Molecular Ecology Resources (2009) 9, 429–430 doi: 10.1111/j.1755-0998.2008.02467.x © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Isolation and characterization of 11 microsatellite primers for a temperate reef fish, the California sheephead (Semicossyphus pulcher) MARLOES POORTVLIET,*† JEANINE L. OLSEN,† KIMBERLY A. SELKOE,‡ JAMES A. COYER† and GIACOMO BERNARDI* *Department of Ecology and Evolutionary Biology, University of California Santa Cruz, 100 Shaffer Road, Santa Cruz, CA 95060, USA, †The Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands, ‡Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106, USA Abstract Eleven microsatellites were characterized for Semicossyphus pulcher (California sheephead) using an enrichment protocol. The number of alleles varied from three to 14 for a sample of 40 individuals from two populations. Expected heterozygosities ranged from 0.311 to 0.891. All loci but one were in Hardy-Weinberg equilibrium. No evidence for linkage disequilibrium was observed. These polymorphic microsatellites will be useful for genetic diversity and connectivity analyses of S. pulcher. Keywords: California sheephead, microsatellite, population structure, Semicossyphus pulcher, simple sequence repeat Received 4 June 2008; revision accepted 30 September 2008 The California sheephead (Semicossyphus pulcher) is a protogynous hermaphroditic wrasse common to the tem- perate coastal waters from Point Conception, California, USA to Cabo San Lucas, Baja California, Mexico, including a disjunct population in the Sea of Cortez (Cowen 1985). Fishing pressure by both recreational and commercial fisheries has caused major changes in the abundance and size distribution of California sheephead in southern California. This pressure resulted in two major issues: declining stocks (California Department of Fish and Game, www.dfg.ca.gov/marine/ sheephead2004/pdfs/summary.pdf), as well as an artificially biased sex ratio (Warner 1975; Platten et al. 2002). Neutral and highly polymorphic microsatellite markers are well suited to assess those issues (Waples 1998; Zane et al. 2002). Here we describe the isolation and characterization of 11 novel microsatellite loci (Table 1). Genomic libraries enriched for microsatellite motifs were constructed by Genetic Identification Services (GIS, www.genetic-id-services.com; Chatsworth, California, USA). Libraries were built using a sample containing 100 μg of genomic DNA extracted from gill tissue of a single individual S. pulcher collected in Palos Verdes, California, USA. The sample was stored in 90% ethanol and extracted following a standard phenol–chloroform procedure (Sambrook et al. 1989). Libraries were enriched for CA, CATC, TACA, TAGA motifs. GIS sequenced 64 microsatellite-containing clones using universal M13 primers. We tested 11 of these microsatellites, which were deter- mined to have flanking sequences of length sufficient for primer design using DesignerPCR version 1.03 (Research Genetics, Inc.). Amplification reactions were carried out using a fluorescently labelled forward primer and a cold reverse primer (Sigma-Aldrich) in an Applied Biosystems GeneAmp PCR 9700 system in a total volume of 13 μL containing 1× PCR Mastermix (2.5 mm TAPS pH 9.5, 5.0 mm KCl, 0.2 mm MgCl 2 , 20.0 μm of each dNTP, Taq 0.5 μ/μL, Thermo Scientific), 12.5 pmol of both primers and approxi- mately 2 ng of DNA template. The following temperature profile was used: 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a final extension of 72 °C7 for 7 min. Forty individuals from two populations (20 from Guadalupe Island, Mexico and 20 from Catalina Island, California, USA) were genotyped to estimate allelic diversity and calculate average observed and expected heterozygosities for these 11 loci. All 11 primer pairs were polymorphic and successfully amplified for all samples of S. pulcher (Table 1). Microsatellite amplifications Correspondence: Marloes Poortvliet, Fax: 831 459 3383; E-mail: M.Poortvliet@rug.nl