ARS | Publication request: DEVELOPMENT AND CHARACTERIZATION OF MICROSATELLITE AND RFLP-DERIVED PCR MARKERS IN OAT

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 30, 2001
Publication Date: N/A

Interpretive Summary: Molecular markers linked to agronomically important genes are valuable tools for understanding the physiology of traits and breeding of more productive plant lines with enhanced disease resistance. As more and more molecular markers are identified, it is possible to construct maps of plant chromosomes, which make the identification of new genes and chromosomal regions easier and more precise. In these studies, we examined how frequently two classes of molecular markers detected differences in the chromosomes of 13 oat species and 13 cultivated oat varieties. First, we isolated, characterized, and made markers specific for highly variable regions of oat chromosomes called microsatellites. However, their identification was laborious and expensive. As a second approach, we designed gene-specific markers for oats using information generated by ongoing genome research projects to isolate and characterize all of the genes of wheat and rice. Our results showed that information produced by these plant genomics projects on different grass species, which is freely available to the public, could be used directly to generate molecular markers for oats and that the resulting markers were at least as informative as microsatellite markers. The information presented here will aide other scientists in their efforts to identify chromosomal regions that contain genes, which in turn will lead to more rapid development of new and improved oat lines.

Technical Abstract: Two sources were evaluated for the production of polymerase chain reactio (PCR) markers for oats. First, nucleotide sequences of 306 randomly selected clones from oat microsatellite-enriched genomic libraries were determined. Fifty of the 68 primer pairs designed were functional, of which 24 (48%) were polymorphic among 13 Avena species and 11 (22%) were polymorphic between 'Kanota' and 'Ogle'. Using these markers, 26 loci wer placed on the hexaploid oat restriction fragment length polymorphism (RFLP) map. Second, primers were designed from the sequences of six cDNA RFLP probes. Primer pairs from all 6 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. 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 level of analysis.