Mapping Of Disease-resistant Gene Against Clubroot In Raphanus Sativus And Response Characteristics Of MYB28 Transcription Factor In Brassica Napus L.to Plasmodiophora Brassicae And Salicylic Acid

Clubroot caused by Plasmodiophora brassicae is a significant soil-borne disease on cruciferous plants.The pathogen can live in the soil for a long time in the form of resting spores,and can be spread by various methods.Traditional methods of agriculture and chemistry are difficult to completely solve the clubroot disease.Cultivating resistant varieties is the most widely used and most effective method.However,due to various pathotypes of P.brassicae,there are still many difficult problems in cultivating disease-resistant varieties.Normally,the resistance controlled by single gene loses easily in practice.Therefore,it is of great significance to make clear the molecular mechanism of resistance in clubroot disease and cultivate resistant varieties that can defense against multiple pathotypes.In this study,on one hand,bulked segregant analysis?BSA?combined with high-throughput sequencing was used to locate the clubroot disease resistance?CR?genes in resistant radish,which can provide resistant resource for breeding resistant varieties.On the other hand,this study analyzed the expression patterns of MYB28 gene after P.brassicae inoculation and salicylic acid?SA?treatment in the susceptible Brassica napus L.The main results are as follows:1.Through disease resistance identification of different radish varieties to P.brassicae collected from ten different regions of China,a radish variety‘JY'was immune to the dominant pathotypes of multiple regions and a radish variety‘MTH'was high sensitivity.Using‘JY'as the female parent and‘MTH'as the male parent for cross-breeding,the hybrid progenies of F₁ and F₂ were generated and inoculated with pathotype 4 of P.brassicae.The results showed that the incidences of the parental‘JY'and the parental‘MTH'were 0 and 100%,and the progenies of F₁ and F₂ were 11.11%and 23.36%,respectively.Among them,the number of F₂ plants resistant to susceptible plants is in accordance with the 3:1 separation ratio.It is speculated that the resistance of radish may be controlled by a single dominant gene.BSA combined with genome resequencing for the mapping and screening of the CR candidate gene was performed.The R-pool was mixed by DNA of 30 disease-resistant plants and the S-pool was mixed by 30 disease-susceptible plants of the F₂ population.The parents together with the R-pool and the S-pool were used for genome resequencing.Based on BSA-seq combined with bioinformatics analysis,we found that the SNP-index in the region?from 39?45 Mb?on chromosome 5showed an imbalance and??SNP-index?of this region was significantly different at 95%significance level.In this region,there were 18 candidate genes,among which 9 genes showed non-synonymous SNPs between the two parents and was probably associated with disease resistance.According to the functional annotation results,the expression patterns of 12 candidate genes were analyzed after P.brassicae inoculation between resistant and susceptible radish.The results showed that the expression patterns of 7 genes changed significantly at 7 and 14 d after inoculation.In the susceptible radish,RSG3778,RSG3860 and RSG23487 were up-regulated at 3 d,and were down-regulated at 14 d;RSG3887,RGS3778 and RGS23487 were up-regulated in infected radish at 3d and 7 d,and were down-regulated at 14 d;In the disease-resistant radish,all of the seven genes have little changes at 3 d after inoculation,but five of them?RSG3559?RSG3860?RSG23484?RSG3887 and RSG10137?were down-regulated at 7 d.RGS3887 and RGS23487 were up-regulated in the disease-resistant radish 14 d after inoculation.RGS3887 and RGS23487 encoded U11/U12 small nuclear ribonucleo protein and TIR-NBS-LRR class disease resistance protei,respectively,which may associated with clubroot resistance in radish.2.MYB28 is a type of R2R3 type MYB transcription factor in plants and is involved in plant growth and stress.MYB28 plays an important role in regulating plant defense-related signal pathway.This study analyzed the MYB28 gene expression patterns after Plasmodiophora brassicae inoculation and salicylic acid?SA?treatment in susceptible Brassica napus L.‘ZS11'.The evolutionary relationship and amino acid sequence analysis of the MYB28 gene showed high homology among B.napus,B.rapa,B.oleracae and Arabidopsis thaliana.MYB28 has two highly conserved repeating sequences.Phylogenetic tree analysis desmonstrated that MYB28 has higher homology among species than within species.Tissues spectiffic expression patterns showed that the expression of BnaMYB28 is at a higher level in stems and seeds than roots and leaves.Bna MYB28 gene had different expression responses after P.brassicae inoculation and SA treatment.The expression of BnaMYB28-1 and BnaMYB28-3 was induced by SA treatment while the expression of BnaMYB28-2 was suppressed.After P.brassicae inoculation,BnaMYB28-1 was significantly induced throughout the infection period,but the expression of BnaMYB28-1 was significantly inhibited after adding SA;the expression of BnaMYB28-3significantly increased at 7 and 28 d after inoculation.The expression level did not change during the key cortical infection period?14 d after inoculation?,but the expression of Bna MYB28-3 increased significantly under the combination treatments of pathogen inoculation and SA.Based on the analysis of expression patterns under different treatments,exogenous SA can positively regulate the expression of BnaMYB28-1 and BnaMYB28-3.BnaMYB28-3 gene may be involved in interaction between B.napus,and P.brassicae through the process mediated by SA.