THE MOLECULAR-BASIS OF A MICROSATELLITE NULL ALLELE FROM THE WHITE SANDS PUPFISH

Microsatellite loci were cloned and characterized from the White Sands pupfish (Cyprinodon tularosa), a New Mexico state-listed endangered s pecies. One locus exhibited a high-frequency nonamplifying allele loca lized to a single population. This null allele was PCR amplified by re design of one of the original primers and multiple individuals homozyg ous for null as well as for nonnull alleles were sequenced using the n ew primer. These molecular dissections revealed that the original fail ure to amplify some alleles from this microsatellite locus was due to a 4 bp deletion in one of the original PCR priming sites. Furthermore, the reamplifications revealed five distinct size classes of alleles t hat had been masquerading as the original null. These null alleles did not overlap in length with the nonnull alleles, and they also differe d consistently by a linked nucleotide substitution. Results suggest th at the original null allele (as well as the nonnull class) has diversi fied considerably since its origin and has not recombined frequently w ith the nonnull class of alleles.