大豆RN型细胞质雄性不育恢复基因Rf1的精细定位与候选基因鉴定

Cytoplasmic male sterility "three-lines" are the basic materials for soybean hybrid breeding. Breeding for strong restorer lines is very complicated and restoring ability can only be identified by testcross with male sterile lines, which is time-consuming and labor-intensive. F2 segregating population, which was obtained by crossing between the gametophyte genetically RN-cytoplasmic male sterile line and its near-isogenic line with strong restorer gene, will be used for the Rf1 gene mapping through determination of the inheritance pattern by sterility identification of each generation and further polymorphism analysis by high density of SSR markers. Then for fine mapping of the Rf1 gene, SNPs and InDels will be developed by target sequence capture microarray and re-sequencing of restorer gene location region of the fertile and sterile DNA library, respectively. Finally, candidate Rf1 gene will be screened in the fine mapping region based on the soybean genome sequencing and re-sequencing database,combined with other plants Rf gene characteristic. In conclusion, the linked markers Rf1 identified in this study will accelerate the breeding process of RN-restorer lines, meanwhile the identified candidate genes will lay the foundation for Rf1 gene cloning and RN-CMS fertility restoration mechanism exploration in future.

细胞质雄性不育"三系"是大豆杂交种选育的基础材料。其中，强恢复系的选育非常繁琐，且恢复力只能通过与不育系的测交来鉴定，既耗时又费力。本研究以配子体遗传的RN型细胞质雄性不育系与其导入强恢复基因的近等基因系杂交获得的F2代分离群体为材料，通过单株育性鉴定,明确恢复基因遗传模式；再利用高密度SSR标记，定位恢复基因Rf1。然后，利用可育和不育DNA文库分别与Rf1基因定位区间特异序列捕获芯片杂交，对杂交片段进行重测序，开发该区间SNPs和InDels标记，进行多态性检测，精细定位Rf1基因。最后，参照大豆基因组测序和重测序数据库，结合其它植物已克隆的Rf基因特征，对精细定位区段候选基因进行筛选分析，并初步鉴定候选Rf1基因。通过本研究所获得的连锁标记，有望加速RN型恢复系的选育进程；同时，筛选鉴定的候选基因，也将为今后克隆Rf1基因和解析RN型细胞质雄性不育育性恢复机制奠定基础。

细胞质雄性不育“三系”是大豆杂交种选育的基础材料。其中，强恢复系的选育非常繁琐，且恢复力只能通过与不育系的测交来鉴定，既耗时又费力。本研究以RN型细胞质雄性不育系与其导入强恢复基因的近等基因系杂交获得的F2代分离群体为材料，通过单株育性鉴定，明确恢复基因遗传模式为显性单基因配子体遗传；再利用高密度 SSR 标记，定位恢复基因至16号染色体上SSR标记SSRBARCSOYSSR-16-1065和BARCSOYSSR-16-1073之间，遗传距离分别为0.9 cM和0.2 cM。然后，利用恢复系和不育系亲本提取基因组DNA进行重测序，开发SNPs和InDels标记，最终将恢复基因定位于40 kb区间。通过比较基因组和生物信息学分析分析预测出1个候选基因。该基因序列长度为1518 bp，编码505个氨基酸残基，分子量为56.75，等电点为8.77。通过实时荧光定量PCR分析发现，恢复系中该基因的表达量在始花期、盛花期和终花期均显著高于不育系。通过生物信息学软件进行定位分析发现该基因是跨膜蛋白，具有线粒体信号肽，能进入线粒体作用于线粒体基因。推测该基因可能是RN型细胞质雄性不育恢复基因Rf1。通过本研究所获得的连锁标记，有望加速RN型恢复系的选育进程；同时，筛选鉴定的候选恢复基因，也将为今后解析育性恢复机制奠定基础。