Microsatellites development in Ostrea edulis and Mytilus edulis

The European flat oyster Ostrea edulis and the blue mussel Mytilus edulis are both very valuable commercial species across Europe. Despite their economical importance, only a few microsatellite markers have so far been developed in those species. The aim of this study was to develop new microsatellites for O. edulis and M. edulis that could be used for population genetics studies, genetic variability assessment of stocks, parentage analysis or genetic and QTL mapping studies. For Ostrea edulis, an enriched library was made by ecogenics GmbH (Zurich, Switzerland) from size selected genomic DNA ligated into SAULA/SAULB-linker and enriched by magnetic bead selection with biotin-labelled (GT)13 and (CT)13 oligonucleotide repeats (Gautschi et al. 2000a,b). Of 758 recombinant colonies screened, 179 gave a positive signal after hybridization. Plasmids from 133 positive clones were sequenced and primers were designed for 94 microsatellite inserts. Optimisation of PCR conditions was first performed on agarose gel by changing the annealing temperature, the MgCl2 concentration and the primers concentration. Successful amplification was achieved on 2% agarose gel for 76 primer pairs. For Mytilus edulis, an enriched library was made by ecogenics GmbH (Zurich, Switzerland) from size selected genomic DNA ligated into SNX forward/SNX reverselinker and enriched by magnetic bead selection with biotin-labelled (GT)13 and (CT)13 oligonucleotide repeats. Of 750 recombinant colonies screened, 157 gave a positive signal after hybridization. Plasmids from 157 positive clones were sequenced and primers were designed for 62 microsatellite inserts. Out of 31 primer pairs tested, successful amplification was achieved on 2% agarose gel for 22 of them after optimisation of the PCR conditions. Further optimisation was performed on a 3130xl Genetic Analyzer (Applied Biosystems), using an economic method for the fluorescent labeling of PCR fragments. This M13-tail protocol consists of using 3 primers during the PCR: a sequence-specific forward primer with M13 tail at the 5' end, a sequence-specific reverse primer and the universal fluorescent-labeled M13 tail. Four different M13 tails were used, each labeled with a different fluorescent dye (FAM, VIC, PET, NED). Test of polymorphism for the optimised microsatellites of O. edulis and M. edulis was performed by genotyping 16 individuals per population, in two wild populations.