Mapping Of Clubroot Resistance Gene Based On Chinese Cabbage "CR-096"

Chinese cabbage,as the main supply vegetable in northern my country in winter,plays an important role in the Chinese diet.However,due to the infection of Chinese cabbage clubroot,the yield of major Chinese cabbage producing areas dropped sharply,which seriously threatened the safe and sustainable production of cruciferous vegetables in our country.Chinese cabbage clubroot is a soil-borne disease that infects the roots of the cruciferous family.This disease is mainly caused by the infection of the flagellate species Plasmodiophora brassicae.The main symptom is Chinese cabbage.Tumors appear at the roots,which can cause yellowing and wilting of Chinese cabbage leaves or even plant death in the later stage.At present,one of the main methods to control the spread of clubroot disease is to carry out resistant breeding by means of molecular biotechnology.Therefore,locating new resistant genes by molecular biology techniques based on the currently available disease-resistant varieties will help to breed new Chinese cabbage varieties resistant to clubroot in the future.At present,more than 20 disease resistance loci have been excavated based on Chinese cabbage varieties with clubroot disease resistance,which are Crr1,Crr2,Crr3,Crr4,CRa,CRb,CRc,CRd,CRk,CRs,Pb Ba3.1,Pb Ba3.3.Rcr1,Rcr4,Rcr8,Rcr9,CRq,Bra A.PB.1.2,Bra A.Pb.8.2,Bra A.Pb.8.4,Bcr1,Bcr2,etc.In addition,researchers have started to develop new resistant varieties based on some CR loci.However,combined with actual production,it is found that due to the continuous evolution and variation of physiological races of Plasmodiophora brassicae,the resistance of most resistant varieties is also decreasing year by year.The relatively single resistant genes possessed by resistant varieties may also be the main reason for the reduction of resistance of Chinese cabbage varieties.Based on the status quo,locating new resistant genes,developing molecular markers linked to them,and then cultivating new resistant varieties through transgenic and other technical means are the keys to safely and sustainably solving the problem of clubroot.At present,the Chinese cabbage variety CR-096 retained by the research group has been proved to be a Chinese cabbage variety with strong resistance to various physiological races after years of inoculation experiments.In order to study its unique disease resistance locus,this study used susceptible cabbage variety 12A to cross with CR-096,and self-crossed generation by generation to establish F₂ generation population,and selected 243 of them to continue self-crossing to obtain F_2:3families.40 seeds of F_2:3families were selected for cultivation and inoculated with strong pathogenic bacteria HBBY-30 to obtain population resistance and susceptibility data.Subsequently,Indel primers were designed by resequencing technology,and a total of3112 pairs of genome wide SSR and Indel primers were used to screen out polymorphic markers with parental DNA.After population verification,a genetic map of molecular markers for Chinese cabbage was constructed and QTL analysis was performed.Finally got the following conclusions:1.After inoculation,72 F₁ generation plants obtained by crossing the two parents showed resistance to infection separation,42 plants were susceptible,and 28 plants showed disease resistance.The 243 F_2:3 families were inoculated and identified.According to the grading standard,the identification results showed that the ratio of all resistant families,isolated families with resistant and susceptible families and all susceptible families was 11:206:26.The population morbidity index was investigated and calculated,and SPSS software was used to analyze the data to find the characteristics consistent with quantitative traits,and the population phenotype presented a skewed normal distribution.2.The genetic linkage analysis of the genotype results of the F₂ population was carried out using Join Map4.0 software,and a genetic map of 10 linkage groups was constructed,with a total of 79 polymorphic molecular markers distributed in the map.The full length of the map is 829.5c M,the average map distance is 10.5c M,and the density is 0.095/c M.3.Combined with molecular marker genetic map and phenotype identification results,QTL analysis found two disease resistance loci,the disease resistance locus on chromosome A05 was named Bra A.Pb.5.1,and the disease resistance locus on chromosome A07 was named Bra A.Pb.7.1.Its physical locations are 0.77Mb～5.8Mb and 21.5Mb～23.6Mb.The LOD values were 6.1 and 3.1,respectively,and the genetic contribution rates were 31.7%and 8.2%.Finally,molecular markers ACMP00842 and sau＿um392 linked to Bra A.Pb.5.1 were developed,with two flanking markers with a genetic distance of 17.9 c M.The molecular markers linked to Bra A.Pb.7.1 were cnu＿ssr341 and cnu＿um179,respectively,and the genetic distance between the two flanking markers cnu＿ssr341 and cnu＿um179 was 5.4 c M.In conclusion,the new resistance genes discovered based on the high-resistance material CR-096 to a variety of physiological races will provide new resistance genes for the subsequent breeding of Chinese cabbage resistant varieties.