Fine Genetic And Physical Mapping Of The CRb Gene Conferring Resistance To Clubroot Disease In Brassica Rapa

Clubroot is a worldwide soil-borne disease. which is caused by the obligate biotroph Plasmodiophora brassicae. It’s one of the most serious diseases in Chinese cabbage(Brassica rapa) and other crucifer all over the world. The use of clubroot resistance (CR) genes is an effective and economical approach for controlling clubroot disease. In a previous study, Piao et al (2004) identified and mapped the CRb locus in the doubled-haploid line’CR Shinki’.’CR Shinki’and a clubroot-susceptible DH line, Pakchoi’702-5’were used as parental lines to construct the F2population. To further confirm the inheritance of CRb, two F2populations (F2-L and F2-S) were tested with the same isolate of P. brassicae. CRb was initially mapped between the two flank markers TCR01and cnu_m413. Futher, CRb was finely mapped in combination with developing markers closely linked to CRb and two F2populations. The detailed results are as follows:1. Through the two F2population survey of disease resistance,2203plants showed resistance, while693was susceptible in F2-L population;4314plants showed resistance, while693was susceptible in F2-S population. The chi square test results are consistent with the genetic segregation ratio, proving once again that the CRb is controlled by a dominant gene.2. A co-dominant flank marker linked to CRb was developed by two high-density integrated genetic maps and BSA(bulked segregant analysis). CRb was initially mapped with two flank markers cnu_m413and TCR01. The genetic distance between cnu_m413and TCR01is1.23cM (physical distance is363kb)3. The sequence between cnu_m413and TCR01was obtained from B. rapa genomes (http://brasicadb.org), and according to this sequence,40pairs of InDel markers developed markers and20to SSR markers. A polymorphic survey of two parental lines gave2polymorphic SSR and5InDel markers.4. The CRb-linked flanking markers TCR01and cnu_m413were used to screen the F2-L and F2-S populations for recombinants.67recombinants were identified between the two markers. Seven newly developed markers were applied to detect the genotypes of the67recombination events. On the basis of phenotypic and genotypic data for the recombinants,genetic distances between CRb and nearby markers were calculated to finely map the CRb locus. The CRb locus was targeted in a0.14cM region defined by TCR108and TCR79.5. The PCR products of the newly developed markers were sequenced. The BAC contig map around the CRb locus was construct using sequencing results and BAC clones. The physical distance between the two nearest markers TCR79and TCR108was83kb.6. Four polymorphic markers linked to CRbKato and CRaim-T were used to genotype67recombinants in order to compare the genetic relationships among the CRa, CRb, and CRbKato loci. The result indicated that CRb is not an allele of CRa. Although four CRbKato-linked markers showed linkage with CRb, they were more closely linked to the CRa locus and were located at the outside of TCR01.7. Fifteen genes were annotated in the target region anchored by two markers closely linked to CRb.