Molecular Ecology Resources (2008) 8, 1503–1505 doi: 10.1111/j.1755-0998.2008.02327.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Characterization of 18 new microsatellite loci in Atlantic cod (Gadus morhua L.) SIGURLAUG SKIRNISDOTTIR,* CHRISTOPHE PAMPOULIE,† SIGURBJORG HAUKSDOTTIR,* INGA SCHULTE,* KRISTINN OLAFSSON,* GUDMUNDUR O. HREGGVIDSSON*,‡ and SIGRIDUR HJORLEIFSDOTTIR* *Matis-Prokaria, Gylfaflöt 5, 112 Reykjavik, Iceland, †Marine Research Institute, Skulagata 4, 101 Reykjavik, Iceland, ‡Department of Biology, University of Iceland, Sturlugata 7, 101 Reykjavik, Iceland Abstract Eighteen new microsatellite loci consisting of 10 di-, 5 tri-, 2 tetra- and 1 heptanucleotide repeats are introduced for the Atlantic cod (Gadus morhua L.). All loci were co-amplified in two polymerase chain reactions (plus two previously published microsatellites) and all products were typed clearly. The number of alleles per locus ranged from six (PGmo130) to 45 (PGmo76) and the observed heterozygosity ranged from 0.356 (PGmo130) to 0.957 (PGmo95). All loci except one followed Hardy–Weinberg expectations. Genetic linkage disequilibrium analysis between all pairs of loci did not yield any significant values. Keywords: Atlantic cod, microsatellite, multiplex assay systems Received 28 May 2008; revision accepted 29 June 2008 During the last century, cod stocks have declined dramatically due to overexploitation (Christensen et al. 2003). The decline of exploited marine fishes has been suggested to result in part from the fact that management units do not necessarily reflect biological units as such (Stephenson & Kenchington 2000). Although sustainable management requires stock abundance and productivity estimates, as well as information on stock dynamics, the assessment of spatial structure remains of primary impor- tance (National Research Council 1998). Microsatellite loci often reveal weak but biologically meaningful population structure of exploited species at small geographical scales which can subsequently be integrated into fisheries manage- ment. Although various methods can be used for assessing stock structure, genetic analyses are the most informative and economical method. Microsatellite loci have revealed high level of structure in cod populations within the Atlantic Ocean (O’Leary et al. 2007). We report the isolation and characterization of 18 new microsatellite loci containing di-, tri-, tetra- and heptanu- cleotide repeats which were co-amplified in two multiplex assay systems of 10 microsatellite each, also containing two previously published loci, PGmo38 and PGmo49 (Jakob- sdóttir et al. 2006). These markers were selected on the basis of size range, ease of amplification and of automated size variation detection by an ABI PRISM 3730 sequencer. Genomic DNA was isolated from gill tissue preserved in 90% ethanol using Puregene DNA isolation kit (Gentra). Microsatellite enrichment was performed using a variation of the protocol available at www.genomics.liv.ac.uk/animal/ MICROSAT.PDF. The blunt-end restriction enzyme RsaI and adaptors were used for the protocol (Mirosevich et al. 2001). Enriched DNA fragments were cloned into Topo TA cloning vectors (see manufacturer’s instructions, Invitro- gen). Sequencing and primer design was according to Jakobsdóttir et al. (2006). A total of 266 individuals were collected at two different spawning grounds located in Iceland (sampling sites 511, 911 and 931, see Pampoulie et al. 2006). DNA was isolated using Chelex 100 Resin (Walsh et al. 1991). Polymerase chain reactions (PCR) were performed in a 10-μL volume containing 3 μL of 1/10 diluted DNA, 200 μm of each dNTP, 1× Teg buffer (100 mm Tris-HCl, pH 8.8; 500 mm KCl; 15 mm MgCl 2 ; 1% Triton X-100), 0.2 U Teg polymerase (Matis-Prokaria, Taq comparable), 0.04–0.10 μL of the labelled forward (100 μm) and 0.04–0.1 μL of the reverse primer (100 μm). PCR were performed on GeneAmp2700 thermal blocks as follows: initial denatura- tion step of 4 min at 94 °C followed by 30 cycles of 50 s at Correspondence: Sigurlaug Skirnisdottir, Fax: +354 4225001; E-mail: sigsk@matis.is