For the European flat oyster, in vitro sequencing investigated 40 loci from two EST libraries (Morga et al. 2011, 2012). Primers were designed using Primer3 software package (Rozen and Skaletsky 2000). A total of 22 oysters, 16 from four different natural populations collected on the Atlantic and Mediterranean coasts and six belonging to the first generations of three selected families for resistance to bonamiosis were used to investigate polymorphisms. The PCR and sequencing protocols used were the same as those given in Harrang et al. (2013). Sequence alignment was performed with ClustalW via the BioEdit interface (Hall 1999). The validity of each SNP was checked individually on nucleotide sequences and sequence alignments. A total of 420 in vitro SNPs were detected in the dataset of 40 sequenced fragments. Among them, the indels (n = 34) were discarded. Moreover, 347 SNPs were also discarded because of neighboring polymorphisms or low functionality scores. However, as we wanted some genes of interest to be represented in the SNP dataset, we kept some (n = 13) that had neighboring polymorphisms. To favor genotyping, those polymorphic nucleotides were treated as degenerated nucleotides. In total, 52 in vitro SNPs were included in the array, representing 35 different gene fragments
