Identification Of Candidate Genes For Clubroot-Resistance In Brassica Oleracea And Exploring The Resistance Mechanism By Using QTL-Seq And RNA-Seq

Cabbage is one of the main vegetable crops in China.It belongs to the annual or biennial herb of the Brassica species.Clubroot disease is a worldwide soil-borne disease caused by Plasmodiophora brassicae Woron and is a threat to all Cruciferous plants.At present,the area of cabbage clubroot disease in China is increasing rapidly,and the disease seriously affects the quality and yield of cabbage in production.Once clubroot occurs in the field,the soil will be contaminated by the bacteria,and carries the bacteria for a long time,which seriously threatens the safe and sustainable production of Cruciferous vegetables.Breeding cabbage clubroot resistant cultivar is one of the most effective measures of controlling clubroot,however,,there are little resistant resources in B.oleracea,so slow progresses had been made in the development of clubtoot resistance breeding and resistance gene in B.oleracea.In order to discover the clubroot-resistant genes in cabbage,an F1 generation,named as“CP”population,was developed by crossing Syngenta’s clubroot-resistant cabbage hybrid material“GZ87”（as the female parent）and susceptible cabbage inbred line“263”（as male parent）.On the one hand,the genome of the extreme resistance and extreme susceptibility pools constructed in the CP population was re-sequenced,and the BSA correlation analysis was used to locate the clubroot resistance zone.On the other hand,the resistance and susceptibility pools were subjected to transcriptome sequencing at different time points before and after inoculation.Expression differences and disease-resistant metabolic network at different stages were analyzed,and candidate genes for clubroot resistance were screened through the comprehensive analysis of re-sequencing and RNA-seq results.The main results of this experiment were listed as follows.（1）Construction of extreme resistant and susceptible pools:vegetative clones were developed from 74 individuals of the CP population using asexual reproduction technology,and each of 20 extremely resistant and extremely susceptible individuals were selected by using molecular marker-assisted selection and artificial inoculation identification.The resistant lines all carried resistance-linked molecular markers and presented an average incidence rate of 7.88%and an average disease index of 1.89,while the susceptible lines did not carry the resistant molecular markers,exhibiting an average incidence rate of 87.55%and an average disease index of 50.21.（2）Mapping of disease-resistant QTL intervals:according to the QTL-seq anlaysis on the geneomic resequencing data of the two pools and the parents,one resistance QTL locus was detected from chromosome C04（17.06-18.37Mb,1.31Mb）where located 75genes.Another three resistant QTLs were found on chromosome C07,with sizes of0.46Mb（6.34-6.80Mb）,0.77Mb（38.70-39.53Mb）and 0.44Mb（41.38-41.82Mb）,carrying a total of 163 genes.（3）Screening of candidate genes in the QTLs:The genes in the QTL range were combined with RNA-Seq results to analyze the difference in the genes expression of two pools.After screening,two and five candidate genes were found on C04 and C07chromosomes respectively.There are obvious differences in expression patterns.（4）Exploration of the disease resistance mechanism of Brassica oleracea:According to the results of RNA-Seq,the Ca²⁺signal,ROS reaction and NO signal in the interaction path-way between plants and pathogenic bacteria were greatly increased in the susceptible pool compared with the resistance pool after inoculation of P.brassicae.Compared with the susceptible pool,the hypersensitivity pathway（ETI）in the resistant pool had been dramatically increased.In the phytohormone synthesis and signal transduction pathways,content of hormones such as IAA,CKT,and JA did not change significantly in the resistance pool,but changed drastically in the susceptible pool,and there was a significant accumulation of these hormones.This indicated that the disease-resistant material had a stronger self-balancing ability,and the susceptible material mainly developed resistance through the PTI reaction,while the resistance pool developed stronger ETI response.