Characterization of 22 microsatellite loci for conservation genetic studies of an endemic anemonefish, Amphiprion latezo
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MICROSATELLITE LETTERS
Characterization of 22 microsatellite loci for conservation genetic studies of an endemic anemonefish, Amphiprion latezonatus Rosemary Steinberg • Martin van der Meer Jean Paul Hobbs • Michael L. Berumen • Lynne van Herwerden
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Received: 31 July 2014 / Accepted: 19 August 2014 / Published online: 9 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Island endemic species are of particular conservation concern as they exhibit a range of vulnerable traits that greatly increases their risk of extinction (McKinney 1997). The wideband anemonefish, Amphiprion latezonatus, is endemic to a range of 4° of latitude (Rushworth et al. 2011) in the subtropical waters off the Eastern Australian coast, with populations occurring at Lord Howe Island (LHI), Norfolk Island, and scattered locations off the east Australian coast between the Sunshine Coast and South-West Rocks. They are hosted by only two species of anemones, Entacmaea quadricolor and Heteractis crispa (Scott and Malcolm 2011). Though there have been several ecological and ornamental aquaculture studies on A. latezonatus (e.g., Scott and Malcolm 2011; Rushworth et al. 2011), none have examined gene flow, population genetic structure, or genetic diversity of this species. This study describes the
R. Steinberg (&) L. van Herwerden Molecular Ecology and Evolution Laboratory, Australian Tropical Sciences and Innovation Precinct, James Cook University, Townsville, QLD 4811, Australia e-mail: [email protected] R. Steinberg L. van Herwerden School of Marine and Tropical Biology, James Cook University, Townsville, QLD 4811, Australia M. van der Meer ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia J. P. Hobbs Department of Environment and Agriculture, Curtin University, Perth, WA 6845, Australia M. L. Berumen Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia
development of 22 polymorphic microsatellite markers for A. latezonatus using 454 shotgun pyrosequencing on a 454 GS FLX Titanium (Roche) at the Biosciences Core Laboratory (BCL), at the King Abdullah University of Science and Technology (KAUST) in Saudi Arabia. Genomic DNA was extracted using a Qiagen Gentra Puregene (Qiagen, Doncaster, Australia) extraction protocol including RNAse treatment. DNA was shotgun sequenced on 50 % of a 454 GS FLX Titanium plate in the KAUST BCL. Resulting sequences (totalling 1,636,335 reads, average read length 399.5 bp) were screened for microsatellite loci as per van der Meer et al. (2012). This process identified 45,228 microsatellite loci (within 2.76 % of sequences obtained); PCR primers were successfully designed for 5,318 (0.3 %) of loci found. Of these, directlylabelled forward primers (FAM, NED, or VIC) were synthesised for 24 loci and grouped into 4 multiplex reactions of 6 loci per multiplex (Table 1). Loci were tested for amplification success and specificity; genotypes were generated as per van der Meer et al. (2012). Primer
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