A method for genotype validation and primer assessment in heterozygote-deficient species, as demonstrated in the prosobranch mollusc Hydrobia ulvae

BACKGROUND: In studies where microsatellite markers are employed, it is essential that the primers designed will reliably and consistently amplify target loci. In populations conforming to Hardy-Weinberg equilibrium (HWE), screening for unreliable markers often relies on the identification of heterozygote deficiencies and subsequent departures from HWE. However, since many populations naturally deviate from HWE, such as many marine invertebrates, it can be difficult to distinguish heterozygote deficiencies resulting from unreliable markers from natural processes. Thus, studies of populations that are suspected to deviate from HWE naturally would benefit from a method to validate genotype data-sets and test the reliability of the designed primers. Levels of heterozygosity are reported for the prosobranch mollusc Hydrobia ulvae (Pennant) together with a method of genotype validation and primer assessment that utilises two primer sets for each locus. Microsatellite loci presented are the first described for the species Hydrobia ulvae; the five loci presented will be of value in further study of populations of H. ulvae. RESULTS: We have developed a novel method of testing primer reliability in naturally heterozygote deficient populations. After the design of an initial primer set, genotyping in 48 Hydrobia ulvae specimens using a single primer set (Primer set_A) revealed heterozygote deficiency in six of the seven loci examined. Redesign of six of the primer pairs (Primer set_B), re-genotyping of the successful individuals from Primer set_A using Primer set_B, and comparison of genotypes between the two primer sets, enabled the identification of two loci (Hulv-06 & Hulv-07) that showed a high degree of discrepancy between primer sets A and B (0% & only 25% alleles matching, respectively), suggesting unreliability in these primers. The discrepancies included changes from heterozygotes to homozygotes or vice versa, and some individuals who also displayed new alleles of unexpected sizes. Of the other four loci examined (Hulv-01, Hulv-03, Hulv-04, & Hulv-05), all showed more than 95% agreement between primer sets. Hulv-01, Hulv-03, & Hulv-05 displayed similar levels of heterozygosity with both primer sets suggesting that these loci are indeed heterozygote deficient, while Hulv-08 showed no deficiency in either primer set. CONCLUSION: The simple method described to identify unreliable markers will prove a useful technique for many population studies, and also emphasises the dangers in using a single primer set and assuming marker reliability in populations shown to naturally deviate from HWE.