The Pacific Oyster Crassostrea gigas, natively distributed around coast of Asia is now one of the most important economic bivalves produced throughout the world. The large demand and exploitation of this marine mollusk accelerated the development and implementation of genetic management practices and selection breeding programmes.

Microsatellites, also known as simple sequence repeats or SSRs, are a small array of tandemly arranged bases (one to six) spread throughout the genomes, which as DNA markers have been useful tools for many studies in the species (Sekino et al. 2003; Li et al. 2003; Hubert and Hedgecock 2004; Li and Kijima 2006; Wang et al. 2008) as they exhibit high polymorphism and are inherited co-dominantly in a Mendelian fashion. Much more novel and useful DNA markers are stilled needed for the increasing genetic studies. Here we report our work on exploitation of microsatellite markers for C. gigas.

All microsatellite loci were identified and isolated by the MISA software (http://pgrc.ipk-gatersleben.de/misa/) from end sequences of an in-house fosmid library and all microsatellite primers were designed using PRIMER3 software (Rozen and Skaletsky 2000). Thirty wild Pacific oysters were used for polymorphism analysis and the extraction of genomic DNA was performed by a standard phenol–chloroform protocol (Sambrook et al. 1989). PCR was carried out in a volume of 15 μl containing 40 ng of template DNA, 0.5 U of Taq polymerase, 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 200 μM each dNTP and 0.3 μM each primer. The thermal cycling conditions were: 95°C for 2 min, then 30 cycles of denaturation at 94°C for 30 s, T a of each primer pair (shown in Table 1) for 30 s and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. The PCR products were separated by 12% non-denaturing polyacrylamide, stained by ethidium bromide and then photoed by ImageMaster VDS (Pharmacia Biotech).

Table 1 Characterization of 33 novel polymorphic microsatellite loci for the Pacific Oyster Crassostrea gigas
Full size table

The data including number of alleles, heterozygosity, tests of Hardy–Weinberg equilibrium (HWE) and pairwise check of linkage disequilibrium (LD) were analyzed using the software POPGENE version 1.32 (Yeh et al. 2000). As was shown in Table 1, the size of repeat motif ranged from 2 to 6 and the number of alleles per locus ranged from 3 to 9. The observed and expected heterozygosity values varied from 0.080 to 0.880 and from 0.174 to 0.876 respectively. Twelve loci did not conform to HWE due to the limited sample size and/or the possible presence of null alleles (P < 0.05). LD was detected for two pairs of loci, i.e. otgfa0_0732_B03 & rotgfa0_069213 and otgfa0_018860 & rotgfa0_1610_B08 (P < 0.05). All the 33 loci were compared with the 407 microsatellite sequences for C. gigas in public database (up to September 3, 2009, NCBI) and no homologies were found, indicating the loci reported here are new microsatellite DNA markers.