Isolating microsatellite loci: looking back, looking ahead.

Microsatellite DNA loci are tandemly repeated simple sequence repeats (SSRs) that are ubiquitous in eukaryotic genomes. When flanked by unique sequences, length variation (driven by high rates of strand slippage during DNA replication) at a given repeat locus can be assayed by PCR and electrophoretic separation of the resulting DNA fragments (representing alleles defined by fragment size or repeat number at that locus). In nonmodel organisms that do not have sequence information at SSR loci (or at SSRs in a closely related taxon), microsatellites must be isolated and sequenced de novo. Traditionally, this has been accomplished with cloning of genomic DNA fragments enriched for SSRs, a protocol described in detail here. PCR primers flanking microsatellite repeats can be used to assay repeat length variation among individuals (typically through fluorescent labeling of one strand and capillary electrophoresis), useful for questions related to population variation, individual assignment, mating studies, selection scans, mapping, and phenotypic traits. High-throughput next-generation sequencing will likely supplant traditional cloning methods for the discovery of microsatellite loci.