Analysis of genome-wide variants through bulked segregant RNA sequencing reveals a major gene for resistance to Plasmodiophora brassicae in Brassica oleracea

Two cabbage ( Brassica oleracea ) cultivars ‘Tekila’ and ‘Kilaherb’ were identified as resistant to several pathotypes of Plasmodiophora brassicae . In this study, we identified a clubroot resistance gene ( Rcr7 ) in ‘Tekila’ for resistance to pathotype 3 of P. brassicae from a segregating population derived from ‘Tekila’ crossed with the susceptible line T010000DH3. Genetic mapping was performed by identifying the percentage of polymorphic variants (PPV), a new method proposed in this study, through bulked segregant RNA sequencing. Chromosome C7 carried the highest PPV (42%) compared to the 30–34% in the remaining chromosomes. A peak with PPV (56–73%) was found within the physical interval 41–44 Mb, which indicated that Rcr7 might be located in this region. Kompetitive Allele-Specific PCR was used to confirm the association of Rcr7 with SNPs in the region. Rcr7 was flanked by two SNP markers and co-segregated with three SNP markers in the segregating population of 465 plants. Seven genes encoding TIR-NBS-LRR disease resistance proteins were identified in the target region, but only two genes, Bo7g108760 and Bo7g109000 , were expressed. Resistance to pathotype 5X was also mapped to the same region as Rcr7 . B. oleracea lines including ‘Kilaherb’ were tested with five SNP markers for Rcr7 and for resistance to pathotype 3; 11 of 25 lines were resistant, but ‘Kilaherb’ was the only line that carried the SNP alleles associated with Rcr7 . The presence of Rcr7 in ‘Kilaherb’ for resistance to both pathotypes 3 and 5X was confirmed through linkage analysis.