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Supplementary Methods Microarray analysis Grains of 7 DAP of the wild-type and gif1 were harvested for RNA preparation. Microarray analysis was performed with the Affymetrix (Santa Clara, CA) GeneChip Rice Genome Array representing 51,279 transcripts with three biological replicates. Raw data were analyzed with Affymetrix GeneChip Operating Software (GCOS, Version 1.4) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500. Data were compared between sample chips from the same biological replicate and the effective expressed probe sets were defined on at least once P (Present) of scaling detection on gif1 or wild-type sample. Reproducibly differentially expressed probe sets were selected from the total normalized data, based on a Signal Log 2 Ratio (SLR) of at least 1.0, a gene expression change call of I (increase), and P-value <0.002; or a SLR of at least -1.0, a gene expression change call of D (decrease), and P-value >0.998 for all three biological replicates of either wide-type and gif1 samples using a stringent selection criteria (Zhang et al. 2007). Probe sets that meet these rigorous selection criteria were further analyzed. The rice data file (rice[1].all.mapman.txt at http://bioinfoserver.rsbs.anu.edu.au/utils/genebins/download.php) with blastx best matches tabular file in MapMan format was downloaded from GeneBins (Goffard and Weiller 2007) (http://bioinfoserver.rsbs.anu.edu.au/utils/genebins/download.php). MapMan version 1.6.0 (Goffard and Weiller 2006) was used for functional classification of the regulated genes using the average SLRs for three biological replicates. References for the Supplementary Methods Zhang, Z., Li, Q., Li, Z., Staswick, P.E., Wang, M., Zhu, Y. & He, Z. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant Physiol. 145, 450 464 (2007). Goffard, N. & Weiller, G. GeneBins: a database for classifying gene expression data, with application to plant genome arrays. BMC Bioinformatics 8, 87 (2007). Goffard, N. & Weiller, G. Extending MapMan: application to legume genome arrays. Bioinformatics 22, 1158 1159 (2006).

Supplementary Figure 1. Grain development of the wild-type and gif1. Rice plants were grown under the paddy field conditions, grains were observed and measured during the grain filling stage. a, b, Grains of the wild-type (a) and gif1 (b) at 5 DAP. c, d, Grains of the wild-type (c) and gif1 (d) at 12 DAP. The fertilized gif1 grains were discolored probably due to slower filling. e, f, Grains at 25 DAP. The wild-type grains (e) were nearly matured with complete filling, whereas the gif1 grains (f) were incompletely filled.

Supplementary Figure 2. Amylase and amylospectin contents of mature grains of the wild-type and gif1. Values are means ±SE (n = 3).

Supplementary Figure 3. Map-based cloning of GIF1. The GIF1 locus was mapped using the F2 population derived from the cross of gif1 and Zhenshan 97 (indica). a, The GIF1 locus was initially mapped on the long arm of chromosome 4 between the SSR markers RM5749 and RM5635. The GIF1 locus was narrowed down to a 32-kb interval between the markers CAPS-4 and CAPS-8, and co-segregated with the marker CAPS-7. Numbers represent recombination events. The GIF1 candidate gene contains one nucleotide deletion at the fourth exon that generates a premature stop codon TAA. b, The GIF1 transcript levels detected by RT-PCR in the wild-type and gif1 grains. Prolonged PCR could detect the transcript in gif1. Ubi-1 was used as a control. The analysis was repeated three times with similar results.

Supplementary Figure 4. Sequence alignments of GIF1 with homologous proteins from other plants. The BLAST search program (http://www.ncbi.nlm.nih.gov/blast/) was used to look for invertase sequences homologous to GIF1. The highest homologous invertase sequences were aligned with GIF1 using a MEGA version 3.1 software. The aligned invertases include the functionally known Mn1 (maize) and LIN5 (tomato), and functionally unknown invertases AJ534447 (barley), X69321 (carrot), ATCWINV2 (Arabidopsis) and AF030420 (wheat). The β-fructosidase motif, the Cys catalysis site and the conserved glycosylation motif are indicated.

Supplementary Figure 5. Complementation test. a, Grain weight of five T1 lines compared with the gif1 mutant and the wild-type. b, Grain weight of twenty-three T2 progenies of H5 complement line. Note that some T2 progenies were segregated with the mutant phenotype. c, The GIF1 transcript levels detected by RT-PCR revealing the transgene segregation in the T2 progenies of line H5 (b) with grain weight indicated. Ubi-1 was used as a control.

Supplementary Figure 6. Cell wall invertase activity. Cell wall invertase activity was measured in insoluble extraction of developing grains at 7 DAP. a, Cell wall invertase activity in the wild-type and gif1. b, Cell wall invertase activity in leaves of the transgenic control with empty vector and four GIF1 overexpression transgenic lines with the 35S promoter (GIF1-OE).

Supplementary Figure 7. Grains and GIF1 expression of GIF1-OE plants. a, Matured grains of the wild-type and two representative GIF1-OE lines, indicating the severely shrunken GIF1-OE grains that hardly germinate. b, The GIF1 transcript levels in leaves detected by RT-PCR showing GIF1 overexpression in GIF1-OE lines. The analysis was repeated twice with similar results. Ubi-1, the loading control.

Supplementary Figure 8. Expression of GIF1 in the root, leaf, uppermost internode and flowering panicle. Ubi-1 was used as a RT-PCR control. The analysis was repeated twice with similar results.

Supplementary Figure 9. Grain weight of different genotypes in the introgression backcross population (BC4F2). GIF1(0R)/GIF1(0R), homozygous plants with the wild rice (O. rufipogon) GIF1 allele; GIF1/GIF1, homozygous plants with the cultivated rice allele; GIF1/GIF1(0R), heterozygous plants. For each genotype, 1000-brown grain weight of more than 6 individuals was statistically analyzed. Asterisk indicates significance at P<0.05.

Supplementary Figure 10. Fine mapping of the wild rice locus decreasing grain weight. Phenotyping and genotyping of fixed recombinant plants in the mapping population (BC4F3) narrowed the wild rice locus corresponding to decreased grain weight to the region flanked by the markers SSLP-1 and CAPS-8 and centered by GIF1 (OR). 1000-brown grain weight of recombinants, SW19 and control Teqing was shown. Regions for wild rice, Teqing and heterozygotes were indicated in balck, empty and gray, respectively.

Supplementary Figure 11. Grain weight of different subspecies introgression plants. Grain weight of the introgression lines Katy (japonica), Suyunnuo (SYN, japonica), Chenglong-shuijingmi (CLSJ, indica) and the recurrent parent control (Huajingxian74, indica).

Supplementary Figure 12. Grain sizes and GIF1 expression of transgenic rice overexpressing GIF1 under its native promoter. a-c, Grain thickness, width and length in the transgenic lines G-2 and G-8, the vector and wild-type controls. d, The GIF1 transcript levels detected by RT-PCR in grains of the controls and lines G-2 and G-8, showing that GIF1 expression was increased in the transgenic lines. Ubi-1, the loading control. The analysis was repeated twice with similar results.

Supplementary Table 1. Yield components of gif1 and wild-type Zhonghua11. ZH11 gif1 gif1/zh11 P-Value Panicles / plant 11.00 (2.30) 9.96 (2.73) 0.91 0.05<P<0.1 Seeds / panicle 122.12 (33.71) 124.82 (30.70) 1.02 P>0.1 Seeds / plant 1343.29 (372.12) 1279.38 (239.05) 0.95 P>0.1 unfilled seeds / panicle 35.56 (10.83) 34.33 (15.68) 0.97 P>0.1 Seed weight / panicle (g) 2.90 (0.73) 2.45 (0.63) 0.84 P<0.01 Seed weight / plant (g) 32.14 (8.60) 25.09 (4.44) 0.78 P<0.01 1000-grain weight (g) 24.00 (0.01) 19.71 (0.01) 0.82 P<0.01 1000-kernel weight (g) 21.33 (0.1) 16.15 (0.15) 0.76 P<0.01 Values are means with SE in parentheses

Supplementary Table 2 (Microsoft Excel file). Summary of 341 reproducibly differentially regulated genes of the gif1 mutant versus wild-type Zhonghua11 at 7 DAF.

Supplementary Table 3 (Microsoft Excel file). Summary of 44 carbohydrate metabolism-related genes reproducibly differentially regulated in the gif1 mutant versus wild-type Zhonghua11 at 7 DAF.

Supplementary Table 4. Rice germplasm used in domestication analysis. Rice germplasm Name/Accession no. Origin O. sativa spp. japonica Nipponbare Japan O. sativa spp. japonica Shiokari Japan O. sativa spp. japonica Murasaki-daikoku Japan O. sativa spp. japonica Kimazi Japan O. sativa spp. japonica Dongjing Japan O. sativa spp. japonica Maratelli France O. sativa spp. japonica NW 2 Yunnan, China O. sativa spp. japonica NW 1 Yunnan, China O. sativa spp. japonica Zhonghua11 Beijing, China O. sativa spp. japonica Zhejing 27 Zhejiang, China O. sativa spp. japonica Hangtian 18 Zhejiang, China O. sativa spp. japonica Zheyou10 Zhejiang, China O. sativa spp. japonica Zhenuo205 Zhejiang, China O. sativa spp. japonica Taichung 65 Taiwan, China O. sativa spp. japonica Taipei309 Taiwan, China O. sativa spp. japonica Zhenuo 4 Jiansu, China O. sativa spp. japonica Xiushsui 63 Jiaxing, China O. sativa spp. japonica Xiushui110 Jiaxing, China O. sativa spp. japonica Zhejing 22 Zhejiang, China O. sativa spp. japonica Zhenuo 5 Jiansu, China O. sativa spp. japonica R5100 Ningxia, China O. sativa spp. japonica NW 3 Yunnan, China O. sativa spp. indica Pi 9 American O. sativa spp. indica pb17 Zhejiang, China O. sativa spp. indica pb24 Hunan, China O. sativa spp. indica pb32 Hubei, China O. sativa spp. indica pb33 Zhejiang, China O. sativa spp. indica pb77 Hainan, China O. sativa spp. indica pb103 Human, Chian O. sativa spp. indica pb114 Beijing, China O. sativa spp. indica Zhenshan 97 Zhejiang, China O. sativa spp. indica Longtepu Fujian, China O. sativa spp. indica R9719 Ningxia, China O. sativa spp. indica pb15 Sichuan, Chian O. sativa spp. indica Gumei4 Sichuan, China O. sativa spp. indica 9311 Jiangsu, China O. rufipogon 100588 Taiwan, China O. rufipogon 103823 Guangxi, China O. rufipogon Hainan 1 Hainan, China O. rufipogon Dongxiang 1 Shansi, China O. rufipogon 103404 Bangladeshi

O. rufipogon 106057 India O. rufipogon 106352 Burma O. rufipogon 105567 Indonesia O. rufipogon 105720 Cambodia

Supplementary Table 5 (Microsoft Excel file). Analysis of recombinant plants in fine-mapping of the IL locus.

Supplementary Table 6. Primers used in this study. Primer name Primer sequence Enzyme Purpose Caps-1-F CGAACCCGACCAAACATAAATC EcoR1 Mapping Caps-1-R GACTAACTAATACACGCATACAC Mapping Caps-4-F CGCAACACGCAAGCACAAGTATCC Xba1 Mapping Caps-4-R CACGCCCTCATTTCGCACAAGTCT Mapping Caps-7-F GCGCAATTTCTTTCACGGCTTAT Xba1 Mapping Caps-7-R GGCTGGGCGCGACGAGGTT Mapping Caps-8-F CATCGCTTATATTGGTGCTGTGCT SpeI Mapping Caps-8-R GAATTCCCGTTGTCCTCTTGTAGTC Mapping SSR-1-F GAGAGGGGAATCAACATACAC Mapping SSR-1-R GACATACCTAGCTCTGAACGAATT Mapping GFP-CIN-F ATCGATACTTCTCGCTCTCACTTCT Clone GFP-CIN-R GGTACCGTTTTGGCTCCATTCATCAT Clone GUS-CIN-F TATAAGCTTGATCGGCCATACTCC Clone GUS-CIN-R TAGGATCCCTTTGCTCTCACACTTG Clone Wax-F GCCAAGCTTATTACAGCCGTGG Clone Wax-R CAATCGATGGTGGTTGTCTAGCTGTTG Clone Real-GIF1-F CATCGCGCAACCCGAACATG Real-time PCR Real-GIF1-R TGTCGATCAGGCTCCTCAGAG Real-time PCR GIF1-RT-F CCCGCCGGCGACGAGCACCACAT RT-PCR GIF1-RT-R CCGCCGGCCTGAACACCCTGAAGA RT-PCR Dom-P-F CATCGTTTGGCTTATTCGTG Sequencing Dom-P-R TACTGTGTGGACGACCTAAC Sequencing Dom-2-F AGCGCCGATGTACTACAAG Sequencing Dom-2-R CAGGACGTGCGGTTAATTAC Sequencing Dom-5-F TTGTGCAGATCGACAGGTC Sequencing Dom-5-R AAGTCGTTATCCAGAAAGA Sequencing

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