Identification and Genetic Mapping of Clubroot Resistance in Two Brassica nigra Lines

Clubroot, caused by the protist Plasmodiophora brassicae Woronin, is one of the most serious diseases to affect members of the plant family Brassicaceae. This biotrophic soil-borne pathogen is an emerging threat to canola and mustard production in western Canada. To manage the disease, it is important to identify and use new sources of clubroot resistance (CR). The purpose of this research is to map CR genes in the mustard species, Brassica nigra, and to analyze host differential gene expression during P. brassicae infection. Lines of Brassica nigra with a broad spectrum of resistance to clubroot were recently identified. Plant materials utilized in this study include resistant (R) lines BRA192/78 and PI 219576; and susceptible (S) line CR2748. The two resistant lines of B. nigra were crossed with CR2748 female susceptible line to produce the F1. F1 plants were self-pollinated to produce F2. F1 plants from CR2748 x PI 219576 and CR2748 x BRA192/78 were backcrossed with the CR2748 plants to produce BC1 populations. Genetic mapping of CR genes was carried out using bulked segregant RNA-Sequencing (RNA-Seq). Validation and genotyping of single nucleotide polymorphism (SNP) markers were carried out using the Kompetitive Allele Specific PCR (KASP) method. Complete resistance to clubroot was found in all F1 plants derived from crosses of CR2748 with PI 219576 or BRA192/78. Evaluation for resistance to clubroot showed ratios of 1R:1S in BC1 and 3R:1S in F2 for both R genotypes, indicating that CR is controlled by a single dominant gene in both PI 219576 and BRA192/78. Short reads from R and S bulked RNA-Seq samples in the BC1 population derived from PI 219576 and BRA192/78 were assembled into the B. rapa, B. oleracea and B. nigra reference genomes. Transcriptome analysis was conducted, with 11 differentially expressed genes (DEGs) identified in PI 219576 and 382 DEGs identified in BRA192/78 at 95% confidence. Several of the DEGs annotated were involved in CR, such as genes encoding Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR)-class disease resistance proteins and pathogenesis-related (PR) transcriptional factors. An ethylene-responsive gene was up-regulated, whereas auxin-responsive genes and a gene associated with cell growth/development were down-regulated in resistant plants. A CR gene designated Rcr6a in PI 219576 was mapped to the region between 14.36 Mb and 14.84 Mb on chromosome B3, in a region homologous to one on chromosome A08 of B. rapa. SNP markers closely linked to Rcr6a were developed. Plants in the BC1 population from BRA192/78 were also analyzed with the SNP markers linked to Rcr6a and results showed that resistance in BRA192/78 was linked on B3 chromosome, in the region between 12.76 Mb and 14.84 Mb, indicating that the CR gene namely Rcr6b in BRA192/78 was likely in the Rcr6a region. This is the first report on mapping of two single dominant CR alleles, Rcr6a and Rcr6b, in B. nigra. In addition, several SNP markers closely linked to the genes were developed and these markers will be useful in marker-assisted breeding for clubroot resistant canola cultivars. There has been limited research done on the B. nigra genome compared to the vegetable species, B. rapa and B. oleracea, and its potential for CR is poorly understood. However, some B. nigra accessions are highly resistant to clubroot, and CR genes in B. nigra may be transferred into canola (B. napus) as well as mustard species, B. juncea and B. carinata. The identification of DEGs is a significant step in better understanding CR mechanisms so CR genes with potentially different modes of action against clubroot can be utilized