Supplemental Table 1 Major Agronomic Traits and Yield Heterosis Ability of Ysa Mutant And

1Disruption of a rice PPR protein causes a seedling-specific albino phenotype and 2its utilization to enhance seed purity in hybrid rice production

3Ning Sua,1, Mao-Long Hub,c,1, Dian-Xing Wud,1, Fu-Qing Wua, Gui-Lin Feia, Ying Lana, 4Xiu-Ling Chena, Xiao-Li Shud, Xin Zhanga, Xiu-Ping Guoa, Zhi-Jun Chenga, Cai-Lin 5Leia, Cun-Kou Qic, Ling Jiangb, Haiyang Wanga, Jian-Min Wana,b,*

6aNational Key Facility for Crop Gene Resources and Genetic Improvement, Institute 7of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. 8China

9bNational Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing 10Agricultural University, Nanjing 210095, P.R. China 11cInstitute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 12210014, P.R.China 13dState Key Laboratory of Rice Biology, IAEA Collaborating Center, Institute of 14Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, P.R. China 15 161These authors contributed equally to this work

17＊For correspondence

20 21 22 23 24 25 26 27 28 29 30 31 2

32 Supplemental Figure 1. 33 Phylogenetic tree of OsYSA and 34 other pentatricopeptide repeat 35 proteins in plants. The phylogenetic 36 tree was constructed using MEGA 37 4.1 programs. Bootstrap values are 38 indicated for major branches as 39 percentages. One thousand replicates 40 were used for all possible tree 41 topology calculations. PPR proteins 42 from the following organisms were 43 used: At, Arabidopsis thaliana; Gm, 44 Glycine max; Gh, Gossypium 45 hirsutum; Mt, Medicago truncatula; 46 Os, Oryza sativa; Ph, Petunia hybrid; 47 Pp, Physcomitrella patens; Pt, 48 Populus trichocarpa; Rs, Raphanus 49 sativus; Sb, Sorghum bicolor; Zm, 50 Zea mays. The first two letters of 51 each protein label represent the 52 abbreviated species name, followed 53 by the gene name or locus tag in the 54 Arabidopsis Information Resource 55 (TAIR). The accession numbers of 56 the genes are as follows: 57 NP_683419.1(At ABO5), 58 NP_172066.3(At CLB19), 59 NP_190263.1(At CRR2), 60 NP_182060(At CRR4), 61 NP_200385.1(At CRR21), 62 NP_196000.2(At EMB175), 63 NP_172498.1(At GRP23), 64 NP_180698.1(At GUN1), 65 NP_187576.1(At HCF152), 3

66NP_181456.1(At LOJ),NP_176416.1(At MEF9), NP_177628.2(At OTP43), 67NP_565382(At OTP51), NP_194257.1(At OTP70), NP_178398.1(At OTP85), 68NP_177599.1(At OTP87), NP_194913.1(At PGR3), NP_187294.1(At PPR2), 69NP_188314.1(At PPR40), NP_564786.1(At PPR336), NP_177623.1(At pTAC2), 70NP_201558.3(At DG1), NP_189313.2(At MEF14), NP_200075.1(At MEF1), 71NP_190938.1(At MEE40), NP_179827.1(AT SLO1), NP_193221.3(At LOI1), 72NP_176459.2(AtNG1), NP_172730.3(At RPF1), NP_176454.1(At RPF2), 73NP_176481.2(At RPF3), NP_176050.1(At PGN), NP_200904.1(At PNM1), 74NP_178481.1(At REME1), NP_196831.1(At RARE1), NP_173004.1(AtECB2), 75NP_174349.1(At EMB2279), NP_195209.2(At MRL1), NP_195672.1(At PPR5), 76NP_196000.2(At EMB1899), NP_568141.2(At PPR4), NP_190486.2(At EMB2261), 77NP_193372.6(At SVR7), NP_190408.1(At BIR6), NP_175740(At1g53330.1), 78JN167987(Os YSA), AAS93059.1(Os PPR1), BAD08214.1(Os Rf-1), 79ABC42330.1(Os Rf1a), ABC42331.1(Os Rf1b), ACL79586.1(Os OGR1), 80ACP39956.1(Gh PPR4), AAM52341.1(Ph Rf-PPR592), AAM52340.1(Ph Rf- 81PPR591), BAF02672.1(Pp PPR531-11), ABO70667.1(Rs Rfo), DQ508419.1(Zm 82PPR4), NP_001105879.1(Zm CRP1), NP_001106062.1(Zm PPR5), 83NP_001157212.1(Zm PPR10), NP_001169662.1(Zm PPR), XP_003637510.1(Mt 84PPR), XP_003542463.1(Gm PPR), XP_002305565.1(Pt PPR), XP_002464340.1(Sb 85PPR).

88Supplemental Figure 2. Expression level analysis of YSA in Pei'ai64S and ysa mutant 89by real-time PCR. The relative expression level of YSA was normalized using Actin1 90as an internal control. The expression level of YSA in Pei'ai64S was set as 1.0. Data 91presented are mean values of three experiments. The bars represent the standard 92deviations. No significant differences were observed between Pei'ai64S and ysa based 93on the Student’s t-test, P<0.05.

109Supplemental Table 1 Segregation of wild-type and mutant plants in the F2 110populations derived from four different crosses

Cross WT Mutant Total χ2 (3:1) P value ysa×Pei'ai64S 745 238 983 0.285 0.644 Pei'ai64S ×ysa 1169 392 1561 0.005 0.950 ysa×Taiziyuzhu 6221 2087 8308 0.058 0.836 ysa×9311 1389 457 1846 0.046 0.852 111

133Supplemental Table 2 The PCR-based molecular markers developed for fine 134mapping of ysa 6

Markers Oligos Size (bp) Type BAC number

P1 ACCCACTTGTCATCACC 259 SSR OsJNBa0010D22 CCCACCCTCAACCTCATC (ctt)13 P2 ACACGGCAAGGCTATCG 174 SSR OsJNBb0056O10 CCATCAACGGTGAGGGA (ga)14 P3 ATGAAATACTTTTGACAGGGAA 184 SSR OsJNBa0087M10 TGGATCTTTGTAATAATTGGCT (aag)13 P4 TCCTTCCCGCCAACCCTCTC 149 SSR OsJNBb0042N11 TACTGCTGGGCTTCCCGTTC (aat)5 P5 CCTCCTCATAGCCCCAATCC 253 dCAPS OsJNBb0042N11 TCACTCTCCCATAGGTGGTC AsuI P6 GTGAAGGCTCTAGCGCGACT 277 dCAPS OsJNBa0087M10 ATGGCGTTGTTTAAGGGGAC SpeI P7 TTTCACCCTCTTTTCCCCCC 430 dCAPS OsJNBa0087M10 TCAGACCCGATACATAGCACGAC NcoI P8 CATTATCATTTGTCACGCTCAG 408 dCAPS OsJNBa0087M10 CTTTGTACTCTTGTGCACTTCCA ScaI P9 GGCGATCCTCATCTTGCACT 262 Indel OsJNBa0087M10 TGCCTTGCAGATCCATTTGA 135

138Supplemental Table 3 Major agronomic traits and yield heterosis ability of ysa mutant and its F1 hybrids Type Lines Days Plant Number Panicle Number of Stigma Self- Cross- 1000-grain Yield per from height of tillers length florets per extrusion pollinated pollinated weight plant sowing to (cm) per plant (cm) plant rate seed setting seed setting (g) (g) heading (%) rate rate (d) (%) (%) T/PGMS ysa 99 82.73.4 10.71.6 17.83.1 89.614.7 39.15.2 37.35.2 30.77.3 20.60.7 NA line Pei'ai64S 96 82.12.7 10.51.7 17.22.7 90.213.5 39.64.3 36.54.7 31.26.7 20.80.5 NA F1 hybrid ysa/93-11 132 103.73.5 12.61.9 21.63.4 197.318.7 NA 90.27.2 NA 24.50.5 49.73.1 Pei'ai64S/ rice 132 103.33.2 12.71.5 21.33.8 198.117.9 NA 89.96.9 NA 24.30.4 49.32.9 93-11 Guangzhan 93 NA T/PGMS 87.33.2 10.41.7 18.31.9 97.912.3 44.35.4 47.94.6 39.66.4 25.60.4 63S+ ysa line Guangzhan 90 86.92.9 10.71.5 18.11.6 97.511.7 43.94.5 49.23.5 38.16.8 25.70.3 NA 63S Guangzhan F1 hybrid 121 107.53.5 12.91.7 25.33.5 183.315.3 NA 87.35.9 NA 28.50.5 48.62.9 63S+ysa / rice 93-11 Guangzhan 121 106.93.9 12.31.5 25.93.1 182.416.5 NA 86.95.7 NA 28.10.7 48.33.2 63S/93-11 139 140 141Note 1421. Mean values and standard deviations (s.d) were based on 30 plants. Statistic analysis (Student’s t-test, P<0.05) didn’t detect any significant differences between 8

143plants that have the ysa mutation and the corresponding plants without the ysa mutation. 1442. The agronomic traits of T/PGMS lines and self-pollinated seed setting rates were investigated in the Winter Nursery in Lingshui County (18°16′N, subtropical 145climate), Hainan province in 2007. The T/PGMS lines were fertile in the early spring season, and were sterile in the summer season for production of hybrid seed; 146self-pollinated seed setting rates should be zero. 1473. The cross-pollinated seed setting rates and yields of hybrid rice seed were investigated in the summer season at Jiande (30°15′N, temperate climate), Zhejiang 148Province in 2008 using the indica rice 93-11 as the restorer line. 149NA, data not available.

152Supplemental Table 4 Comparison of the male sterility trait between ysa mutant and 153Pei'ai64S grown in the phytotrons (PAR 384 µmol m-2s-1) Photoperiod Seed setting rate (%) Number of plants Temperature (°C) ysa Pei'ai64S （Hours） 11.5 24 10 18.13±0.69 18.05±0.65 23 10 19.29±0.71 19.23±0.69 12.5 24 10 6.17±0.54 6.21±0.43 28 10 0.95±0.28 0.97±0.31 13.5 24 10 0.25±0.09 0.26±0.06 23 10 1.39±0.11 1.35±0.14 14.5 24 10 3.57±0.21 3.65±0.25 28 10 0.00±0.00 0.00±0.00 154 155Note 1561. Ten plants were used for each comparison. No differences in seed setting between ysa and 157Pei'ai64S in the same growing conditions were detected by the Student’s t-test, P<0.05.

158 159 10

160Supplemental Table 5 Primers used in quantitative real-time RT-PCR 161 Primer Sequence (5'-3') YSA-F： AAGGGGATATCTCAAGATCCGC YSA-R CCATCCACTCTTTGAGATTTCCC Actin1-F TGGCATCTCTCAGCACATTCC Actin1-R TGCACAATGGATGGGTCAGA HEMA1-F： CGCTATTTCTGATGCTATGGGT HEMA1-R： TCTTGGGTGATGATTGTTTGG PORA-F： TGTACTGGAGCTGGAACAACAA PORA-R： GAGCACAGCAAAATCCTAGACG CAO1-F： GATCCATACCCGATCGACAT CAO1-R： CGAGAGACATCCGGTAGAGC CAB1R-F： AGATGGGTTTAGTGCGACGAG CAB1R-R： TTTGGGATCGAGGGAGTATTT CAB2R-F： TGTTCTCCATGTTCGGCTTCT CAB2R-R： GCTACGGTCCCCACTTCACT psaA-F： GCGAGCAAATAAAACACCTTTC psaA-R： GTACCAGCTTAACGTGGGGAG psbA-F： CCCTCATTAGCAGATTCGTTTT psbA-R： ATGATTGTATTCCAGGCAGAGC rbcL-F： CTTGGCAGCATTCCGAGTAA rbcL-R： ACAACGGGCTCGATGTGATA rbcS-F： TCCGCTGAGTTTTGGCTATTT rbcS-R： GGACTTGAGCCCTGGAAGG 162