TECHNICAL NOTE Isolation and characterization of twenty-six, polymorphic microsatellite loci for the tropical sea urchin, Colobocentrotus atratus Benjamin J. Wainwright • Irma S. Arlyza • Stephen A. Karl Received: 8 October 2012 / Accepted: 29 October 2012 Ó Springer Science+Business Media Dordrecht 2012 Abstract Twenty-six, polymorphic microsatellite loci were developed for the tropical sea urchin Colobocentrotus atratus. Microsatellite enriched genomic libraries were constructed and subsequently sequenced using Roche 454 technology. The 26 loci were characterized in 21 individ- uals from Pulau Asu, Nias, Indonesia. Observed levels of heterozygosity ranged from 0.52 to 1.00 with a mean of 11.9 alleles per locus. No pairs of loci showed evidence of significant linkage disequilibrium and 3 loci significantly deviated from Hardy–Weinberg expectations. Keywords Connectivity Á Gene flow Á Indonesia Á Conservation Marine protected areas are considered important tools in the conservation of marine ecosystems (Palumbi 2003; Mous et al. 2005; Planes et al. 2009) and population connectivity is an important component of protected area design (Sala et al. 2002). In the marine environment it is extremely difficult to directly track or tag pelagic larvae through to the point of metamorphosis and settlement. Because of these difficulties genetic approaches have been developed and are important tools in the efforts to under- stand and quantify connectivity in marine ecosystems. The microsatellite loci developed here will have applications throughout the range of C. atratus and will be useful tools in inferring connectivity and gene flow, thereby helping to facilitate future conservation strategies and policies. Genomic DNA (gDNA) was extracted from the tube feet of one individual and microsatellite enriched libraries were constructed following the method of Glenn and Schable (2005) using four separate mixes of biotinylated oligonu- cleotides (Toonen 1997). Microsatellite enriched DNA was recovered via PCR (Glenn and Schable 2005), visualized on an agarose gel and pooled into a single sample of approximate equimolor concentration. Enriched fragments were submitted for sequencing on a Roche GS-FLX sequencer (454 Life Sciences, Branford, CT, USA) using titanium chemistry. The DNA was tagged with unique multiplex identifiers (MIDS) and placed on 1/4 of a Pico- TiterPlate with three other, unrelated libraries. Sequencing was performed at the Center for Advanced Studies in Genomics, Proteomics and Bioinformatics (University of Hawai‘i at Ma ¯noa). Sequence reads were separated by MID tags and all sequences trimmed to remove regions with a greater than 0.5 % chance of error per base using GENEIOUS v 5.6 (Drummond et al. 2010). Sequences were searched for microsatellites and suitable priming regions with QDD v1.3 (Meglecz et al. 2010). Primers were designed with PRIMER 3 (Rozen and Skaletsky 1999) and synthesized by Eurofins MWG Operon (Huntsville, AL, USA). Screening was performed using a modified M13-tailed primer method described by Boutin-Ganache et al. (2001) to produce fluorescently-labeled amplicons. This method uses three primers in a single reaction: a forward tailed primer at a low concentration with a modified 5 0 tail corresponding to the same sequence as one of four fluorescently labeled primers (FAM, VIC, PET, or NED; Applied Biosystems, Carlsbad, CA, USA), an unmodified reverse primer, and a dye labeled sequence matching the tail on the modified B. J. Wainwright Á S. A. Karl (&) Hawai‘i Institute of Marine Biology, University of Hawai‘i, Ma ¯noa, P.O. Box 1346, Ka ¯neo `he, HI 96744, USA e-mail: skarl@hawaii.edu I. S. Arlyza Research Center for Oceanography, Indonesian Institute of Sciences, Jl. Pasir Putih I Ancol Timur, Jakarta 14430, Indonesia 123 Conservation Genet Resour DOI 10.1007/s12686-012-9808-1