Development and characterization of microsatellite and RFLP-derived PCR markers in oat

Two sources were evaluated for the production of polymerase chain reaction (PCR) markers for oat (Avena spp.). First, nucleotide sequences were determined for 250 unique clones from oat microsatellite-enriched genomic libraries. Forty-four of the 63 primer pairs designed were functional, of which 18 (41%) were polymorphic among 13 Avena species and six (14%) were polymorphic between oat cultivars Kanota and Ogle. Second, primers were designed from the sequences of six cDNA fragment length polymorphism (RFLP) probes. Primer pairs from ali six cDNA clones were polymorphic among the 13 Avena species, three were polymorphic between Kanota and Ogle, but only one was polymorphic directly between 'Clintland 64' and IL86-5698. However, by cloning and sequencing the PCR products from Clintland 64 and IL86-5698, it was possible to identify nucleotide sequence differences at restriction enzyme cutting sites in DNA fragments from two other primer pairs. Using the two types of markers, we placed nine loci on the hexaploid oat restriction RFLP map. The RFLP-derived markers often mapped to the same or similar positions as the corresponding RFLP markers within and between mapping populations. The sequence analysis also revealed single nucleotide polymorphisms (SNPs) not at restriction enzyme cutting sites. Since multiple SNPs could be detected even within genes and the techniques for the development of SNPs and microsatellites are similar, it may be possible to identify more informative SNP markers than microsatellites from the same type of analysis.