Characterization of equine microsatellites and microsatellite-linked repetitive elements (eMLREs) by efficient cloning and genotyping methods

We performed efficient cloning and genotyping methods for isolation of a la rge number of polymorphic microsatellites. The methods contain the time-eff icient cloning method of constructing microsatellite-enriched libraries and the economic genotyping method of fluorescent labeling of PCR products. Ei ghty novel equine microsatellites cloned were efficiently isolated from the enrichment library and analyzed for genotype polymorphism. Of these, 72 mi crosatellites were analyzed with a good resolution. The average heterozygos ity of all loci was 0.52, and the number of alleles ranged from one to 9 wi th an average of 4.5 alleles. The other eight loci showed multiple bands of PCR products, suggesting the occurrence of microsatellites in a repetitive element, in which the number of microsatellite repeats varies among differ ent members of the repetitive element. We found five homologous groups at flanking regions in comparison with the flanking regions of microsatellites from DNA databases. One of them showed homology to equine repetitive element-2. In the other four homologous group s, the two groups were named equine microsatellite-linked repetitive elemen t-1 (eMLRE-1) and equine microsatellite-linked repetitive element-2 (eMLRE- 2) as novel equine repetitive elements identified from equine genome. These data should help the analysis of equine DNA sequences and the design of eq uine genome markers.