Abstract
BACKGROUND: Clubroot disease, caused by the soil-borne protist Plasmodiophora brassicae, is a major threat to Brassica crops worldwide, leading to significant yield losses. Genetic resistance is the most effective and sustainable management strategy; however, the identification and characterization of clubroot resistance (CR) genes remain a challenge, particularly in Brassica nigra. Despite its abundant CR resources, only one CR gene, Rcr6, has been identified in the B genome of B. nigra, leaving much of its genetic potential unexplored. Understanding the genomic distribution and diversity of CR genes in B. nigra is crucial for expanding resistance breeding options, especially for canola (B. napus). RESULTS: This study identified Rcr12, a single dominant CR gene on chromosome B3 of the highly resistant B. nigra line BRA19278. Using bulked segregant RNA sequencing (BSR-seq) and fine mapping in segregating populations derived from a cross between CR2748 (a susceptible B. nigra line) and BRA19278, together with single-root protoplast-derived isolates (SPIs) of P. brassicae and comparative analysis across multiple reference genomes, we established that Rcr12 is distinct from Rcr6 despite their close physical proximity. Evidence supporting its distinctiveness includes differential resistance patterns against various SPIs, unique SNP marker associations, and pangenomic analyses. Fine mapping refined the Rcr12 locus to a 0.33 Mb region on chromosome B3, containing multiple resistance gene candidates, in contrast to the single candidate identified for Rcr6. This study is the first to report an nucleotide-binding leucine-rich repeat (NLR) cluster-type CR locus near an NLR singleton in Brassica crops, underscoring the evolutionary and functional significance of this gene arrangement. CONCLUSION: The discovery of Rcr12 expands our understanding of NLR gene organization and its role in host resistance evolution. Beyond advancing clubroot resistance breeding, this discovery lays the groundwork for studying functional interactions between NLR singletons and clusters in plant immunity. Additionally, the use of purified SPIs as a pathogen differentiation tool offers a novel approach to resolving ambiguities in clubroot research, addressing the complexity of host-pathogen interactions and facilitating future investigations, especially with the anticipated release of a new pathogen classification system.