Fine Mapping Of Clubroot Resistance QTLs In Cabbage And Creation Of Clubroot Resistance Materials

Cabbage（Brassica oleracea L.）is an important leafy vegetables that is widely cultivated all over the world.Clubroot is a global soil-borne disease caused by Plasmoliophora brassicae,which seriously threatens cabbage production.Due to the scarcity of resistant materials and the complex genetic mechanism of clubroot resistance（CR）in cabbage,exploring CR Quantitative trait loci（QTLs）in cabbage is of great significance for elucidating the CR mechanism of cabbage and using Marker-assisted selection to breed CR varieties.In this study,CR QTL mapping of cabbage was carried out by constructing a genetic linkage map with high-density molecular markers.In addition,the Ougra cytoplasmic male sterile（CMS）restorer and the method of microspore culture were used to create CR cabbage materials.The main results of this study are as follows:1.The cabbage inbred line 2358-Z3 was inoculated by P.brassicae from 6 different regions,and the results showed that 2358-Z3 was resistant to all P.brassicae.Infection process of P.brassicae from Changyang in highly resistant inbred line 2358-Z3 and highly susceptible inbred line 21-3 was observed by using Optical Microscopy and Transmission Electron Microscopy,and the result showed that the root hair and cortex infection stages of P.brassicae in the two lines occurred at 3 and 7 days after inoculation（DAI）,respectively,and the primary plasmodia of P.brassicae were formed at 14 DAI.Compared with 2358-Z3,the reproduction rate of P.brassicae in 21-3 was faster and the content of P.brassicae in21-3 was higher than that in 2358-Z3 during the whole infection process.In the late infection stage of P.brassicae,the cell wall of 21-3 was ruptured and a large number of dormant spores appeared in the cells,while the cell structure of 2358-Z3 was intact,where only a few secondary plasmodia appeared.2.Six generations,F_2:3and recombinant inbred lines（RILs）genetic populations were constructed.Genetic analysis of 2358-Z3 showed that the CR of 2358-Z3 was considered to be controlled by polygenes.Nine genetic linkage groups were constructed using 152molecular markers developed based on the resequencing information of the parents.Combined with the inoculation test results of the F_2:3and RILs populations,2 CR QTLs,q Bo CR2.1 and q Bo CR8.1,were mapped on C02 and C08 of 2358-Z3,respectively.Their physical intervals were 326.8 Kb and 1.61 Mb,and the highest rate of phenotypic variation they can explained were 15.6%and 7.55%,respectively.Twenty-four genes were included in q Bo CR2.1 and 132 genes were included in q Bo CR8.1.According to the gene function annotation information,q Bo CR2.1 contained 3 disease resistance-related genes,including 1CKX gene,1 protein kinase gene and 1 LRR-like gene;q Bo CR8.1 contained 11 disease resistance-related genes,including 4 protein kinase genes,1 NBS-LRR-like genes and 6LRR-like genes.Amplification results of the coding regions of 14 candidate genes in both parents showed that in q Bo CR2.1,2 amino acid substitutions occurred in the 9th exon in the protein kinase gene Boc02g03437.1.In q Bo CR8.1,12 amino acid substitutions occurred in the NBS-LRR-like gene Boc08g03240.1;the stop codon of the LRR-like gene Boc08g03249.1 in the susceptible parent was changed,and the coding region was terminated early;and for the protein kinase gene Boc08g03309.1,1 amino acid substitution and 1 amino acid insertion occurred in the first exon,and 1 amino acid substitution occurred in second exon.3.RNA-seq was used to analyze the transcriptome of the roots of 2358-Z3 and 21-3infected with P.brassicae.The results showed that in cabbage,3,708,5,959,and 1,500differentially expressed genes（DEGs）were identified at 3,7,and 14 DAI,respectively.KEGG analysis showed that plant hormone signal transduction was the major enrichment pathway among the DEGs.Through comprehensive analysis,72 DEGs associated with CR were identified,including 4 LRR-like candidate genes identified from q Bo CR2.1 and q Bo CR8.1.In the transcripts of P.brassicae,there were 27 up-and 51 down-regulated genes at 7 DAI and 26 up-regulated genes at 14 DAI.Not only that,14 proteins were identified as candidate effectors that may be associated with virulence of P.brassicae.Thirty-six CKX genes were identified in the cabbage genome,which were unevenly distributed on 9chromosomes of cabbage and encoded proteins ranging in length from 355 to 594 aa.The results of collinearity analysis showed that 13 CKX genes in Arabidopsis had no homologous copies in cabbage,and other CKX genes contained at least 1 homologous copy in cabbage.Combined with transcriptome data,4 CKX genes whose expression was up-regulated after inoculation were identified,and their roles in cabbage in response to P.brassicae invasion need to be further analyzed.4.The cabbage doubled haploid（DH）lines containing the homozygous CR gene CRa were rapidly obtained by using Ougra CMS cabbage restorer combined with microspore culture technology.It was found that there was an exogenous fragment from the A03chromosome of B.rapa in CR cabbage DH lines by using high-throughput sequencing technology combined with first generation sequencing technology.The length of the exogenous fragment was 3.42 Mb and contained CRa.Further analysis confirmed that the fragment was inserted into the homologous region of C07 chromosome in cabbage.The final analysis result showed that the exogenous fragment from B.rapa may have been inserted into the cabbage genome by homologous exchange during the process of distant hybridization.