The great scallop (Pecten maximus) is a highly valuable shellfish resource that is widely exploited in France and United-Kingdom (Beaumont and Gjedrem 2007). This bivalve has been significantly manipulated with many transfers of individuals reported among natural populations since the 1970’s (Beaumont 2000). In particular, some stocks have been regularly enhanced with hatchery-raised spat in order to ensure the sustainability of scallop fisheries. Such manipulations necessitate a good knowledge of the population genetics of the species to prevent any alteration of the genetic diversity of wild populations. However, the genetic structure of great scallop populations remains poorly documented, with studies based only on allozymes or mitochondrial DNA (Beaumont et al. 1993; Rigaa et al. 1997; Wilding et al. 1997; Saavedra and Peña 2005). Only nine microsatellites have been isolated so far (Watts et al. 2005), but two of these markers do not seem usable because of significant heterozygote deficiencies and possible null alleles. As a consequence, new microsatellites associated with expressed sequence tags (ESTs) were developed to complete the existing set of markers.

A total of 1,122 EST sequences (DN793124–DN794245) were downloaded from the Genebank database (http://www.ncbi.nlm.nih.gov). Firstly, overlapping and contiguous sequences were assembled with CAP3 (Huang and Madan 1999), using the online interface available on the PBIL website (http://pbil.univ-lyon1.fr/cap3.php). Microsatellites were identified with TANDEM REPEAT FINDER (Benson 1999), and primers were designed manually for 22 loci showing a minimum of five repeats. A M13 tail (5′-CACGACGTTGTAAAACGAC) was added to the 5′-end of each forward primer to reduce the genotyping cost (Schuelke 2000). In addition a PIG-tail (5′-GTTTCTT) was added to the 5′-end of each reverse primer to avoid genotyping errors due to “plus-A” PCR artefacts (Brownstein et al. 1996).

The markers were tested in three populations: Baie de Seine (BS; France), Rade de Brest (RB; France) and Galicia (GA; Spain). Genomic DNA was extracted from the adductor muscle (GA) using a classical phenol–chloroform method, or from the gills (BS and RB) with a CTAB phenol–chloroform protocol (Jolly et al. 2003). Each marker was amplified by polymerase chain reaction (PCR) in 10 μL volume, containing 1× reaction buffer (Uptima), 1.5–2.5 mM MgCl2, 0.074 mM each dNTP, 0.05 μM M13 primer end-labelled with IRDye 800 (Li-Cor), 0.05 μM forward primer, 0.4 μM reverse primer, 0.025 U μL−1 Uptitherm DNA polymerase (Uptima) and 1 μL DNA template. PCR amplifications were performed in a Geneamp PCR System 9700 (Perkin Elmer). A touchdown procedure was incorporated in the thermal cycling regime to increase the stringency of the PCRs: 94 °C for 3 min, Ta for 2 min, 72 °C for 30 s, (94 °C for 30 s, Ta − 1 °C for 30 s [−1 °C per cycle until 50 °C], 72 °C for 30 s) × 5–6 cycles, (94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s) × 19–23 cycles, 72 °C for 5 min. Five μL of denaturing loading buffer were added to the PCR products and 1 μL was loaded on 8 % denaturing polyacrylamide gels of 25 cm with 1 % TBE buffer. The gels were run on an automated sequencer IR2 (Li-Cor) during 1–3 h at 1,500 V and 50 °C, and were analysed with GENE PROFILER (Scan Analytics). Each gel was scored by two independent readers to limit genotyping errors. Seventeen of the 22 tested markers produced clear bands that were confidently scored, and nine of the loci were polymorphic (Table 1).

Table 1 Polymorphic EST-linked microsatellites for P. maximus
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The number of alleles (Na), and the level of observed (Ho) and expected (He) heterozygosities were calculated for each polymorphic locus in each population with GENETIX v.4.05 (Belkhir et al. 1996). The variability was low to moderate, with the total Na ranging from two to 13 (Table 2). All loci were polymorphic in each population, except PmGC06 in GA, and PmGC18 in BS and GA. On average, accoss all three populations, Na varied from 1.333 to 8.667, and Ho and He ranged respectively from 0.029 to 0.703 and 0.028 to 0.711. The lowest heterozygosity was observed for PmGC06 in RB (Ho = 0.040; He = 0.040) and the highest was displayed by PmGC05 in BS (Ho = 0.844; He = 0.832). Departures from Hardy–Weinberg equilibrium were tested for each locus and location by estimating FIS values according to Weir and Cockerham with GENEPOP v.4.0.11 (Rousset 2008) and using the Markov chain method (10,000 dememorisation steps, 100 batches, 5,000 iterations). A significant deficit in heterozygotes was found only for PmGC20 in RB after applying a sequential Bonferroni correction for multiple comparisons (Rice 1989), and the other eight loci conformed to Hardy–Weinberg equilibrium. No significant linkage disequilibrium was detected with GENEPOP v.4.0.11 using the Markov chain method (10,000 dememorisation steps, 100 batches, 5000 iterations) and Fisher’s exact test.

Table 2 Number of alleles (Na), observed (Ho) and unbiased expected (He) heterozygosities, and FIS for each location and each polymorphic locus
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This new set of microsatellites will be useful for further population genetics studies on P. maximus. Moreover, given the aquaculture perspectives for this species, combining these new markers with the existing ones might provide a valuable tool for the genetic monitoring of the possible impacts of hatchery practices on the genetic variability of native scallop populations.