ABSTRACT
Microsatellite loci are highly informative genetic markers useful for population genetic studies, linkage mapping and parentage determination. Methods to identify novel microsatellite loci commonly use subtractive hybridization to enrich small-insert genomic libraries for repeat sequences. A critical step in enrichment is attachment of an oligonucleotide linker to genomic DNA fragments so that repeat-containing sequences can be recovered by PCR for cloning. Current linkers and ligation methods rely on single restriction enzymes to size-fraction genomic DNA and generate complementary ends. These restriction enzyme/linker combinations are often species-specific, give poor recovery of repeat-enriched DNA and yield library inserts that are not a broad sample of the genome. We have developed a blunt-end linker, named SNX for its restriction sites, that allows the use of combinations of restriction enzymes to digest the majority of genomic DNA into the 200-1000-bp range. SNX is attached to genomic DNA with a simultaneous ligation/restriction reaction that is highly efficient and improves recovery of sequences after subtractive hybridization. SNX can be used for microsatellite enrichment in any species, since ligation is independent of the restriction enzymes used to size-fraction genomic DNA. These methods improve current repeat-enrichment strategies, resulting in representative small-insert libraries with a very high proportion of positive clones.