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

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