Screening Of Germplasm Resources For Resistance To Clubroot Disease And Identification Of Resistance-related Genes In Cabbage

Cabbage(Brassica oleracea var.capitata L.),member of the Brassicaceae family,is an economically important vegetable crop and has an important role in domestic vegetable supply and export.In recent years,clubroot disease caused by Plasmodiophora brassicae Woronin poses a great threat to the sustainable production of crucifer crops such as cabbage.The rapid spread of clubroot has caused sharp decrease of the yield and quality of cabbage.However,the lack of the resistance resources and the rapid variation of pathogens,which makes it challenging to breed clubroot-resistant cabbage cultivars.Therefore,in the current study,we first collected samples from clubroot-infested regions for identification of the pathotype.Then these strains were used as inoculum to screen the cabbage germplasm for clubroot-resistant sources.The resulting clubroot-susceptible and clubroot-resistant accessions were then subjected to transcriptomic and overexpression analysis to reveal candidate genes involved clubroot resistance.The results are shown below.(1)Diseased soil samples and root galls were collected from clubroot-infested areas in Wangying Town,Lichuan City,Hubei Province,and Huoshaoping Town,Yichang City,Hubei Province,respectively.The morphological characteristics were examined by microscopy,and the primers of targeting Internal Transcribed Sequence(ITS)were designed to identify the pathogent of P.brassicae.The specific primers of Plasmo3-4 can be used for rapid detection of P.brassicae in B.oleracea,B.rapa,B.napus and other Brassicaceae family crops.We adopted the European Clubroot Differential set(ECD)with the method of fungus soil inoculation to characterize the pathotype of P.brassicae.The pathogen of Wangying Town was ECD16/4/0,and Huoshaoping Town was ECD 17/31/13.ECD 16/4/0 was less virulent while ECD 17/31/13 was highly virulent.Then,we screened 88 varieties of cabbage germplasm by artificial inoculation of seedlings with ECD17/31/13 in the field combined with the natural induced identification in the field.Most germplasms showed clubroot-susceptible or-resistant phenotypes in both seedling and adult stages,of whih CR21 showing the most stable and strong resistance and CR55 and CR54 showing the remarkable clubroot susceptibility in the seedling stage and adult stage respectively.(2)Using the identified high-generation inbred lines CR21(-resistant)and CR54(-susceptible),the seedling roots were collected at 3 days after inoculation,because root hair penetration occurs mainly at this time by microscopic examination,so when the root tissues were samples for RNA-Seq.After transcriptome sequencing analysis of the root tissue,1,057 and 4,741 differentially expressed genes(DEGs)were found in CR21 and CR54,respectively.Furthermore,541 DEGs were up-regulated in CR21 but showed either no significant change or down-regulated pattern in CR54.GO functional enrichment analysis of the 541 DEGs indicated that most DEGs were involved in metabolism and transport,signal transduction and defense.A total of 165 defense-related DEGs were retrieved,including pathogen-associated molecular patterns(PAMPs)-triggered immunity,PTI-related genes and effector-triggered immunity(ETI)-related genes.The shared defense-related genes,wound response gene(Bo3g135780),respiratory burst oxidase(RBOHB),Transcription factor(WRKY28),TIR-NBS-LRR(RPS4,etc.),pathogenicity-related(PR)genes(Bo7g005050),lipid transporters(LTP1,etc.),chitinase(Bo4g020930),plant hormone response-related genes,ethylene signal transduction(HRE1 and EIN3),ABA signaling(PP2C,PYL13)and so on,cell wall modification-related genes(EXP 17 and PME44,beta-1,3-glucanase and cytochrome P450 genes).In summary,this study provided a comprehensive insight into transcriptomic responses to P.brassicae infection in two different cabbage lines,and identified several candidate genes that might contribute to clubroot resistance in cabbage and other Brassica crops.(3)A total of 61 BonsLTPs were identified in B.oleracea and unequally distributed on nine chromosomes,59 of which were divided into ten types according to the similarity of residues between eight cysteine motifs(ECMs).According to the organs RNA-Seq results,with the exception of BonsLTPIX.2 and BonsLTPXI.3,the other 59 BonsLTP genes were differentially expressed in tissues and organs,while some genes are specifically expressed in buds,siliques,stems,leaves and callus.Seven BonsLTPs genes associated with clubroot resistance were obtained,including BonsLTPI.1,BonsLTPI.5,BonsLTPI.9,BonsLTPI.10,BonsLTPI.14,BonsLTPIV.1 and BonsLTPIV.6,through the comparative transcriptomic data of CR21 and CR54.As expected,BonsLTPI.5 and BonsLTPI.14,overexpressed in Arabidopsis,can enhance the resistance of host to P.brassciae.Notably,BonsLTPI.5 overexpression significantly reduced the disease index of host,and BonsLTPI.14 highly significantly reduced the disease rate and disease index of host..Analysis of cis-acting element in the promoter region of these genes suggested that,BonsLTPI.5 and BonsLTPI.14 contribute to disease resistance presumbly by strengthening the first defense line,intracellular signal transduction pathways,or indirect response to water or nutrient deficiency.(5)A total of 40 BoChiAs were identified in B.oleracea and unequally distributed on seven chromosomes,which were divided into five(?-?)types according to the sequence and structure of putative proteins.According to the tissue-specific expression profiles,transcripts of eleven BoChiAs(nine members in type ?)genes were not detectable,BoChiAIV.12 was specifically expressed in leaves,and a total of 16 BoChiAs genes were differentially expressed in seven tissues and organs.Three BoChiAs genes associated with clubroot resistance were obtained,including BoChiA?.6,BoChiA?.14 and BoChiA?.22,through the comparative transcriptomic data of CR21 and CR54.Furthermore,heterlogous expression of BoChiA?.6 and BoChiA?.14 in Arabidopsis,confers partial resistance to P.brassicae,as indicated by reduced disease incidence and disease index,in which BoChiA?.14 highly significantly alleviated the disease of host.In agreement with these results,chitin oligosaccharides treatment also alleviates disease severity in Arabidopsis.Subcellular localization and promoter response element analysis revealed that these genes might improve the disease resistance by degrading chitin.Taken together,this study identified the pathotype of P.brassicae in one of the most clubroot-infested areas in China,which can facilitate the development of resistance resources.In addition,the combination of transcriptomic analysis,genome-wide identification of gene families of BonsLTPs and BoChiAs,and overexpression of candidates genes,revealed some genes related to clubroot disease in cabbage,which could provide a theoretical basis for the development of proper control strategies against clubroot disease.