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

Abstract

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) 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 Rcr2. 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 Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2.

Keywords: Brassica rapa; Plasmodiophora brassicae; RNA-Seq; SNPs; clubroot; fine mapping.

FIGURE 1

Phenotypes of the Brassica rapa parents: Chinese cabbage cv. “Jazz” (R) and susceptible canola breeding line ACDC. Plants were inoculated with pathotype 3 of Plasmodiophora brassicae.

FIGURE 2

BSR-Seq to map Rcr2 according to the method described by Liu et al. (2012). (A) The physical position of each SNP marker (x-axis) was plotted versus the probability of each SNP marker being in complete linkage disequilibrium with the causal gene (y-axis). (B) Chromosome A03 was scanned by using a window containing 50 SNPs with a step size of five SNPs. Within each window, the median linkage probability obtained from a Bayesian BSA analysis across all the 50 SNPs was determined and was plotted against the middle physical position of the window.

FIGURE 3

Linkage maps of the regions in which the Rcr2 gene is located. Broken lines drawn regions defined by different molecular markers on B. rapa linkage group A03. (A) Fine mapping of Rcr2 based on a F₁ population consisting of 675 plants derived from ACDC × Jazz. The genetic distance is shown on the left. (B) Physical locations in bases (right) of the SNP markers.

FIGURE 4

Number of unique poly variants in the four TIR-NBS-LRR genes from the pooled R samples.