Giant clams are an important part of coral reef habitats, providing reef stability and settlement substrate. Individuals are harvested for the aquarium trade, subsistence food, and for the shell trade (Copland and Lucas 1988) resulting in population decline due to overfishing. All species are listed in Appendix II of CITES (UNEP-WCMC 2007) and on the IUCN Red List of Threatened Species (Mollusk Specialist Group 1996). Restocking efforts are ongoing, especially in the Philippines (Gomez and Mingoa-Licuanan 2006). In order to provide genomic tools for inferring fine-scale population genetic structure and demographic parameters on recent time scales, we developed 9 new microsatellite markers for the boring giant clam (Tridacna crocea) and tested for cross-amplification in three congeners.

We isolated genomic DNA from fresh muscle tissue of one individual obtained legally from the aquarium trade using the QIAGEN DNeasy Tissue Kit and digested with a combination of AluI, XmnI, and RsaI. STR enrichment followed Glenn and Schable (2005) except for our choice of biotinylated oligonucleotide probes: (AC)13 (AAC)6, (ACAT)8, (ACTC)6, (AGAT)8, and (ATCC)5. Plasmids were cloned with PROMEGA pGEM-T Vector System I cloning kit according to the manufacturer’s protocols and grown on LB agar plates containing ampicillin and X-gal. Positive clones were recovered in PCR reactions using T7 and M13R universal primers and sequenced using the BigDye Terminator method (ABI) on an ABI 3730. Sequences were edited in Sequencher 4.6 (Gene Codes) and short tandem repeats (STRs) were identified with the Simple Sequence Repeat Identification Tool (Temnykh et al. 2001). We designed primers in PRIMER3 (Rozen and Skaletsky 2000) and screened them using AUTODIMER (Vallone and Butler 2004).

We assessed allelic variation in 20 individuals from Ulugan Bay, Philippines. Cross-priming was tested on 8 individual T. maxima and T. squamosa and 3 individual T. gigas. Genomic DNA was extracted using 10% Chelex (Walsh et al. 1991). PCR amplification was in 10 μl reactions containing 1.0 μl DNA template, 0.25U Taq Gold polymerase (ABI), 1 μl 10 × PCR Buffer, 1 μl dNTPs (8 mM), 0.8 μl MgCl2 (25 mM), and 0.5 μl of each primer (10 mM). For loci Tc004 and Tc160 the amount of MgCl2 was doubled and water reduced to a final volume of 10 μl. Thermocycling parameters were: 1 × 94°C (10 min), 30× [30 s at 94°C, 30 s Ta °C, 40 s at 72°C], and 1 × 72°C (60 min). Annealing temperatures (Ta) for each locus are in Table 1.

Table 1 Microsatellite loci and thermocycling parameters
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Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were assessed in ARLEQUIN 3.11 (Excoffier et al. 2005). Locus Tc157 amplified well in all samples, but was only scoreable in some individuals due to multiple peaks. Locus Tc092 showed a significant departure from HWE after Bonferroni correction (Table 1). Linkage disequilibrium was only significant in 1 out of 36 pairwise comparisons at the P < 0.01 level (Tc074 vs. Tc092) indicating virtually no linkage among loci.

Since no species-specific PCR optimization was attempted, the results reported here represent a minimum rate of cross-species primer conservation. The test resulted in 5 loci amplifying in T. maxima (5 polymorphic); 4 loci amplifying in T. squamosa (3 polymorphic); and 4 loci amplifying in T. gigas (1 polymorphic) (Table 2). The T. gigas were from cultured stocks, therefore the lack of polymorphism is expected and may not represent diversity in natural populations.

Table 2 Cross-priming success in 3 additional Tridacna species
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A technical challenge to developing microsatellites in this species is targeting the host genome rather than that of symbiotic dinoflagellates (Symbiodinium spp.). Symbionts are restricted to mantle tissue in giant clams, therefore we extracted genomic DNA from muscle tissue for primer development. To verify that none of our primers amplify symbiont DNA, we tested them on 7 symbiont types using PCR conditions as described above (Table S1). Excepting Tc004, no significant amplification was achieved in the range of allele sizes reported for T. crocea and we are confident that the loci developed are specific to giant clam DNA. For Tc004, a 128 bp fragment was amplified in two isolates. This is a common allele in T. crocea (T.S. DeBoer unpublished data); therefore, caution should be employed when using this locus.

Population genetic studies using mtDNA show strong structure in T. crocea (DeBoer et al. 2008, Kochzius and Nuryanto 2008). Microsatellites are expected to be more variable, and one application where this would be useful is forensic identification of clams sold in the aquarium trade. It should be possible to identify source regions for traded clams, as well as distinguish wild from cultured stocks. This would assist efforts to stop illegal trade in these internationally protected species. These markers will also be useful for inferring modern genetic connectivity and gene flow among natural and restored populations.