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利用cDNA抑制差减杂交技术建立了水稻减数分裂时期穗部特异表达的SSH文库,从中筛选121个cDNA片段作为EST(expressedsequencetags)标记,用RFLP(restrictionfragmentlengthpolymorphism)方法分析了温敏不育系安农S-1和正常品种安农N之间的多态性,其中一个EST标记HN57可以检测到亲本间的多态性.共分离分析结果表明,HN57与安农S-1的温敏不育性完全共分离,进而将安农S-1的温敏不育基因tms5定位于RGP(ricegenomeresearchprogram)遗传图的第二染色体的31.2cM处.为了进行精细定位,在该区域设计了80对SSR(simplesequencerepeat)引物对,用12对多态性引物将tms5定位于181kb区间
SSH cDNA library was constructed based on cDNA subtractive hybridization (SSH). At the meantime, 121 cDNA fragments were screened for EST (expressed sequence tags) markers. An RFLP (restriction fragment length polymorphism) method was used to analyze the thermo-sensitive sterile line Annong S-1 and the normal variety Annong N. One of the EST markers, HN57, could detect the polymorphism among parents.The co-segregation analysis showed that the thermo-sensitive sterility of HN57 and Annong S-1 Completely separated, and then the thermo-sensitive sterile gene tms5 of Annong S-1 was located at 31.2 cM of the second chromosome of RGP (genetic map of RGP) .For the fine mapping, 80 pairs of SSRs (simplesequencerepeat ) Primer pair, 12 pairs of polymorphic primers will be located in the tms5 181kb interval