Isolation and characterization of twenty-three polymorphic microsatellite loci for Linckia laevigata
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TECHNICAL NOTE
Isolation and characterization of twenty-three polymorphic microsatellite loci for Linckia laevigata Benjamin J. Wainwright • Irma S. Arlyza Stephen A. Karl
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Received: 13 August 2012 / Accepted: 22 August 2012 / Published online: 1 September 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Twenty-three polymorphic microsatellite loci were developed for the tropical sea star, Linckia laevigata. Microsatellite enriched genomic libraries were constructed and subsequently sequenced using Roche 454 technology. The 23 loci were characterized in 21 individuals from Pulau Derawan, Indonesia. Observed levels of heterozygosity ranged from 0.20 to 1.00 with a mean of 8.0 alleles per locus. No pairs of loci showed evidence of significant linkage disequilibrium and 4 loci significantly deviated from Hardy–Weinberg expectations. Keywords Connectivity Gene flow Indonesia 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 understand and quantify connectivity in marine ecosystems. The microsatellite loci developed here will have applications throughout the range of Linckia laevigata and will be B. J. Wainwright 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: [email protected] I. S. Arlyza Research Center for Oceanography, Indonesian Institute of Sciences, Jl. Pasir Putih I Ancol Timur, Jakarta 14430, Indonesia
useful tools in inferring connectivity and gene flow, thereby helping to facilitate their integration into future conservation strategies and policy. Genomic DNA (gDNA) was extracted from the tube feet of one individual and enriched gDNA libraries were constructed following the method of Glenn and Schable (2005) using four separate mixes of biotinylated oligonucleotides (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 PicoTiterPlate 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 5.5 (Drummond et al. 2010). Sequen
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