Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing

Clubroot, caused by <i>Plasmodiophora brassicae</i>, is an important disease of canola (<i>Brassica napus</i>) in western Canada and worldwide. In this study, a clubroot resistance gene (<i>Rcr2</i>) was identified and fine mapped in Chinese cabbage cv. "Jazz" using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene <i>Rcr2</i>. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and <i>Rcr2</i> was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from <i>Rcr2</i>, respectively. Five SNP markers co-segregated with <i>Rcr2</i> in this region. Variants were identified in 14 of 36 genes annotated in the <i>Rcr2</i> target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them <i>Bra019410</i> and <i>Bra019413</i>, had high numbers of polymorphic variants and so are the most likely candidates of <i>Rcr2</i>.