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Abstract
A procedure is described for developing single-nucleotide polymorphism (SNP) markers linked to Pc68, a gene conferring resistance to crown rust [Puccinia coronata f. sp. avenae] in many oat (Avena sativa) cultivars currently grown in Canada. Three restriction-fragment length polymorphism (RFLP) markers, located close to the resistance gene Pg9 to stem rust [Puccinia graminis f. sp. avenae] through comparative mapping, were used as sources of DNA-sequence information for SNP identification, since Pc68 is tightly linked or allelic to Pg9. Specific primers designed from the RFLP-marker sequences were used to amplify the target genomic region from recombinant inbred lines with and without Pc68. Putative SNP sites were identified by means of comparative sequence alignment of the polymerase chain reaction (PCR) fragments and were validated by the single-base extension method, a non-gel-based assay for genotyping SNPs. The 774-bp PCR fragment amplified by primers derived from the RFLP marker cdo309 was a sequence-tagged site (STS) marker linked to Pc68, and only the SNPs derived from a region within the STS were linked to Pc68. These SNPs and STS cosegregated in two genetic populations. The map distance between these markers and Pc68 was 4.2 and 6.7 cM (centimorgans), depending on population. The SNP markers identified in the present study can distinguish plants homozygous for Pc68 from heterozygotes, a useful feature for eliminating heterozygous plants in early generations. As SNP markers for other resistance genes or other important traits become available, breeders can benefit from using the technology with high-throughput, automation, and multiplexing capabilities, such as single-base extension assay, in breeding applications, including resistance gene pyramiding.