Isolation and characterization of twenty-one polymorphic microsatellite loci for Polycarpa aurata using third generation
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TECHNICAL NOTE
Isolation and characterization of twenty-one polymorphic microsatellite loci for Polycarpa aurata using third generation sequencing Benjamin J. Wainwright • Irma S. Arlyza Stephen A. Karl
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Received: 13 February 2013 / Accepted: 15 February 2013 / Published online: 27 February 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Twenty-one microsatellite loci were developed for the tropical ascidian, Polycarpa aurata. Microsatellite enriched genomic libraries were constructed and sequenced using Pacific Biosciences single molecule real time sequencing technology. The 21 loci were characterized in 20 individuals from Alor, Indonesia. Observed levels of heterozygosity ranged from 0.30 to 1.00 with a mean of 8.2 alleles per locus. Loci PA21 and PA45 showed evidence of significant linkage disequilibrium and three 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
B. J. Wainwright S. A. Karl (&) Hawai‘i Institute of Marine Biology, University of Hawai‘i, Ma¯noa, P.O. Box 1346, Ka¯ne‘ohe, 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
microsatellite loci developed here will have applications throughout the range of Polycarpa aurata and will be useful tools in inferring connectivity and gene flow, thereby helping to facilitate their integration into future conservation strategy and policy. Genomic DNA (gDNA) was extracted from the mantle of one individual collected from Bali, Indonesia. Microsatellite enriched 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 Pacific Biosciences RS third generation sequencing platform employing C2 chemistry and Circular Consensus Sequencing (CCS) was performed over two 45 min movies on one SMRT cell. Sequencing was performed at the Yale Center for Genomic Analysis. Sequences were trimmed using GENEIOUS v 5.5 (Drummond et al. 2010) to contain a maximum of 5 low quality bases (Q \ 20). Sequences were then searched for microsatellites and suitable priming regions with QDD v1.3 (Meg
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