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大豆细胞质雄性不育育性恢复基因Rf3的精细定位
贾顺耕, 郭凤兰, 林春晶, 孙妍妍, 张 颖, 雷 蕾, 彭 宝, 赵丽梅, 张春宝
0
(吉林省农业科学院大豆研究所)
摘要:
在植物细胞质雄性不育/恢复系统中,一个不育基因能够被不同的恢复基因所恢复。在利用“三系”法进行杂交大豆选育过程中同样发现,作为父本的恢复系恢复能力不尽相同,可能存在不同的育性恢复基因。本研究以大豆细胞质雄性不育系JLCMS84A为母本,以恢复系JLR1为父本配制杂交组合所获得的F2代分离群体为试验材料,在大豆盛花期(R2期)根据花粉育性鉴定及显著性分析结果,明确其为显性单基因遗传模式;结合集群分离分析法(BSA,bulked segregant analysis)和高通量测序技术,将JLR1所含恢复基因定位在9号染色体上;进一步利用简单重复序列(SSR,simple sequence repeat)分子标记将该基因定位在分子标记BARCSOYSSR_09_1151和BARCSOYSSR_09_1183之间,遗传距离分别为0.6 cM和0.5 cM。其在ZH13 v2.0参考基因组中的物理位置位于9号染色体上的41.152 Mb~41.674 Mb区间,物理距离约为522 kb。研究结果表明该基因是一个不同于已有研究的新恢复基因,将其命名为Rf3。对定位区间进行分析发现,区间内的51个基因中SoyZH13_09G151700、SoyZH13_09G152000和SoyZH13_09G154200具备恢复基因典型特征,即为PPR蛋白家族编码基因。对上述三个PPR基因进行单拷贝克隆和测序分析发现,SoyZH13_09G152000在亲本基因序列间有3处单核苷酸突变,2处发生单核苷酸插入缺失,产生了移码突变,SoyZH13_09G154200在亲本基因序列间有47处单核苷酸突变,推测这两个基因中的一个可能为Rf3基因的候选基因。本研究为Rf3基因的精细定位及鉴定提供了研究基础,同时也为开发与该基因紧密连锁的分子标记,并利用分子标记辅助选择(MAS,marker-assisted selection)含有此恢复基因的新恢复系提供了可靠的理论依据。
关键词:  细胞质雄性不育  恢复基因  集群分离分析法  简单重复序列  大豆
DOI:10.13430/j.cnki.jpgr. 20210204003
投稿时间:2021-02-04修订日期:2021-02-27
基金项目:吉林省科技发展计划项目(20190101007JH);吉林省农业科技创新工程项目(C02100415)
Fine Mapping of Fertility Restorer Gene Rf3 of Cytoplasmic Male Sterility in Soybean
JIA Shun-geng, GUO Feng-lan, LIN Chun-jing, SUN Yan-yan, ZHANG Ying, LEI Lei, PENG Bao, ZHAO Li-mei, ZHANG Chun-bao
(Soybean Research Institute, Jilin Academy of Agricultural Sciences)
Abstract:
In cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) system, the male sterility might become fertile once the fertility restorer gene(s) is present. However, different restorer genes may represent levels of restorative ability in the three-line hybrid soybean breeding. In this study, the CMS line JLCMS84A and its restorer line JLR1 were used as female and male parents, respectively, to construct F2 population used for genetic mapping. Segregation analysis during R2 stage indicated a typical inheritance pattern of gametophytic CMS with fertility restorer being controlled by a single dominant gene. allocated The restorer gene was allocated using BSA-seq on chromosome 9 between SSR markers BARCSOYSSR_09_1151 and BARCSOYSSR_09_1183 with the distances of 0.6 cM and 0.5 cM, respectively. Based on the soybean reference genome ZH13 v2.0, this physical interval spans 522 kb between 41.152 to 41.674 Mb and hosts a novel restorer gene named Rf3. Out of 51 annotated genes in the target region, three genes (SoyZH13_09G151700, SoyZH13_09G152000 and SoyZH13_09G154200) are encoded for putative pentatricopeptide repeat (PPR) protein. By PCR amplification and Sanger sequencing of their full-length DNA sequences between the CMS line JLCMS84A and its restorer line JLR1, three SNPs and two InDels, which lead to frame-shift mutations in SoyZH13_09G152000, have been identified, while 47 SNPs in SoyZH13_09G154200 have been detected. Therefore, both genes were the strongest candidates for Rf3. These results lay a foundation for fine mapping and function identification, as well as marker-assisted selection of new restorer lines which contained this restorer gene.
Key words:  cytoplasmic male sterility  restorer gene  BSA  SSR  soybean

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