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. and Melthide Sinama, Vincent Dubut, Caroline Costedoat, André Gilles, Marius Junker, Thibaut Malausa, Jean-François Martin, Gabriel Nève, Nicolas Pech, Thomas Schmitt, Marie Zimmermann, Emese Meglécz.
Novel microsatellite markers for the spruce bark beetle (Ips typographus L.) are identified and characterized using next generation sequencing technology. 18 polymorphic loci were obtained by screening 10,684 reads and tested on 35 bark beetle samples from different locations in Bavaria, Germany. Allelic richness ranged from two to 38 alleles, observed heterozygosity from 0.03 to 0.66 and expected heterozygosity from 0.08 to 0.97. Four loci showed significant deviation from the Hardy-Weinberg equilibrium and no linkage between loci was detected. The 18 loci, along with another six loci previously described, provide effective analytical tools for analyzing the fine-scale genetic structure of bark beetle populations. The result of this study demonstrates that next generation sequencing technology is a valuable method for isolating microsatellites of a coleopteran species. and Bernhard C. STOECKLE, Ralph KUEHN.
Novel microsatellite markers for the solitary bee, Anthophora plumipes, were identified and characterised using 454 GS-FLX Titanium pyrosequencing technology. Thirty seven loci were tested using fluorescently labelled primers on a sample of 20 females from Prague. The number of alleles ranged from 1 to 10 (with a mean of 4 alleles per locus), resulting in an observed heterozygosity ranging from 0.05 to 0.9 and an expected heterozygosity from 0.097 to 0.887. None of the loci showed a significant deviation from the Hardy-Weinberg equilibrium and only two loci showed the significant presence of null alleles. No linkage between loci was detected. We further provide information on a single multiplex PCR consisting of 11 of the most polymorphic loci. This multiplex approach provides an effective analytical tool for analysing genetic structure and carrying out parental analyses on Anthophora populations. Most of the 37 loci tested also showed robust amplification in five other Anthophora species (A. aestivalis, A. crinipes, A. plagiata, A. pubescens and A. quadrimaculata). The result of this study demonstrates that next generation sequencing technology is a valuable method for isolating quality microsatellites in non-model species of solitary bees. and Obsahuje seznam literatury