Brassica rapa includes a variety of important vegetable and oilseed crops, yet it is significantly challenged by clubroot disease. Notably, the majority of genotypes of B. rapa with published genomes exhibit high susceptibility to clubroot disease. The present study presents a high-quality chromosome-level sequence of the genome of the DH40 clubroot-resistant (CR) line, a doubled haploid line derived from the hybrid progeny of a European turnip (ECD01) and two lines of Chinese cabbage. The assembled genome spans 420.92 Mb, with a contig N50 size of 11.97 Mb. Comparative genomics studies revealed that the DH40 line is more closely related to the Chinese cabbage Chiifu than to the turnip ECD04. The DH40 genome provided direct reference and greatly facilitate the map-based cloning of the clubroot resistance gene Crr5, encoding a nucleotide-binding leucine-rich repeat (NLR) protein. Further functional analysis demonstrated that Crr5 confers clubroot resistance in both Chinese cabbage and transgenic Arabidopsis. It responds to inoculation with Plasmodiophora brassicae and is expressed in both roots and leaves. Subcellular localization shows that Crr5 is present in the nucleus. Notably, the Toll/interleukin-1 receptor (TIR) domain of Crr5 can autoactivate and trigger cell death. In addition, we developed two Crr5-specific Kompetitive allele-specific PCR (KASP) markers and showcased their successful application in breeding CR Chinese cabbage through marker-assisted selection. Overall, our research offers valuable resources for genetic and genomic studies in B. rapa and deepens our understanding of the molecular mechanisms underlying clubroot resistance against P. brassicae.

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