ACTA AGRONOMICA SINICA SSR. Classification for Some Sterile Lines and Their Restorers of Hybrid Rice with SSR Markers

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1 ACTA AGRONOMICA SINICA Vol132, No12 pp Feb1, 2006 SSR 1 1,2, # , Ξ ( 1, ; 2,,100094) : ( Oryza sativa L. ) SSR(simple sequence repeats), , SSR : (1) SSR 66 65,, (2) GD = 0. 73, ; GD = 0. 63, SSR ; 10 (3) GD = 0. 56,8,,, : ;;SSR ; ;DNA : S511 Classification for Some Sterile Lines and Their Restorers of Hybrid Rice with SSR Markers HE Hao2Hua 1, LUO Xiao2Jin 1,2, #, ZHU Chang2Lan 1, HE Xiao2Peng 1, FU Jun2Ru 1, SUN Jun2Li 1, 3, ZHANG Hong2Liang 2 and LI Zi2Chao 2 ( 1 Group of Crop Genetics and Breeding, Jiangxi Agricultural University, Nanchang , Jiangxi ; 2 Key Laboratory of Crop Genomics and Genetic Improvement, Ministry of Agriculture, and Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing , China) Abstract : Abundant heterosis is manifested from the cross of two genetically diverse parent lines. Therefore, it is essential to reveal genetic relationships among varieties with unknown pedigree. In the present study, genetic divergences among 66 rice ( Oryza sativa L. ) varieties, including 5 photoperiod/ thermo2sensitive genic male sterile lines, 7 cytoplasmic male sterile lines, 53 restorers and one wild rice variety, were analyzed by using 36 SSR ( simple sequence repeats) primer pairs which disperse on 12 chromosomes. A total of 300 polymorphic bands were detected among 66 varieties, and on average distinguishable bands were observed after amplification with each primer pair. Genetic divergences between all possible pairs of varieties were defined by Nei M method with genetic distance ( GD), calculated from SSR data by the equation GD = - ln[2 M XY / ( M X + M Y ) ], where M XY was the total number of bands common to varieties X and Y, and M X and M Y were the total number of bands present in X and Y respectively. 66 cultivars were clustered based on GD by Unweighted Pair Group Method using Arithmetic Average (UPGMA). The results were concluded as follows : (1) Sixty2five out of sixty2six varieties were distinguished obviously. Different ecotypes were also defined using SSR markers, which were consistent with the relationships of their pedigrees, and were similar with the result of phenotype analysis. ( 2) Indica, japonica and wild rice were discriminated based on GD = 0. 73,and some indica cultivars bearing japonica or wild rice consanguinity were discriminated based on GD = 0. 63, indicating that SSR markers are sensitive and precise for classification on indica, japonica and wild rice. Otherwise, ten specific primers were screened and selected to identify Ξ : ( ) : (1964 ),,,,, E2mail : com #, 3 Received( ) : ; Accepted( ) : 转载

2 subspecies between indica and japonica. (3) Twelve male sterile lines and more than fifty restorers were divided into eight ecotypes based on GD = 0. 56,and many parents of hybrid rice used in large scale were classified into multiple ecotypes, suggesting there are great genetic divergences between the parents of elite hybrid rice. Parents selection of hybrid rice will get benefit from the classification on both male sterile lines and restore lines. Key words :Hybrid rice ; Classification ; SSR marker ; Heterosis ecotype ; DNA fingerprint,,, [1 ] [2 ] [3 ] ( ) [4 ],SSR, RFLP RAPD,, DNA [529 ],,SSR ( ) 1 [5,10215],,SSR SSR [16218],SSR [8] SSR,, SSR Table 1 Materials tested and their origins 64S63S 360S S24S S 5, A A A 23A A 1A 9A 7,54 (1) Type of Type of No. Name Origin No. Name Origin I2J I2J 1 64S Pei ai 64S Hunan I Liaoning J 2 S Tianfeng S Jiangxi I Gui 99 Guangxi I 3 360S Guizhou I Jiayu 948 Zhejiang I 4 63S Guangzhan 63S Liaoning I Jiangxi I 5 S24S Jiangxi I Jiangxi I 6 A DongA Jiangxi I 39 F6 Jiangxi I 7 A Fei A Philip. I Jiangxi I 8 9A Zhong 9A Zhejiang I Hunan I 9 1A Chang 1A Jiangxi I Jiangxi I 10 23A Jin 23A Hunan I Jiangxi I 11 A Xinxiang A Hunan I 44 Lemont USA J 12 A Bo A Guangxi I 45 IR36 IRRI I 13 5 Ganyouzao 5 Jiangxi I Jiangsu I Zhongjian 100 Zhejiang I 47 Maiyingdao Jiangxi I 15 HR2195 Sichuan I 48 T070 Jiangxi I 16 7 Yuanzao 7 Zhejiang I Zhongzao 14 Zhejiang I 17 4 Yuanjing 4 Jilin J Xiangzaoxian 19 Hunan I 18 2 Zhuzaoxian 2 Hunan I 51 4 Simiao 4 Jiangxi I 19 Zigui Jiangxi I 52 R1722 Jiangxi I 20 8 Simiao 8 Jiangxi I Jiangxi I 21 1 Heihui 1 Jiangxi I 54 R402 Hunan I 22 R256 Jilin J Jiangxi I 23 T168 Jiangxi I Ce 35 Hunan I 24 B115 Jiangxi I 57 B215 Jiangxi I Jiangxi I 58 R355 Jiangxi I 26 E32 Jiangsu I 59 R120 Jiangxi I 27 R4480 Guangdong I Mianhui 501 Sichuan I Zhongfu 906 Zhejiang I Xiangxian 160 Jiangxi I Zhe 941 Zhejiang I 62 A156 Jiangxi I Jiangxi I 63 A157 Jiangxi I 31 1 Fengtian 1 Jiangxi I 64 A166 Jiangxi I Zhejiang I 65 A174 Jiangxi I 33 5 Zhongyouzao 5 Zhejiang I 66 Dongxiang wild rice Jiangxi :I =,I =, J =,J =. Notes :I = Indica, I = Indica like,j = Japonica, J = Japonica like.

3 2 : SSR ,3, 6 8, 2,15, cm 20 cm,,, ;, 5,, 8, DPS, 1. 3 SSR DNA Rogers Bendich CTAB (Cetyl triethyl ammonium bromide) 36 SSR 12, 3 ( 2) 2 Table 2 The primers used in the study 1 RM23 5 RM249 9 RM215 RM35 RM122 RM219 RM237 RM164 RM242 2 RM207 6 RM RM222 RM208 RM225 RM228 RM211 RM253 RM216 3 RM231 7 RM82 11 RM206 RM232 RM214 RM224 RM16 RM234 RM229 4 RM226 8 RM42 12 RM247 RM241 RM256 RM17 RM261 RM223 RM PCR 15 L 2. 5 L DNA (20 ng/ L), L Taq polymerase (5 U/ L),3. 0 L 10 buffer ( Mg 2 + ),1. 4 L primer,0. 18 L dntp(10 mol/ L), L H 2 O 95 5 min ; s, 55 1 min,72 2 min,30 ;72 10 min PTC2200 PCR,8 % 1. 5 SSR 1, 1 ; 1,1,0, 2 GD = - ln[2 M XY / ( M X + M Y ) ] [19 ] M X M Y X Y, M XY, UPGMA ( unweighted pair2group method with arithmetic average, ) [20 ], SSR 66, , , UPGMA ( 1) 1,, GD = 0. 73, 3, R , 0. 25,,,, ( ), GD = 0. 63, 3,3 ( ),64S Lemont T168, 64S,T168,Lemont ; 1 A,, ;, GD = 0. 56, 3 ( ) A166 A174 A156 A157 E R355 4 A 458, A166 A174 A156 A157,E32 ;2374 R3554 A 458 E32,E32

4 UPGMA Fig11 Dendrogram of 66 varieties of rice based on UPGMA method R120 F6 HR2195 R S S 360S B B115 T S24S 941

5 2 : SSR R1722 9A 23A A 1A A, R120 F6 752, 1A A, 9A 23A A A,B115 T ,B IR R ,IR36 R IR SSR, (2 ) 12 (1 ) 6 (1,3) 66, 65,,, 3 6 Table 3 Distribution of six primers on chromosome RM RM242 9 RM217 6 RM207 2 RM16 3 RM A166 A174 A156 A157,,36 A166 A174, RM217 ( RM224) RM207 RM241 RM226 RM35 RM164 RM17 RM211 RM23 RM256 RM261 A156 A157 A166 ( A174),66 ( ) SSR, ,, 9311 E32 64S,R402 F AS, 99 9A 23A,752 A A,,,,,9A 23AA A 1A,, ; 63S S 360S, 2. 4 SSR 40,DPS 8 ( 2),2,, SSR, Fig. 2 Dendrogram of 8 characters of 40 varieties clustered by mean distance 1 = 9520, 2 = 4, 3 = 35, 4 = 14, 5 = E32, 6 = R402, 7 = , 8 = F6, 9 = R1722, 10 = 906, 11 = 4, 12 = 19, 13 = 1, 14 = Lemont, 15 = 941, 16 = 7, 17 = R4480, 18 = 92206, 19 = 948, 20 = 2, 21 = 8, 22 = 2374, 23 = 5, 24 = 974, 25 = 647, 26 = 2923, 27 = 99, 28 = 752, 29 = 138, 30 = , 31 = 100, 32 = HR2195, 33 =, 34 = 5, 35 = 458, 36 = 651, 37 = R355,38 = IR36, 39 = R256, 40 = B215 1 = 9520, 2 = Yuanjing 4, 3 = Ce 35, 4 = Zhongzao 14, 5 = E32, 6 = R402, 7 = , 8 = F6, 9 = R1722, 10 = Zhongfu 906, 11 = Simiao 4, 12 = Xiangzaoxian 19, 13 = Fengtian 1, 14 = Lemont, 15 = Zhe 941, 16 = Yuanzao 7, 17 = R4480, 18 = 92206, 19 = Jiayu 948, 20 = Zhuzaoxian 2, 21 = Simiao 8, 22 = 2374, 23 = Zhongyouzao 5, 24 = 974, 25 = 647, 26 = 2923, 27 = Gui 99, 28 = 752, 29 = 138, 30 = , 31 = Zhongjian 100, 32 = HR2195, 33 = Zigui, 34 = Ganyouzao 5, 35 = 458, 36 = 651, 37 = R355, 38 = IR36, 39 = R256, 40 = B215.

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