A chromosome-level reference genome facilitates the discovery of clubroot-resistant gene Crr5 in Chinese cabbage

Abstract

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.

Acknowledgements

This study was financed by the National Natural Science Foundation of China (grant No. 32 202 485), Zhongyuan Sci-Tech Innovation Leading Talents (grant No. 244 200 510 041), the Key Sci-Tech R&D Project of Joint Foundation in Henan Province (grant No. 232 301 420 024), the Fund for Distinguished Young Scholars from Henan Academy of Agricultural Sciences (grant No. 2024JQ02), and the Taishan Scholars Program and Natural Science Foundation for Distinguished Young Scholars of Shandong Province (grant No. ZR2023JQ010).

Author contributions

X.Z., L.G., and Y.Y prepared the project design; X.W. performed the genome assembly; S.Y., W.Z., and H.S. conducted experimentations; X.W. and Z.W conducted data analyses; X.W., Y.Z., and Z.W prepared the manuscript. The manuscript was prepared by S.Y and X.W. and all the authors reviewed and approved the manuscript.

Data availability

The genome sequence and annotation file of Chinese cabbage DH40 were submitted to the National Genomics Data Center under (accession ID: GWHERQJ00000000) and Figshare ( 10.6084/m9.figshare.25532824.v1). The Hi-C and transcriptome data have been submitted to the National Genomics Data Center (accession ID: PRJCA024893), and the HiFi data are available at NCBI SRA (accession ID: SRR28482076).

Conflict of Interests

The authors have no conflicts of interest to declare.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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