Fine Mapping and Functional Analysis of Major QTL, CRq for Clubroot Resistance in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Abstract

Clubroot disease caused by Plasmodiophora brassicae is one of the major threats to Brassica crops. New clubroot resistant varieties of Chinese cabbage (B. rapa ssp. pekinensis) have been developed through breeding, but the underlying genetic mechanism of clubroot resistance is still unclear. In this study, two Chinese cabbage DH lines, clubroot-resistant Y635-10 and susceptible Y177-47 were crossed to develop F2 population for fine mapping and cloning resistance gene CRq. After sequence analysis, the expression vector was constructed by gateway technology and transferred into Arabidopsis thaliana for functional characterization. Bulked segregant analysis sequencing (BSA-seq) confirmed that CRq is located in the 80 kb genomic region on chromosome A03 between markers GC30-FW/RV and BGA. In silico tools confirmed that the gene length was 3959 bp with 3675 bp coding sequences (CDs), and it has three exons and two introns. In addition, we found 72bp insertion in the third exon of CRq in the susceptible line. We developed and verified functional marker Br-insert1, by which genotyping results showed that 72bp insertion might lead to the destruction of the LRR region of Y177-47, resulting in a loss of resistance relative to clubroot. The results of genetic transformation showed that the roots for wild-type Arabidopsis thaliana were significantly enlarged compared with T2 generation transgenic Arabidopsis after treatment by P. brassicae spores, and transgenic Arabidopsis had certain resistance. Therefore, CRq is a candidate gene of clubroot disease resistance in Chinese cabbage, which could be used as a reference for elucidating disease resistance mechanisms and the marker-assisted breeding of clubroot resistant varieties.