Pecten maximus supports an important commercial fishery. France and the UK land the largest annual catches with total UK scallop landings of 34,000 tonnes in 2009, equating to a value of £47 million (FAO 2012). Despite its commercial importance, the genetic structure of P. maximus stocks remain poorly defined. Watts et al. (2005) developed nine microsatellite markers but to date no studies of population structure have been published using them and our testing found that only four produced reliable results. Consequently, new microsatellite markers were developed using 454 transcriptome sequencing. Since the development of the microsatellites in this study, nine further microsatellite markers have been published from expressed sequence tags (Charrier et al. 2012). The power of many genetic analyses is increased more rapidly by the addition of more loci (and more variable loci) than by increasing the sample size (Felsenstein 2006; Wang and Santure 2009; Landguth et al. 2012). As such, the microsatellite markers from all three studies could be combined to create a very powerful suite of genetic markers.

Microsatellite markers were identified using next generation sequencing of the scallop transcriptome. Briefly, cDNA was isolated from gill, muscle, and mantle tissue from four individual scallops and sequenced on Roche’s Titanium Flex 454 sequencer. Sequences were cleaned of vectors and primers using Seqclean (http://compbio.dfci.harvard.edu/tgi/software/) before contig assembly using iAssembler (http://bioinfo.bti.cornell.edu/tool/iAssembler/). Microsatellite containing contigs were then identified using microsatcommander (Faircloth 2008) and primers designed using Primer3 (Rozen and Skaletsky 2000). Following initial testing twelve markers which were polymorphic and amplified reliably were then multiplexed. Primers were labelled with one of four different fluorescent tails (PET®, VIC®, NED®, FAM®). Oligonucleotide tails were added to the 5′ end of the forward primer and fluorescently labelled oligonucleotides complementary to the oligonucleotide tail were then incorporated during PCR with the addition of cycles using an annealing temperature of 50 °C (Schuelke 2000).

The markers were tested on three populations: Galicia in Spain, Norway and the Isle of Man (GAL, NOR, IOM). DNA was extracted using CTAB extraction buffer (Doyle and Doyle 1987). Each 12 μl PCR contained 1 μl of DNA (~10 ng), 6 μl of Qiagen Type IT multiplex PCR mix, 0.5 μl of BSA (10 mg/ml), 2.5 μl of water and 2 μl of Primer mix. The primer mix contains the forward and reverse primers for all loci in the multiplex plus the appropriate fluorescent tails (PET®, VIC®, NED®, FAM®) (Table 1). There were two different thermocycling profiles for the multiplex reactions. Profile one used a touchdown PCR regime; 95 °C for 5 min, [95 °C 30 s, 64 °C (−1 °C per cycle) 90 s, 72 °C 30 s] × 6 cycles, (95 °C 30 s, 58 °C 90 s, 72 °C 30 s) × 9 cycles, (95 °C 30 s, 50 °C 90 s, 72 °C 30 s) × 15 cycles with a 45 min extension at 72 °C. Profile two; 95 °C 5 min, (95 °C 30 s, 51 °C 90 s, 72 °C 30 s) × 16 cycles, (95 °C 30 s, 50 °C 90 s, 72 °C 30 s) × 19 cycles, final extension of 45 min at 72 °C. PCR products were resolved on an ABI 3130XL sequencer and Genemapper® software (Applied Biosystems) to size alleles.

Table 1 Polymorphic microsatellite markers for P. maximus with locus characteristics and multiplex PCR primer concentrations
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Data was checked for null alleles, stuttering and large allele dropout using Microchecker (Van Oosterhout et al. 2004). Genepop v4.0 (Rousset 2008) was used to test each pair of loci in each population for evidence for linkage disequilibrium and conformity to Hardy–Weinberg Equilibrium (HWE) using Fisher’s exact tests (Markov chain parameters: burn-in = 10,000 followed by 100 batches of 5,000 iterations each). The alpha value was corrected following multiple testing using the False Discovery Rate method (Benjamini and Hochberg 1995). GENALEX 6 (Peakall and Smouse 2006) was used to estimate observed and expected heterozygosity (Ho and He respectively) and the number of alleles.

Marker PmNH9 in GAL had very low amplification success of 43 % and therefore data from this population was not included in the analysis of this marker. No evidence of stuttering or large allele dropout was detected. Marker PmNH20 showed a significant deficit of heterozygotes in all populations. PmNH19 showed a deficit of heterozygotes in IOM and NOR but not GAL, and PmNH68 had a deficit of heterozygotes in IOM only. Microchecker indicated that this could be due to null alleles at these loci/population combinations. All other markers showed no significant deviation from HWE in any population following correction for multiple testing (Table 2). No significant linkage disequilibrium was detected between any pair of loci. The number of alleles ranged from two to 13 and all loci were polymorphic in all populations. The lowest observed heterozygosity was 0.02 at locus PmNH70 in NOR, with the highest observed heterozygosity of 0.77 at locus PmNH59, also in NOR.

Table 2 Number of alleles (Na), observed (Ho) and expected heterozygosity (He)
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Markers PmNH20, PmNH19 and PmNH68, whilst highly polymorphic, show evidence of null alleles and should therefore be used with caution. Marker PmNH9 did not amplify reliably in the Galicia samples but was reliable in the other populations. The remaining eight markers conformed to HWE in all three populations and amplified reliably. When combined with previously developed markers (Watts et al. 2005; Charrier et al. 2012) these loci will constitute a large and powerful suite of markers for P. maximus.