The hawksbill turtle (Eretmochleys imbricata) is a globally critically endangered marine species. Demand for the ornately patterned carapace scutes, known as tortoiseshell or bekko, by many cultures and over several centuries has significantly reduced the size and distribution of nesting populations of hawksbill turtles (Parsons 1972). In the Greater Caribbean region, several major rookeries were extinguished or severely depleted by overharvesting (Meylan 1999; McClenachan et al. 2006). Resolving connectivity among remnant rookeries and between nesting and foraging sites is essential for conservation planning and genetic data from both mitochondrial and nuclear markers are needed. Dinucleotide microsatellite loci have been previously isolated from hawksbill turtles (FitzSimmons et al. 1995; Lin et al. 2008; Miro-Herrans et al. 2008). In addition, several tetranucleotide microsatellite markers designed from loggerhead turtles amplify well and conform to Hardy–Weinberg equilibrium expectations in hawksbill turtles (Shamblin et al. 2009). However, additional markers are needed for fine-scale studies such as relatedness analyses. We address this need through development of 14 tetranucleotide markers to complement currently existing markers.

Genomic DNA was enriched for microsatellite loci using the methods described by Glenn and Schable (2005) with minor modifications. DNA was extracted from blood samples taken from two juvenile green turtles captured while foraging in the Indian River Lagoon east of Sebastian, Florida, USA using a Qiagen® DNEasy blood and tissue kit. DNA was digested using RsaI endonuclease (New England Biolabs). Double stranded SuperSNX linkers (Glenn and Schable 2005) were ligated to the digested genomic DNA overnight at 16 °C. Linker-ligated DNA was recovered using PCR on an Applied Biosystems 9700 thermal cycler and verified by agarose gel electrophoresis. Recovered DNA was hybridized to two different mixtures of biotinylated oligonucleotide probes (Integrated DNA Technologies, mix 1: (AAAC)6, (AAAG)6, (AATC)6 (AATG)6, (ACAG)6, (ACCT)6, (ACTC)6, (ACTG)6; mix 2: (AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8). Probes and hybridized DNA were captured using streptavidin-coated magnetic beads (DynaBeads®, Invitrogen®). Enriched DNA was washed and eluted at 95 °C and recovered through PCR. The enrichment procedure was repeated to increase the efficiency of microsatellite array capture. DNA from the second enrichment was used as template for amplification by two sequential rounds of PCR. The re-amplified PCR products were ligated into a pCR®2.1-TOPO® vector and transformed into TOP 10® competent cells (Invitrogen).

A total of 285 colonies were screened by colony PCR, treated with ExoSap-IT® (New England Biolabs) and sequenced using BigDye v3.1 (Applied Biosystems). Sequencing reactions were purified using Sephadex G-50 Fine (Sigma Aldrich) and analyzed on an Applied Biosystems 3730xl DNA Analyzer. Sequences were aligned and edited using Sequencher 4.2 (Gene Codes). Contigs were queried for microsatellite loci using MSATCOMMANDER (Faircloth 2008). BLAST alignments confirmed high sequence identities (>85 %) with loci previously isolated from loggerhead turtles (Cc7E11, Genbank DQ917773; Cc7G11, Genbank DQ917774; Shamblin et al. 2007; Cc2F11, Genbank EU125406; Cc7A08, Genbank EU125397; Shamblin et al. 2009), green turtles (CHMY30, Genbank JQ728662, Shamblin et al. 2012), and leatherback turtles (Derm13, Genbank GU592728; Derm30, Genbank GU592732; Genbank36, Genbank GU592736; Alstad et al. 2011). We designed primers for 37 novel loci using MSATCOMMANDER (Faircloth 2008) or manually with PRIMER3 (Rosen and Skaletsky 2000) when automated primer design failed.

Amplification was performed in 10 μl reactions consisting of 10 mM Tris pH 8.4, 50 mM KCl, 0.5 μM GTTT “pig-tailed” locus specific primer, 0.05 μM CAG (CAGTCGGGCGTCATCA) or M13 (GGAAACAGCTATGACCAT) tagged locus specific primer, 0.45 μM fluorescently labeled CAG or M13 tag (Boutin-Ganache et al. 2001), 1.5 mM MgCl2, 0.125 mM dNTPs, 0.5 U Taq polymerase, and approximately 2–5 ng of template DNA. Fluorophores used were VIC, PET, NED (Applied Biosystems) and FAM (Integrated DNA Technologies). Touchdown cycling parameters (Don et al. 1991) 95 °C for 5 min, 20 cycles of 95 °C for 30 s, 60 °C minus 0.5 °C per cycle for 30 s and 72 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min and a final extension of 72 °C for 10 min. Amplicon fragment sizes were analyzed on an Applied Biosystems 3730xl DNA Analyzer using LIZ500 size standard (Applied Biosystems). Allele sizes were scored using GENEMAPPER (Applied Biosystems).

Thirty-seven primer pairs were screened for amplification consistency and polymorphism in 36 hawksbill turtles captured on foraging grounds in Bocas del Toro Province, Panama, and 13 hawksbills captured while foraging in Bermuda waters. Fourteen loci amplified consistently and were polymorphic (Tables 1 and 2). Data were formatted for analysis using GMCONVERT (Faircloth 2006), and CERVUS 3.0 (Kalinowski et al. 2007) was used to calculate allelic richness (k), observed heterozygosity (HO), expected heterozygosity (HE), and deviations from Hardy–Weinberg equilibrium (HWE). We calculated linkage disequilibrium (LD) using GENEPOP (Raymond and Rousset 1995). ERIM18 and ERIM22 were out of HWE in the Panama foraging aggregation, and ERIM32 was out of HWE in the Bermuda foraging sample. No loci pairs exhibited linkage disequilibrium in either foraging aggregation. The number of alleles per locus ranged from 7 to 25, with an average of 15 alleles per locus across both foraging aggregations. This microsatellite suite has a combined non-exclusion probability of identity of 8.26 × 10−23. These markers will complement those already available for use in individual identification and other fine-scale genetic analyses.

Table 1 Characterization of microsatellite loci amplified in 36 hawksbill turtles captured on foraging grounds in Panama
Full size table
Table 2 Characterization of microsatellite loci of 13 hawksbill turtles captured on foraging grounds in Bermuda
Full size table