Identification of RAPD markers linked to a Rhynchosporium secalis resistance locus in barley using near-isogenic lines and bulked segregant analysis

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Abstract
Three hundred random sequence 10-mer primers were used to screen a pair of near-isogenic lines of barley and their donor parent for markers linked to genes conferring resistance to Rhynchosporium secalis. One primer was identified which reproducibly generated a product, SC10-65-H400, from the donor parent and the Rhynchosporium-resistant near-isogenic line but not from the recurrent parent. Segregation analysis on a barley doubled haploid population and examination of a further three near-isogenic lines, their donor and recurrent parents confirmed that this marker was linked to the Rhynchosporium resistance locus (Rh) on chromosome 3L. The presence or absence of SC10-65-H400 was subsequently used along with the resistance phenotype to identify two groups of individuals in the doubled haploid population which possessed alternative alleles at both loci and defined a genetic interval between these two markers. Based on that information two bulked DNA samples were constructed by combining equal amounts of DNA from five individuals from each group. The two bulks and doubled haploid parental lines were screened with 700 10-mer primers. Seven products were identified which were present in the 'resistant' bulk and parent and were absent in the susceptible samples. Segregation analysis established their association with Rh. In addition co-segregation of the linked markers with a set of chromosome arm specific RFLPs confirmed the location of the Rh locus on the long arm of barley chromosome 3.