RAPD identification of microsatellites in Daphnia.

Simple sequence repeats (SSRs, or microsatellites) have been constantly gaining importance as single-locus DNA markers in population genetics and behavioural ecology. We tested a PCR-based strategy for finding microsatellite loci in anonymous genomes, which avoids genomic library construction and screening, and the need for larger amounts of DNA. In the first step, parts of a genome are randomly amplified with arbitrary 10mer primers using RAPD fingerprinting. Labelled SSR-oligonucleotides serve as probes to detect complementary sequences in RAPD products by means of Southern analyses. Subsequently, positive RAPD fragments of suitable size are cloned and sequenced. Using GA and GT probes, we applied this approach to waterfleas (Daphnia) and revealed 37 hybridization signals in 20 RAPD profiles. Thirteen positive RAPD fragments from three Daphnia species and two hybrid 'species' were cloned and sequenced. In all cases simple sequence repeats were detected. We characterized seven perfect repeat loci, which were found to be polymorphic within and between species.