摘要
水稻籼粳亚种间存在着强大的杂种优势 ,但杂种育性普遍偏低成为这一杂种优势利用的主要障碍。研究证明 ,花粉不育是导致杂种不育的主要原因之一。张桂权和卢永根等 (1987- 1994 )鉴定了 6个花粉不育基因座位 (S a ,S b ,S c ,S d ,S e和S f)。其中S a基因座位已被庄楚雄等 (1996 )初步定位在水稻第一染色体着丝粒附近。本论文是在此基础上 ,利用美国Cornell大学、日本RGP构建的水稻高密度遗传图和日本构建的BAC/PAC物理图及其基因组测序资料 ,对S a作进一步的精细定位 ,建立了包含该基因的TAC重叠群 ,并通过序列分析发现S a候选基因 ,为分离鉴定S a基因及研究该基因在水稻杂种不育中的分子作用机理打下了基础。本研究主要结果如下 :1 构建了台中 6 5 (含S aj)及其近等基因系TILS4 (E4 ,含S ai)杂交的F2 群体 ,观察了 70 6株F2 个体的花粉育性分离状况。其中可育株 36 7株 ,半不育株 339株 ,分离比为 1∶1。2 利用前人筛选的多态性标记R2 15 9、R192 8和本研究新获得的多态性标记GR2、GR1、AR1、D2 3M、D1 5S、F12M 1,用 70 6株F2 群体对S a进行了的精细定位。结果表明S a与分子标记R2 15 9、GR2、GR1、AR1、R192 8、F12M 1、D1 5S和D2 3M之间的遗传距离分别为 2 0 7cM ,1 2 1cM ,0 6 5cM ,0 4
The partial sterility in hybrids between indica and japonica rice is the major problem for rice breeding in utilization of inter-subspecies heterosis. For understanding the molecelur mechanism of hybrid sterility, it is necessary to clone the genes and study their functions using molecular approaches. In this study, fine mapping of S-a locus was carried out by used a large number of molecular markers and a large F 2 population. A physical map covering the S-a locus was constructed by using transformation-competent artificial chromosome (TAC) libraries. Candidate genes for S-a were searched by sequence analysis. The main results are as follows: 1. The pollen fertility of 706 plants of an F 2 population developed from a cross of T65×E4 was investigated. The number of plants with full-fertile and semi-sterile pollens was 367 and 339, respectively. The segregation ratio fit to 1:1. The results confirmed that the genetic interaction of S-aj/ S-ai fits the "one locus sporo-gametophytic interaction model". 2. Fine mapping of S-a was carried out with RFLP, SSR and SNP markers. Eight polymorphic markers closely linked to S-a locus were found. Five markers AR1?R1928?F 12M1?D1.5S和D2.3M were mapped to S-a locus with closely genetic distances of 0.45cM, 0.42cM and 0.42cM, 0.14cM, 0cM respectively. This result indicated that S-a is located near the centromere of chromosome 1, between molecular markers R1928 and D1.5S. Since the physical distance per recombinant event on the region is about 12-17kb, the S-a locus should be located on a region of ca. 30kb around the marker D2.3M. 3. Three TAC genomic libraries were constructed by using an indica variety Guang Lu Ai 4, a japonica variety Taichong 65 and its isogenic line E4 as plant materials. The average size of insert of the libraries was about 43kb. The total genome coverage of each library was about 10 haploid genome equivalents. By using the probes located on the S-a locus region to screen TAC libraries, a TAC contig covering the S-a locus was constructed. Two TAC clones covering the candidate genes were selected. 4. Based on the sequence annotation by RGP GAAS system, several ORFs were found on the mapped S-a locus region. Three of the ORFs with HLH DNA binding domain, protein kinase domain (PK), and DW40 repeats, respectively, were analyzed and selected as the candidates of S-a gene. 5. Using RT-PCR with gene-specific primers, cDNAs were amplified from panicle RNA of T65 and E4. The WD40 repeat gene is 17kb in genomic and 3615bp in ORF with 31 exons, with a DENN (Differentially expressed in normal and neoplastic cells) domain and 8 WD40 repeats in the N and C terminals, respectively. Further sequencing and expression studies of the PK and WD40 repeat genes are underway. The results obtained in this study open the door to identify the S-a gene and study the molecular mechanism of rice hybrid sterility.
出处
《分子植物育种》
CAS
CSCD
2003年第5期757-758,共2页
Molecular Plant Breeding
