白菜型油菜根肿病抗性基因的精细定位及候选基因分析

Clubroot disease is caused by a biotrophic soil-borne pathogen Plasmodiophora brassicae. In recent years, this disease is rapidly spreading in the main growing areas of rapeseed in China, which causes huge losses of rapeseed production. However, there are a few clubroot resistance (CR) resources of Brassica napus in China. Fortunately, many CR materials have been found in the closely related species of rapeseed, such as Brassica rapa, Brassica oleracea and so on. In this study, the mechanism of clubroot resistance was studied in a CR B. rapa line Xu3211. Bulked segregant RNA-Seq (BSR-Seq) was employed to identify and characterize single-nucleotide polymorphisms (SNPs). Kompetitive Allele Specific PCR (KASP) technology was used to develop SNP markers linked to the CR gene, construct high resolution genetic maps and a physic map, and finely map the CR gene. Based on the genomic information of the CR gene, molecular markers linked to the CR gene were developed until the cosegregated molecular markers were identified. According to the annotations of the genes, candidate genes of clubroot resistance were identified, and the structure and expression of these candidate genes were analyzed. In order to verify the function of the candidate genes, these genes were inserted into an expression vector, which were transformed into the wild-type Arabidopsis thaliana. This study will provide a solid foundation for studying clubroot resistance mechanism and CR molecular breeding in rapeseed.

根肿病是由芸薹属根肿菌引起的一种土传病害，近年来该病在我国油菜主产区迅速蔓延，给我国油菜生产带来巨大损失。目前我国甘蓝型油菜中缺乏抗根肿病的资源，而其近缘物种白菜（白菜型油菜）、甘蓝等中存在抗根肿病的材料。因此本研究以一个抗根肿病的白菜型油菜Xu3211为研究对象，利用经典遗传学的方法研究根肿病抗性的遗传规律；采用RNA-Seq（BSR-Seq）技术获取根肿病抗性相关的SNPs，利用KASP技术开发与根肿病抗性相关的SNP标记，建立根肿病抗性基因的高密度遗传及物理图谱，精细定位根肿病的抗性基因；利用定位区域内的基因组信息，开发抗性基因连锁的分子标记，直至获得共分离的分子标记，结合定位区域内基因功能注释，确定根肿病抗性基因的候选基因；对候选基因进行结构及表达分析，构建表达载体转化野生型拟南芥，验证候选基因的功能。该研究将会为研究油菜根肿病的作用机理以及抗根肿病分子育种奠定基础。

本研究利用正向遗传学的方法精细定位了白菜型油菜Xu3211的根肿病抗性基因，获得Xu3211根肿病抗性相关的候选基因，并初步对候选基因的功能进行了验证：1）开发了12个与根肿病抗性基因连锁的分子标记，建立了抗病基因高密度遗传连锁图谱，将候选基因的区域定位到了A03：23,339,019-23,465,030bp之间。利用转录子分析以及候选区域内的基因的结构分析、功能注释，获得了3个根肿病抗性基因的候选基因，并将其转化野生型拟南芥，在T3代，对过表达株系进行了抗病性鉴定，其中基因Bra019409的过表达株系，表现出抗我国根肿菌P4生理小种，因此初步判断该基因是本研究的候选基因。