Development Of KASP Molecular Markers And Identification Of Germplasm Resources For Brassica Rapa

Chinese cabbage(Brassica.rapa,2n=20)is one of the important crops in the Brassica family,among which Chinese cabbage(Brassica campestris ssp.pekinensis)is an important vegetable crop in my country with a long history of cultivation and extensive Cultivation area.Cruciferous clubroot is a soil-borne disease caused by Plasmodiophora brassicae Woronin.It has a great impact on the production of Chinese cabbage and even cruciferous crops.Compared with other control methods,cultivating disease-resistant breeding has become the most economical and effective control method at present.Therefore,stable and reliable molecular markers suitable for high-throughput detection and breeding materials with multiple disease resistance genes are the two prerequisites for disease resistance breeding.In this study,related work was carried out on the design and screening of KASP molecular markers for clubroot resistance loci,identification of genotypes of cabbage resource materials and commercial cabbage materials for clubroot disease resistance,and identification of disease resistance by inoculation of resource materials.The results are as follows:According to the information of 4 cloned clubroot disease resistance genes(CRa,CRb,CRd,Crr1a),the SNP loci located inside the genes were designed as KASP molecular markers;using the published clubroot disease resistance material(ECD04)sequencing information and the genomes of susceptible cabbage materials(Chiffu)were compared,and 302,290,1 SNP loci were obtained in the whole genome.The SNP loci in the whole genome were filtered and screened,and 3 were selected on each chromosome of A01,A02,A03,and A08.KASP marker design was carried out at each SNP site.The designed KASP markers were tested with a test population containing various types of cabbage,and finally 8 KASP molecular markers that were stable,reliable and could accurately identify the materials in the natural population were obtained.Convert other types of molecular markers used clubroot-resistance related research into KASP markers,and test together with the clubroot-resistance KASP molecular markers developed by predecessors.It is not possible to accurately identify disease-resistant materials in natural populations when used in the relative population of the locus.Using the developed 8 KASP markers,865 cabbage resources and 257 commercial cabbage materials were genotyped.The proportion of disease-resistant genotypes in the cabbage resources was 0.81%-7.05%.The proportion of disease-resistant genotypes in commercial cabbage materials is 0-6.61% and the proportion of untyped samples was less than 1%.Among the cabbage resource materials,the homozygous disease-resistant genotype materials were mainly turnip and oil cabbage.A total of 132 materials containing a single disease resistance gene were identified in the cabbage resource group,and 34 materials containing two or more disease resistance loci were identified.Indicating that in most natural populations,clubroot disease resistance genes have been single form of the gene is present in the disease-resistant material.From the physical distance and marker detection results of the four disease resistance loci on chromosomes of CRa,CRb,CRd and CR-03-12,it can be seen that although the four disease resistance loci are on chromosome A03,they are different disease resistance loci.The identification results of markers for commercial cabbage materials show that among commercial cabbage varieties,only a few materials have clubroot disease resistance genes and the disease resistance genes exist in the cabbage materials in a heterozygous.Beijing Xin No.3 was used as a susceptible control,and 31 resource materials containing disease resistance genes in the Chinese cabbage resource population were inoculated and identified by using the field Phytophthora rhizogenes from Changyang,Hubei Province.Among them,20 were resistant materials,accounting for 64.52%,and 11 were susceptible materials,accounting for 35.48%.Among the resistant materials,15 materials were immune(I)and 5 materials were resistant(R);11 susceptible materials were all of high sensitivity(HS)type.The accuracy rate of disease resistance identification using marker CRa-3.1 alone was 66.67%,the accuracy rate of identifying disease resistance using marker CRBr G3-8alone was 40%,and the accuracy rate of identifying disease resistance using marker CR-03-12 alone was75%.CRd-2G,CRd-3,and CRd-4 were all derived from CRd.The accuracy rate of the three markers in the identification of disease resistance was 100%.Disease resistance of materials could not be accurately identified when used alone.The markers CR-02-13 and Crr1-10 could not effectively identify this physiological race.The 31 inoculation materials can be divided into single-resistance genetic material,double-resistance genetic material,and triple-resistance genetic material.The proportions of materials with disease resistance were 66.67%,62.50% and 50% respectively.Indicating that for the physiological race of clubroot,the increase in the number of non-targeted disease resistance loci in the same material has no significant effect on the disease resistance of the material.