M8 R M9 F M9 R CCGACAAATCTGATAATGACTG CGACCAAATGCCAATGTAAG GTGCTACAGATGTTTGGATGC GAAGTGAACAGCATTGAACAGT CCTACCTTTGAACACTCACTGA GCAGGACGACAGATTGAGAA GGAGGAAGCAAATCATTAGTG CTGAGTAAAATGAATCGTCGAA GAGTAGTCTTGACTTTGACCA GATGCCTGGTTGATTTAGT CGTGACACCCTAAATAAACC CCAAACACCCCATTAGTTAC AAGAACTCACTCACAAACACTCC GAACAGCAAGAACAACCCAC ATCTTGGGGCTTGTGCTT TGAACTTGGTCTGAGAGGTTTC ACCAGTAGCAGTAGCATCATTG CTGTTCTTTGATTCTCCTCTCG Primers used to generate DNA constructs gipa11f AGGGCCCGCTTACCAGCTATTGGTTACACATATT gipa11r AGGTACCGCAATGTAGAGCCACGTAGGCAAG RNAi1F AGGATCCCCAGCCATGGGATACTACTACC RNAi1R AGGTACCCAGCATTAACACTGATACTTAAA RNAi2F AGAGCTCCCAGCCATGGGATACTACTACC RNAi2R AACTAGTCAGCATTAACACTGATACTTAAA IPA1GFP1R CCCTCCGCCGCGTCCACCAC IPA1OE1F AGGATCCATGGAGATGGCCAGTGGAGGAG IPA1GFP2R ATCTAGACAGAGACCAATCCATCGTGTTGC gipa11f CGAGCTGTGCTATCTCTCTTCTGTC gipa11r GACAGAAGAGAGATAGCACAGCTCG 7mIPA1GFP1F TGCGCCCTGTCCCTGCTCTCCACCCAGCCATGGGATACTAC 7mIPA1GFP1R GGAGAGCAGGGACAGGGCGCAGCTCGAGTCGGTGGCGGCAC 156OE1F AGGATCCATCCAAATCAACAGTAATGTTCTAC 156OE1R AGAGCTCAAACTGAAAGCCTAAATTGATTTATC IPA1P1R AGAGCTCTTACGGTGGCGCCGCCGCGGCCACGCCCTTC IPA1GFPACF AGGGCCCATGGAGATGGCCAGTGGAGGAGG Nature Genetics: doi:1.138/ng.591 1
2 Primers used for 5 modified RACE, real-time PCR, RT-PCR and detection of OsmiR156 IPA1-157R GGAGAGTTACATAGATGGATGCGG IPA1rtime1F GGATATGGTGCCAACACATACAG IPA1rtime1R GACATGGCTGCAGCCTGGTTGTG UiRMF AACCAGCTGAGGCCCAAGA UiRMR ACGATTGATTTAACCAGTCCATGA IPA1RT1F CGGTCGACTAGCTGCATCTGTTGG IPA1RT1R CATCGTGTTGCTGGTTTGGTCGAAG Os8g3987F CGGCACTGACGAGGCGAATAAC Os8g3987R TTTGGGCAGCACACTGCACACA Os8g3988F CACAGTGAAAAAGAGTCGTGTC Os8g3988R CCATCATTGCATTGAAGATAGA Os8g3991F GTTTAAGCGTCTCAACGTGGGA Os8g3991R AATCGTGACCTGATGATGTCCG Os8g3992F CCTGCATCCGCTCTCTCCTTC Os8g3992R GCGTCCTCCTGCTCATCACCA Os8g3993F CTTTCAACCATCAGCCCGTCC Os8g3993R TTGCCACTCCTCTAGCATCCA Os8g3995F TCATAAAGATGGGAACTAGGCAC Os8g3995R TATTAGGGGAGAAAAAAAGAGAA Os8g3996F CTTATCAAGACAAACCTCACAGG Os8g3996R GAGAAGAGAATATTGGAACACCA Os8g3997F CGGTGGGTGAAGATGGAAACTCA Os8g3997R ACGGATGGCACAGAAGCAAATAA Os8g3998F GGTGTAAATGTTTGTAGGGGTTG Os8g3998R ATGTGTCCTGATTCAGTGTAGGT UiRTF CCCTCCACCTCGTCCTCAG UiRTR AGATAACAACGGAAGCATAAAAGTC Anti-OsmiR156 GTGCTCACTCTCTTCTGTCA Nature Genetics: doi:1.138/ng.591 2
3 Supplementary Tale 2. Predicted genes/orfs in the region etween markers M4 and M5 Accession numer LOC_Os8g3987 LOC_Os8g3988 LOC_Os8g3989 LOC_Os8g399 LOC_Os8g3991 LOC_Os8g3992 LOC_Os8g3993 LOC_Os8g3994 LOC_Os8g3995 LOC_Os8g3996 LOC_Os8g3997 LOC_Os8g3998 Annotation eta-glucosidase homologue G-patch domain containing protein SBP-ox gene family memer hypothetical protein retrotransposon protein retrotransposon protein hypothetical protein polygalacturonase inhiitor 1 precursor potassium transporter 3-oxoacyl-reductase, chloroplast precursor ENT domain containing protein DNA-inding romodomain-containing protein 3 Nature Genetics: doi:1.138/ng.591
4 /IPA1 /ipa1 /ipa1 No. of tillers /IPA1 /ipa1 /ipa1 c No. of primary ranches (per main panicle) /IPA1 /ipa1 /ipa1 d Diameter of third internode (cm) /ipa1 /ipa1 /IPA1 Supplementary Figure 1. Phenotypic characterization of /ipa1 plants. Comparison of gross morphologies (a), tiller numer (), primary ranch numer (c) and internode diameter (d) among /IPA1, NIL OsSPL14 IPA1/ipa1 and /ipa1 plants at the maturity stage. Scale ar, 1 cm. Values are means ± s.d. (n = 12). Nature Genetics: doi:1.138/ng.591
5 ttccgtctctttcctctctcttctctctccccctctcctggaggagagagaggagaagaggagggggggccgcgccaaga 8 gccacgcgcgctacagtctccttcccacccgcgaccgcgagcaatggagatggccagtggaggaggcgccgccgccgccg 16 M E M A S G G G A A A A CCGGCGGCGGAGTAGGCGGCAGCGGCGGCGGTGGTGGTGGAGGGGACGAGCACCGCCAGCTGCACGGTCTCAAGTTCGGC 24 A G G G V G G S G G G G G G G D E H R Q L H G L K F G AAGAAGATCTACTTCGAGGACGCCGCCGCGGCAGCAGGCGGCGGCGGCACTGGCAGTGGCAGTGGCAGCGCGAGCGCCGC 32 K K I Y F E D A A A A A G G G G T G S G S G S A S A A GCCGCCGTCCTCGTCTTCCAAGGCGGCGGGTGGTGGACGCGGCGGAGGGGGCAAGAACAAGGGGAAGGGCGTGGCCGCGG 4 P P S S S S K A A G G G R G G G G K N K G K G V A A CGGCGCCACCGCCGCCGCCGCCGCCGCCGCGGTGCCAGGTGGAGGGGTGCGGCGCGGATCTGAGCGGGATCAAGAACTAC 48 A A P P P P P P P P R C Q V E G C G A D L S G I K N Y TACTGCCGCCACAAGGTGTGCTTCATGCATTCCAAGGCTCCCCGCGTCGTCGTCGCCGGCCTCGAGCAGCGCTTCTGCCA 56 Y C R H K V C F M H S K A P R V V V A G L E Q R F C Q GCAGTGCAGCAGGTTCCACCTGCTGCCTGAATTTGACCAAGGAAAACGCAGCTGCCGCAGACGCCTTGCAGGTCATAATG 64 Q C S R F H L L P E F D Q G K R S C R R R L A G H N AGCGCCGGAGGAGGCCGCAAACCCCTTTGGCATCACGCTACGGTCGACTAGCTGCATCTGTTGGTGAGCATCGCAGGTTC 72 E R R R R P Q T P L A S R Y G R L A A S V G E H R R F AGAAGCTTTACGTTGGATTTCTCCTACCCAAGGGTTCCAAGCAGCGTAAGGAATGCATGGCCAGCAATTCAACCAGGCGA 8 R S F T L D F S Y P R V P S S V R N A W P A I Q P G D TCGGATCTCCGGTGGTATCCAGTGGCACAGGAACGTAGCTCCTCATGGTCACTCTAGTGCAGTGGCGGGATATGGTGCCA 88 R I S G G I Q W H R N V A P H G H S S A V A G Y G A ACACATACAGCGGCCAAGGTAGCTCTTCTTCAGGGCCACCGGTGTTCGCTGGCCCAAATCTCCCTCCAGGTGGATGTCTC 96 N T Y S G Q G S S S S G P P V F A G P N L P P G G C L GCAGGGGTCGGTGCCGCCACCGACTCGAGCTGTGCTCTCTCTCTTCTGTCAACCCAGCCATGGGATACTACTACCCACAG 14 A (SNJ) A G V G A A T D S S C A L S L L S T Q P W D T T T H S I (SNJ) TGCCGCTGCCAGCCACAACCAGGCTGCAGCCATGTCCACTACCACCAGCTTTGATGGCAATCCTGTGGCACCCTCCGCCA 112 A A A S H N Q A A A M S T T T S F D G N P V A P S A TGGCGGGTAGCTACATGGCACCAAGCCCCTGGACAGGTTCTCGGGGCCATGAGGGTGGTGGTCGGAGCGTGGCGCACCAG 12 M A G S Y M A P S P W T G S R G H E G G G R S V A H Q CTACCACATGAAGTCTCACTTGATGAGGTGCACCCTGGTCCTAGCCATCATGCCCACTTCTCCGGTGAGCTTGAGCTTGC 128 L P H E V S L D E V H P G P S H H A H F S G E L E L A TCTGCAGGGGAACGGTCCAGCCCCAGCACCACGCATCGATCCTGGGTCCGGCAGCACCTTCGACCAAACCAGCAACACGA 136 L Q G N G P A P A P R I D P G S G S T F D Q T S N T TGGATTGGTCTCTGTAGaggctgttccagctgccatcgatctgtcgtcccgcaaggcgagtcatggaactgaagaacctc 144 M D W S L * atgctgcctgcccttattttgtgttcaaattttcctttccagtatggaaaggaaattctaaggtgactggcgattaatct 152 ccctgtgatgaataataatgcgcgcccttgaactcaattaattgctgtgccgcatccatctatgtaactctccatgaatt 16 tttaagtatcagtgttaatgctgt 1624 Supplementary Figure 2. Sequences of the OsSPL14 cdna and its deduced protein. Blue letters indicate the 5 - and 3 -untranslated regions (UTRs). The underlined letters refer to the conserved SBP domain, red asterisks to the OsmiR156 target sites, and red letters to the nucleotide and amino acid changes in the Shaoniejing (SNJ) rice line. Nature Genetics: doi:1.138/ng.591
6 LOC_Os8g3987 LOC_Os8g3995 LOC_Os8g3988 LOC_Os8g3989 (OsSPL14) LOC_Os8g3991 LOC_Os8g3992 LOC_Os8g3996 LOC_Os8g3997 LOC_Os8g3998 Uiquitin LOC_Os8g3993 Supplementary Figure 3. Expression levels of predicted genes/orfs etween markers M4 and M5 revealed y RT-PCR in and NIL OsSPL14 ipa1 plants. Total RNAs were prepared from two-weeks seedlings. Expression of Uiquitin was used as a loading control. The expressions of LOC_Os8g399 and LOC_Os8g3994 were undetectale in the experiment. Nature Genetics: doi:1.138/ng.591
7 c OsSPL14 NP gosspl14 d NO. of tillers g Diameter of third internodes (cm) NP NP gosspl e gosspl14 No. of primary ranches (per main panicle) NP gosspl h NO. of Grains per main panicle NP gosspl14 Supplementary Figure 4. Confirmation of the OsSPL14 function y overexpressing OsSPL14 in Nipponare. (a) Morphological phenotypes of Nipponare (NP) and OsSPL14- overexpression transgenic (gosspl14) plants after heading. Scale ar, 1 cm. () Panicles of NP (left) and gosspl14 plants (right). Scale ar, 1 cm. (c) The OsSPL14 transcript levels detected y RT-PCR in NP and gosspl14 plants. UBI, Uiquitin. (d) Comparison of the tiller numer etween NP and gosspl14 plants. (e) Comparison of primary ranch numer per main panicle etween NP and gosspl14 plants. (f) Comparison of secondary ranch numer per main panicle etween NP and gosspl14 plants. (g) Comparison of diameter of third internodes etween NP and gosspl14 plants. (h) Comparison of grain numer per main panicle etween NP and gosspl14 plants. Values in (d) to (h) are means ± s.d. (n = 1). The Nature single Genetics: and doule doi:1.138/ng.591 asterisks represent significance difference determined y the Student s test at P <.5 and P <.1, respectively f UBI NO. of secondary ranches (per main panicle) NP gosspl * NP gosspl14
8 c OsSPL14 RNAi OsSPL14 d g Plant height (cm) NO. of secondary ranches (per main panicle) OsSPL14 RNAi RNAi RNAi h e No. of tillers No. of grains (per main panicle) Supplementary Figure 5. Confirmation of the OsSPL14 function y the down-regulation of OsSPL14 under the ackground. (a) Morphological phenotypes of and OsSPL14 RNAi transgenic plants. Scale ar, 1 cm. () Panicles of (left) and OsSPL14 RNAi transgenic plants (right). Scale ar, 5 cm. (c) OsSPL14 IPA1 expression detected y RT-PCR in and OsSPL14 RNAi transgenic plants. UBI, Uiquitin. (d) Comparison of plant height etween and OsSPL14 RNAi transgenic plants. (e) Comparison of the tiller numer etween and OsSPL14 RNAi transgenic plants. (f) Comparison of primary ranch numer per main panicle etween and OsSPL14 RNAi transgenic plants. (g) Comparison of secondary ranch numer per main panicle etween and OsSPL14 RNAi transgenic plants. (h) Comparison of grain numer per main panicle etween and OsSPL14 RNAi transgenic plants. (i) Comparison of Nature diameter Genetics: of internodes doi:1.138/ng.591 etween and OsSPL14 RNAi transgenic plants. Values in (d) to (i) are means ± s.d. (n = 1). The doule asterisks represent significance difference determined y the Student s test at P <.1. RNAi RNAi f UBI No. of primary ranches (per main panicle) i Diameter of third internodes (cm) RNAi RNAi
9 OsSPL14 IPA1 MEMASGGGAAAAAGGGVGGSGGGGGGGDEHRQLHGLKFGKKIYFEDAAAAAGGGGTGSGS 6 AtSPL9 MEMGSNSGP----GHGPGQAESGGSSTESSSFSGGLMFGQKIYFED-----GGGGSGSSS 51 OsSPL14 IPA1 GSASAAPPSSSSKAAGGGRGGGGKNKGKGVAAAAPPPPPPPPRCQVEGCGADLSGIKNYY 12 AtSPL9 SGG-----RSNRRVRGGGSGQSGQ IPRCQVEGCGMDLTNAKGYY 9 OsSPL14 IPA1 CRHKVCFMHSKAPRVVVAGLEQRFCQQCSRFHLLPEFDQGKRSCRRRLAGHNERRRRPQ- 179 AtSPL9 SRHRVCGVHSKTPKVTVAGIEQRFCQQCSRFHQLPEFDLEKRSCRRRLAGHNERRRKPQP 15 OsSPL14 IPA1 ---TPLASRYGRLAASVGEHRRFRSFTLDFSYPRVPSSVRNAWPAIQPGDRISGGIQWHR 236 AtSPL9 ASLSVLASRYGRIAPSLYENG---DAGMNGSFLGNQE---IGWPSSR---TLDTRVMRRP 21 OsSPL14 IPA1 AtSPL9 OsSPL14 IPA1 AtSPL9 NVAPHGHSSAVAGYGANTYSGQGSSSSGPPVFAGPNLPPGGCLAGVGAATDSSCALSLLS 296 VSSPSWQINPMNVFSQGSVGGGGTSFSSPEIMD-TKLES---YKGIG---DSNCALSLLS 254 * --TQPWDTTTHSAAASHNQAAAMSTTTSFDGNPV-----APSAMAGSYMAPSPWTGSRGH 349 NPHQPHDNNNNNNNNNNNNNNTWRASSGFGPMTVTMAQPPPAPSQHQYLNP-PWVFK--D 311 OsSPL14 IPA1 EGGGRSVAHQLPHEVSLDEVHPGPSHHAHFSGELELALQGNGPAPAPRIDPGSGSTFDQT 49 AtSPL9 NDNDMSPVLNLGRYTEPDNCQISS---GTAMGEFELSDHHHQ-SRRQYMEDENTRAYDSS 367 OsSPL14 IPA1 SNTMDWSL 417 AtSPL9 SHHTNWSL 375 Supplementary Figure 6. Alignment of the rice OsSPL14 IPA1 and Araidopsis SPL9 (At2g422) protein sequences. The sequences were aligned with the BioEdit software. The underlined letters refer to the SBP domain and the red letters to the amino acid sequences corresponding to the OsmiR156 target site. Nature Genetics: doi:1.138/ng.591
10 .8.8 OsSPL14 IPA1 /UBI Nipponare mirna156oe OsSPL14 IPA1 /UBI Nipponare MiM156 Supplementary Figure 7. Confirmation of OsmiR156-directed regulation of OsSPL14 IPA1 in vivo. (a) OsSPL14 IPA1 transcript levels revealed y real-time PCR in Nipponare and OsmiR156 overexpression transgenic (mirna156oe) plants. Total RNAs were extracted from shoot apexes of plants at the tillering stage. () OsSPL14 IPA1 transcript levels revealed y real-time PCR in Nipponare and MIM156 transgenic plants. UBI, Uiquitin. Total RNAs were extracted from 2-week-old seedlings. Values are means ± s.d. of three independent experiments. Nature Genetics: doi:1.138/ng.591
11 S L SA R S LSAR S SA OsSPL14 IPA1 OsSPL14 IPA1 RNA Ponceau S Supplementary Figure 8. Levels of OsSPL14 IPA1 transcripts and OsSPL14 IPA1 proteins in and plants. (a) Comparison of OsSPL14 IPA1 transcripts y Northern lot etween and NIL OsSPL14 ipa1 in various organs, including seedlings (S), young leaves (L), shoot apexes (SA) and seedling roots (R). () Comparison of OsSPL14 IPA1 protein levels etween and in seedlings (S) and shoot apexes (SA) y Western lot. Nature Genetics: doi:1.138/ng.591
12 Plant height (cm) No. of tillers c Diameter of third internodes (cm) Supplementary Figure 9. Comparison of plant architecture etween NIL OsSPL14 IPA1 and plants. (a) Comparison of the plant height. () Comparison of the tiller numer. (c) Comparison of the diameter of third internodes. Values are means ± s.d. (n = 12). The doule asterisks represent significance difference determined y the Student s test at P<.1. Nature Genetics: doi:1.138/ng.591
13 Numer Leaf numer Tiller numer Days after Germination No. of vascular undle B S Supplementary Figure 1. (a) Kinetic comparison of leaf and tiller numers etween and plants at different developmental stages. Values are means ± s.d. (n = 2). Blue color stands for the and red for the NIL OsSPL14 ipa1. The squares indicate the leaf numer and the triangles indicate the tiller numer. () Comparison of the numer of vascular undles etween and culms. B, ig vascular undles; S, small vascular undles. The doule asterisks represent significance difference determined y the Student s test at P<.1. Nature Genetics: doi:1.138/ng.591
14 M UBI::GFP OsSPL14 IPA17m -GFP M UBI::GFP OsSPL14 IPA17m -GFP OsSPL14 IPA17m -GFP OsSPL14 IPA17m -GFP GFP WB: anti-gfp WB: anti-osspl14 IPA1 Supplementary Figure 11. Determination of OsSPL14 IPA1 antiody specificity y Western lot. (a) Western lot with anti-gfp (Acam,Code:a29). () Western lot with anti-osspl14 IPA1. Nature Genetics: doi:1.138/ng.591
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