Creation Of Clubroot-Resistant Rapeseed Resources And Mapping Of Resistance Genes

The clubroot disease is a soil-borne fungal disease caused by Plasmodiophora brassica Wor..At present,it has occurred in more than 60 countries around the world,causing an average annual reduction of 10%-15%on cruciferous crops,which has become one of the main factors restricting the production of cruciferous crops.Brassica napus is the most widely grown oil crop in the world.It is also the main type of rapeseed in China,which has the advantages of high yield,resistance to pests and diseases,and strong resistance to stress,but most of them are not resistant to clubroot disease,causing an increase in the incidence area year by year,and now it has become the main threat factor for rapeseed production in China.At present,most researchers believe that breeding and promoting the clubroot resistance（CR）cultivars is the most effective way to solve the clubroot disease,therefore it is necessary to screen the CR germplasm resources,resynthesize CR germplasm,and identify CR genes.This study was mainly focused on the screening of CR germplasm,the analysis of physiological and biochemical indicators related to clubroot disease,the creation of CR germplasms and the mapping of CR genes.1.Fourty cruciferous gerplasms were used for CR germplasm identification using the pathogen from Chengdu.The resistance analysis,disease index analysis,correlation analysis between disease incidence and disease index were carried out on the tested gerplasms.There was significant difference in disease resistance among different gerplasms;cluster analysis divided the tested gerplasms into four types,resistance,moderate,susceptible and high-sensitivity;There was a significant correlation between the disease incidence and the disease index（P<0.05）.Finally,ten high-resistance gerplasms were identified,including three Chinese cabbages,one cabbage,three European phthalocyanines,one Brassica juncea,one Brassica carinata and one Radish,which provided the basic materials for the follow-up work.2.Ninteen susceptible B.napus were used as experimental materials,and the non-inoculated B.napus were used as the control.The four physiological indexes related to disease resistance including the relative water content,malondialdehyde content,soluble sugar and cell membrane relative permeability were analyzed.The results showed that the relative water content,malondialdehyde content and soluble sugar did not show significant difference after inoculation,and the relative permeability of the cell membrane was increased under stress,and showed significant difference.Therefore,the relative permeability of the cell membrane can be used as a physiological auxiliary index for identification of clubroot disease in the early development stage.3.A high-sensitivity B.napus line Xu09（female）crossed with a CR gerplasm Debra（male,European turnip）using a method of distant hybridization combined with embryo rescue technology.There hybrided seeds were obtained.The morphological identification,SSR molecular marker identification and resistance inoculation identification were used to identify the true and false hybrids,the results showed that all of them were the true hybrids,which can be used as the"bridge materials"for CR breeding.4.A B.napus resistant sterilie line（female）crossed with the susceptible B.napus restorer lines（male）in the field,harvested the F₁ generation and inoculated the seeds using the pathtype 4 of P.brassica.The resistance classification was analyzed,and the ratio of resistant and susceptible was about 2:1.The susceptible B.napus restorer lines were continue to crossed with the clubroot resistance individuals to obtain the BC₁ for the subsequent study.5.By collecting and searching ten published molecular markers closely linked to the CR genes in Chinese cabbage,and were amplified in the 15 gerplasms to identify the CR genes.As a result,the number of gerplasms containing the CRa,CRb,CRc,CRk,Crr1,Crr2,and Crr3 were 15,3,4,4,6,5,and 14,respectively.Among them,a Chinese cabbage Genkang 518 can amplify the polymorphic bands at CRa,CRb,CRc,Crr1,Crr2 and Crr3locus.6.An F₂ population derived from Shaanbei yellow mustard（susceptible）and CT19（resistance）was used as experimental materials.A total of 24 pairs of IP markers were designed based on the sequences of candidate genes published by others,and amplified the two parents,five of which showed polymorphsm.The marker,IP09-4 was used to amplify29 F₂ individuals,it was showed that the results of marker amplification were not consistent with those of resistance identification,which suggested that the genes in our study were not located in the region published by the previous study..7.The F₂ population including seventy individuals derived from Shaanbei yellow mustard and CT19（41 resistant,29 susceptible）were used to map the CR gene using a simplified genome sequencing method.According to the information of SNP/InDel markers obtained by screening,calculated the frequency difference of different genotypes,analyzed the candidate region of target gene.As a result,a significant QTL locus was detected in a region of 29.3-40.7Mb on A03 chromosome,which was predicted to be a candidate gene interval.