水稻F1花粉不育基因的精细定位及其遗传分化研究

Fine Mapping and Genetic Differentiation Analysis of F_1 Pollen Sterility Genes in Rice (Oryza sativa L.)

水稻籼粳亚种间杂种的不育性限制了亚种间的遗传交流和杂种优势利用。本研究通过发展位置特异性的微卫星标记将F1花粉不育基因S b座位进行了精细定位 ;通过分析近等基因系中代换片段的遗传效应 ,鉴定出了F1花粉不育基因S d座位 ,利用位置特异性的微卫星标记将S d进行了定位 ;根据基因组的序列资料和利用较大的作图群体对S b和S d两个座位进行了物理作图 ;通过分子标记辅助选择培育了一批复等位基因近等基因系 ,对育性基因的遗传分化进行了研究。取得了如下主要结果 :1、根据S b座位初步定位的结果发展位置特异性的微卫星标记 ,将F1花粉不育基因座S b进行了精细定位。结果表明多态性标记均与S b座位紧密连锁 ,其中R830STS、PSM 7、PSM8、PSM 9、PSM 5 9和PSM 6 0位于S b座位一端 ,与S b座位遗传距离分别为 1 5cM、 1 2cM、 0 9cM、 0 9cM、0 9cM和 0 9cM ,而PSM 2 0 2、PSM 2 0 6、PSM 2 0 8、RM13、R2 2 13SSTS和RM 4 13位于S b座位的另一端 ,与S b座位的遗传距离分别为 0 9cM、 2 1cM、 3 8cM、 4 1cM、 4 4cM和 5 3cM。2、根据S b座位精细定位的结果 ,从IRGSP下载了S b座位所在区域克隆的序列 ,将克隆的序列进行了拼接 ,同时将与S b座位紧密连锁的分子标记与序列拼接图进行了电子整合。根据?

The hybrid sterility between two subspecies hampered utilization of heterosis in rice In this research, F 1 pollen sterility locus S d was identified through analysis of genetic effect of substitution segments in near isogenic line, which was subsequently genetically mapped by developing postion specific microsatellite markers. Two pollen sterility genes, namely S b and S d , were physically mapped based on the genome sequences by using large mapping populations. The differentiation of pollen sterility genes was investigated by developing multiple near isogenic lines through marker assisted selection. The main results are as follows. 1 Based on the primary mapping results, F 1 pollen sterility locus S b was fine mapped by developing postion specific microsatellite markers. The results indicated twelve polymorphic markers were tightly linked with locus S b . R830STS, PSM7, PSM8, PSM9, PSM59, PSM60 were located on the one side of locus S b and the genetic distance between S b and each marker was 1 5cM, 1 2cM, 0 9cM, 0 9cM, 0 9cM, 0 9cM, respectively, while PSM202, PSM206, PSM208, RM13, R2213SSTS, RM413 were located on the other side and the genetic distance between S b and each marker was 0 9cM, 2 1cM, 3 8cM, 4 1cM, 4 4cM, 5 3cM, respectively 2 According to the results of fine mapping, sequences of PAC or BAC clones around the region of locus S b were downloaded from IRGSP and were assembled into one contig. The markers tightly linked with S b were also anchored to the physical map by the use of in silico hybridization. Postion specific microsatellite markers and STS markers were designed based on the integration results of genetic and physical map. Locus S b was finally delimitated to the region of 182.2kb between PSM8 and PSM215 using one mapping population of 500 plants, while there have four markers, viz. PSM214, T17, T18 and T19, cosegregated with S b . 3 One new F 1 pollen sterility gene, S d , was identified on chromosome 1 through analysis of the genetic effect of substitution segments in near isogenic line E11 5. S d was genetically mapped by developing postion specific microsatellite markers based on the genome sequences. The results indicated that microsatellite markers PSM27, PSM24, PSM26, PSM23, PSM31, PSM25, PSM37, PSM41, PSM42, PSM43, PSM44, PSM12, PSM13 were located on the one side of locus S d and the distance between S d and each marker was 10 6cM, 7 2cM, 7 2cM, 6 8cM, 6 8cM, 6 4cM, 6 4cM, 4 8cM, 4 8cM, 3 2cM, 1 6cM, 0 4cM, 0 4cM, respectively, while RM84, RM86, RM323, RM1, RM283 were located on the other side and the distance between S d and each marker was 3 8cM, 4 6cM, 6 7cM, 7 5cM, 8 7cM, respectively. 4 According to the results of genetic mapping, sequences of PAC or BAC clones around the region of locus S d were downloaded from IRGSP and were assembled into one contig. The markers tightly linked with S d were also anchored to the physical map by the use of in silico hybridization. Postion specific microsatellite markers and STS markers were designed based on the integration results of genetic and physical map. Using one mapping population of 2160 plants, locus S d was finally delimitated to the region of 67 8kb between PSM93 and PSM74, while there have four markers, viz. PSM95, PSM96, T1 and T2, cosegregated with S d . RiceGAAS analysis indicated that there have 17 ORFs on the region, among with three ORFs may have relation with hybrid sterility. BLAST analysis indicated that homology on the region was low between indica and japonica subspecies, which may be one of reasons of hybrid sterility. 5 A series of multiple near isogenic lines were developed on F 1 pollen sterility loci S a,S b, S c and S d through the marker assisted selection. Test cross analysis indicated that the multiple genes on each locus not only differentiated into opposite S i and S j , the differentiation of the multiple genes with same genotype also