Identification of high-resolution microsatelliteswithout apriori knowledge of genotypes using a simple scoring approach

1. Large numbers of microsatellite loci for many species are increasingly made available from public data bases or through de novo isolation with next generation sequencing methods. However, the challenge remains to identify the subset of loci with suitable polymorphism for the populations of interest. This normally requires genotyping individuals in a representative subset of populations.

2. Here, we present an approach that does not require individual genotypes, but instead uses the presence and absence of alleles in pooled DNA samples to estimate within-population variation (WPV, i.e. polymorphism) and among-population variability (APV, i.e. rare alleles). From these, we developed a scoring procedure to rank individual loci and thereby identify thosemost suitable for individual genotyping.

3. We used multiplex PCR of 20 pooled individuals from each of 12 populations to screen a total of 80 published and unpublished microsatellite loci in the Northern pike Esox lucius, a species known to exhibit low genetic variability, thus predisposing it as a model species to test our approach. The screening of pooled samples revealed extremely different levels of microsatellite variability, ranging from 1
0 to 27
5 alleles per microsatellite (WPV) and from 1
4 to 12 populations per allele (APV). Scoring placed microsatellites with high WPV and medium APVat the highest ranks.

4. We validated this outcome by genotyping individuals of two pike populations from geographically close areas. A strong differentiation potential (measured as Weir’s & Cockerham’s FST) as well as a better resolving power of higher over lower-ranked microsatellites (measured asNei′sDA and probability of identity) proved that the method presented here is suitable for rapid and inexpensive identification of appropriate microsatellites without a priori knowledge of genotypes.

5. Our scoring procedure is generally applicable to all population genetic studies based on microsatellites and is particularly recommended, if species with low genetic variability are to be investigated.