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 species. One locus exhibited a high-frequency nonamplifying allele localized to a single population. This null allele was PCR amplified by redesign of one of the original primers and multiple individuals homozygous for null as well as for nonnull alleles were sequenced using the new primer. These molecular dissections revealed that the original failure 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 that had been masquerading as the original null. These null alleles did not overlap in length with the nonnull alleles, and they also differed consistently by a linked nucleotide substitution. Results suggest that the original null allele (as well as the nonnull class) has diversified considerably since its origin and has not recombined frequently with the nonnull class of alleles.