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.
Highlights
Plasmodiophora brassicae Woronin is a biotrophic soil-borne pathogen in the Infrakingdom Rhizaria (Nikolaev et al, 2004) that causes clubroot disease in Brassica oil and vegetable crops
Selected single-nucleotide polymorphisms (SNP) identified in the target region were confirmed using the Kompetitive Allele Specific PCR (KASP) method2 following the manufacture’s instruction
RNA sequencing (RNA-Seq) is a robust approach for quantifying gene expression that has been widely used for transcriptome analysis in a range of organisms (Liu et al, 2012)
Summary
Plasmodiophora brassicae Woronin is a biotrophic soil-borne pathogen in the Infrakingdom Rhizaria (Nikolaev et al, 2004) that causes clubroot disease in Brassica oil and vegetable crops. Sources of clubroot resistance in canola are limited, but genotypes with resistance to a broad range of pathotypes of P. brassicae have been identified in the canola progenitor species B. rapa (Hasan et al, 2012; Peng et al, 2014). These lines could be used to broaden the genetic base of clubroot resistance in canola (Yu et al, 2016). Introgression of important agronomic traits such as clubroot resistance from B. rapa into canola is possible through conventional breeding (Yu et al, 2012; Gautam et al, 2013; Li et al, 2013), but it is important to identify and map the resistance genes in B. rapa so that genes can be transferred into canola efficiently
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