The Atlantic cod (Gadus morhua L.) is very common along the entire Norwegian coast. Based on their life history characteristics and migration patterns, Atlantic cod in Norwegian and adjacent waters have traditionally been divided into three stocks, i.e. the northeast Arctic cod (NEAC), the Norwegian coastal cod (NCC) and the North Sea cod (NSC). Local populations exist in many fjords that do not interact extensively with populations living in the open sea (Fevolden and Sarvas 2005; Sarvas and Fevolden 2005a; Sarvas and Fevolden 2005b). Microsatellite markers have been successfully applied in studies of population structure of NEAC (Beacham et al. 2002; Bentzen et al. 1996; Ruzzante et al. 1996; Ruzzante 1998) and NCC (Hutchinson et al. 2001; Knutsen et al. 2003; Knutsen et al. 2004; Nielsen et al. 2001; Nielsen et al. 2003). However, Atlantic cod has a narrow genetic base and more genetic markers should be developed. Recently, we have published a number of microsatellite markers derived from both genomic library (Wesmajervi et al. 2006; Westgaard et al. 2006) and expressed sequence tags (ESTs) (Stenvik et al. 2006). In contrast to genomic microsatellites, EST-derived microsatellite markers have the potential to be functional markers (Varshney et al. 2005). In this paper we report the development of 10 EST-derived microsatellite loci from Atlantic cod. The new microsatellites presented in this work and those previously reported (Brooker et al. 1994; Jakobsdottir et al. 2006; Miller et al. 2000; O’Reilly et al. 2000; Wesmajervi et al. 2006; Westgaard et al. 2006; Stenvik et al. 2006) can be used for conservation and enhancement of wild stocks for commercial harvesting.

In an ongoing genomics program, we are generating ESTs from cDNA libraries of different developmental stages of Atlantic cod (unpublished results). Approximately 1,300 ESTs from Cod embryos were investigated for the presence of microsatellite motifs using Tandem Repeats Finder software (Benson 1999). A set of 32 flanking primer pairs were designed for EST sequences containing di- tri- tetra- and pentanucleotide motifs using Primerselect (DNASTAR). For fluorescent detection the forward primers were dye labelled (PET, NED, VIC or 6-FAM) (Applied Biosystems; AB). A PIG-tail sequence 5′-GTTTCTT (Brownstein et al. 1996) was included in the reverse primers (Table 1). Genomic DNA from 96 unrelated individuals was analysed to evaluate the efficiency of amplification and polymorphism of the markers by polymerase chain reaction (PCR) in a 2720 Thermal Cycler (AB). The amplification reaction was carried out in 10 μl volume containing 10–50 ng template, 1 × PCR-buffer (10 mM Tris–HCl; pH 8.8 at 25°C, 50 mM KCl and 0.1% Triton X-100), 2.25 mM Mg2+, 400 μM each dNTP, 100 nM each primer and 0.4 U Dynazyme® II polymerase. Thermal cycling conditions were: 5 min at 94°C, followed by 30 amplification cycles at 94°C for 20 s, 58°C for 20 s and 72°C for 30 s and then a final extension at 72°C for 10 min. Genotyping was performed using a 3130xl Genetic Analyzer (AB). Data were collected automatically and sized with GeneMapper software (AB). Out of 32 primer pairs only 10 were readily scorable and polymorphic (Table 1). Statistical calculations were performed using the program Genepop version 3.4 (Raymond and Rousset 1995). The number of alleles per locus (A), observed heterozygosity (H o), expected heterozygosity (H E) and inbreeding coefficient (F IS) were calculated. The number of alleles varied from 2 to 13 per locus and the observed- and expected heterozygosity ranged from 0.03 to 0.69 and 0.03 to 0.74, respectively (Table 1). The loci were further tested for both Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium and significance levels were determined after 100 batches of 5,000 interactions each. Three of the loci (Gmo-C131, C132 and C136) deviated from HWE after sequential Bonferroni correction (Rice 1989), probably due to the small sample size, genetic drift and the presence of null alleles. Two of 45 pairwise exact tests rejected genotypic equilibrium between loci (Gmo-C131 and Gmo-C132 and C128 and Gmo-C133) after sequential Bonferroni correction. BLAST analysis gave highly significant hits for five loci (Table 2). These represent well-characterized genes thus defining the associated microsatellites as Type I markers, all located to the 3′UT region of the genes. In conclusion, these 10 polymorphic microsatellite loci provide useful tools for studies of population genetics, reproductive ecology and for constructing linkage maps of Atlantic cod.

Table 1 Ten microsatellite loci isolated from Atlantic cod (Gadus morhua L.) and PCR primers that were employed to screen 96 individuals. Significance level was adjusted following sequential Bonferroni correction
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Table 2 Annotation of gene-associated microsatellites based on BLAST searches against the NCBI GenBank non-redundant database. The putative location of the microsatellite within the gene is indicated and the accession no. of the matching sequence is given
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