Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.