Mapping,Cloning Of Clubroot Resistance Gene And The Comparative Transcriptome Analysis Between The Resistant And Susceptible Accessions In Brassica Oleracea

Cruciferae clubroot,a soil-borne disease caused by Plasmodiophora brassicae Woron.,has been a serious threat to the industry development of broccoli and other morphotypes.Cultivatingdisease-resistant varieties is the most effective method to prevent clubroot.In this study,we mainly focused on screening resistant sources,mapping and cloning the resistant gene and studying the molecular mechanism of resistance.The results are as follows:1?For race 4 of P.brassicae,176 B.oleracea materials were used to screen resistant sources.A total of 6 showed high resistance?HR?and 48 showed resistance?R?,which need repeated trials.Hybrids derived from Xiangan 336/Tuoni×broccoli/cabbage were used to identified their clubroot resistance?CR?.Most of hybrids showed reduced disease index compared to the male parents,but some F₁ materials significantly enhanced their disease resistance.2?With cabbage 02-12 and TO1000DH as genome references,re-sequencing analysis were performed for the wild cabbage?B2013?and broccoli inbred lines?90196?.Then the polymorphic SNPs and InDels variation were detected between two parents.The homozygous InDels were used for marker development.A total of 20,009 InDel primers designed and81.78%showed polymor-phism between two parents.3?The resistance/susceptibility of B2013 and 90196 were stable.The disease index of F₁plants fell somewhere in the middle and was tendentious to B2013.The resistant grades of segrega-tion populations showed continuous variation,suggesting that CR trait in B2013 showed quantita-tively inherited and the major CR allele in B2013 might be dominant.4?Based on QTL-seq combined with bioinformatics analysis,we found that the SNP-index in the region?from 22.86Mb to 26.31 Mb?on chromosome 9 showed an imbalance and??SNP-index?of this region was significantly different from 0 at 99%significance level.So we designated the QTL locus in this region as BoCR9.1.Using traditional QTL analysis,we narrowed BoCR9.1 to a560 kb genomic region.5?In this 560kb region,there are 108 annotated genes based on the cabbage 02-12 reference genome,among which Bol044005 shows non-synonymous SNPs between the two parents and is associated with disease resistance.This gene belonged to the TIR-NBS-LRR type?TNL?R gene family and may be a candidate gene for BoCR9.1.According to the sequence information of Bol044005,we obtained the cDNA sequences through the segmented amplification and cloning sequencing.26 SNPs in its coding region resulted in 14 amino acid mutations between resistant and susceptible parent.These mutations were located in TIR and LRR domain.6?Microscopic observations revealed that root–hair infection and cortical infection were both present at 7 and 14 DAI in B2013 and 90196.Therefore,samples from three time points?0,7,14DAI?were performed for transcriptome sequencing.The results showed that host defense responses against P.brassicae were induced earlier,and related pathways were repressed at T14,including cell wall components,glucosinolate synthesis and plant signal transduction pathways.The genes related to NBS-LRR proteins,SA signal transduction,cell wall synthesis and chitinase were diffe-rently expressed between B2013 and 90196,which may associated with different clubroot resis-tance.