利用BSA-Seq筛选甘蓝型油菜菌核病抗性候选基因

Identification of Major Sclerotinia Resistance Genes by BSA-Seq of Brassica napus

油菜菌核病居油菜3大病害之首,它所引起的茎腐烂是导致油菜减产的重要原因之一,因此筛选出调控菌核病的主效基因,对利用分子辅助育种手段提高油菜产量具有重要意义.该研究以甘蓝型油菜高抗材料21Y490为父本与高感材料21Y689杂交,在F2分离群体终花期截取油菜茎秆进行核盘菌室内接种,培养3 d后测量菌斑大小鉴定菌核病抗性.筛选抗性极端表型个体构建DNA混池,与亲本一起重测序提取差异SNP,InDel信息,利用BSA-seq技术、ED算法和Δ-index算法获得两个显著的关联区域,分别位于C09号染色体的17.8~21.2 Mb和31.6~39.1 Mb区间,区间内共有399个基因.最后,利用拟南芥和甘蓝型油菜基因组注释信息对关联候选区间对应的基因功能进行分析,共筛选出与油菜菌核病抗性相关的8个候选基因(BnaC05g50540D,BnaC09g33900D,BnaA04g09130D,BnaA01g29170D,BnaC09g15640D,BnaC09g35310D,BnaA03g09220D,BnaCnng15460D),通过候选基因同源分析、代谢通路分析及共表达基因分析发现,这些基因主要参与植物激素生长调控及免疫系统的信号转导,为下一步抗性基因的克隆及功能研究奠定了基础.

Sclerotinia sclerotiorum is ranked the first among the three major diseases of rapeseed,and stem rot caused by it is one of the important reasons for the reduction of rapeseed yield.Therefore,screening out the major genes regulating sclerotinia is very important for improving rapeseed yield with molecular assisted breeding.In this study,the high-resistant material 21Y490 was crossed with the high-susceptible material 21Y689.The rape stalks of F2 population were cut at the final flowering stage for indoor inoculation of Sclerotinia sclerotiorum.After culturing for 3 days,the size of the plaque was measured to identify the resistance to Sclerotinia sclerotiorum.DNA mixing pools were constructed with screened extreme resistant and susceptible individuals,respectively,and sequenced with parents to extract differential SNP and InDel information.Using BSA-seq,ED algorithm andΔ-Index algorithm,two significant association regions were identified in the 17.8-21.2 Mb and 31.6-39.1 Mb regions of chromosome C09,respectively.There were 399 genes in the region.Combined with gene annotation,8 candidate genes related to Sclerotinia sclerotiorum resistance were screened out(BnaC05g50540D,BnaC09g33900D,BnaA04g09130D,BnaA01g29170D,BnaC09g15640D,BnaC09g35310D,BnaA03g09220D,BnaCnng15460D).The results of candidate genes homology analysis,metabolic pathway analysis and co-expression analysis showed that these 8 candidate genes are mainly involved in plant hormone growth regulation and signal transduction of the immune system,which laid a foundation for the cloning and functional research of resistance genes in the further work.