Challenges of microsatellite development in Lepidoptera: Euphydryas aurinia (Nymphalidae) as a case study

Currently it remains difficult to obtain robust microsatellite markers for Lepidoptera. In an attempt to overcome the problems associated with developing microsatellite markers for this insect order we combined (i) biotin-enrichment protocol, (ii) next generation pyrosequencing (through 454 GS-FLX Titanium technology) and (iii) the use of individuals collected from eight geographically distant European populations representing three subspecies of Euphydryas aurinia. Out of 96 stringently designed primer pairs, 12 polymorphic microsatellite loci amplified without obvious evidence of null alleles in eight individuals from different subspecies. Between five and seven of these loci showed full within population applicability and three revealed to be robust and transferable between populations and sub-species, providing a first step towards the development of a valuable and robust tool for studying conservation issues and evolution in E. aurinia populations. Nevertheless, as in most studies dealing with Lepidoptera microsatellites, null alleles were detected in most of the developed markers. Our results emphasize the need for further research in order to better understand the complex evolution and organization of Lepidopteran genomes.