Computational protocol: The spatial scale of genetic subdivision in populations of Ifremeria nautilei, a hydrothermal-vent gastropod from the southwest Pacific

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[…] The mitochondrial COI (404-bp segment) region was amplified with the species-specific primers COI-3 and COI-6 [] as follows: 10-100 ng DNA template, 10 × PCR Buffer (20 mM Tris, pH 8.8; 50 mM KCl; 0.01% Triton X-100; 0.02 mg/ml BSA), 2 mM MgCl2, 0.2 mM dNTP's, 0.5 μM each primer, and 1 unit Taq polymerase (Bioline: Taunton, MA) in a 20 μl final volume. Reaction conditions were as follows: 94°C for 1 minute; 30 cycles of 92°C for 40 s, 50°C for 60 s, 72°C for 90 s; final extension of 72°C for 5 min. Amplicons were verified on 1.8% agarose gels. To remove unincorporated nucleotides, 14 μl of PCR product was incubated with 0.2 μl 10 × ExoAP buffer (50 mM Bis-Tris, 1 mM MgCl2, 0.1 mM ZnSO4), 0.05 μl Antarctic Phosphatase (New England Biolabs: Ipswich, MA), 0.05 μl Exonuclease I (New England Biolabs: Ipswich, MA) at 37°C for 60 min followed by 85°C for 15 min to inactivate the enzymes. Bi-directional sequencing reactions were performed using the manufacturer's protocol for Big Dye Terminator Reaction (Applied Biosystems: Foster City, CA). Sequenced PCR product was purified using AMPure magnetic bead system (Agencourt: Morrisville, NC) following manufacturer's protocol, analyzed on an ABI 3730xl DNA Analyzer (Applied Biosystems International), and edited with Sequencher version 4.7 (Gene Codes: Ann Arbor, MI). Consensus sequences were compared against the NCBI GenBank database to confirm species identity [] and aligned using the MUSCLE alignment algorithm []. A sequence for each unique haplotype was deposited in GenBank (North Fiji and Lau haplotypes - accession # JQ074110 to JQ074134; Manus Basin haplotypes - accession # JQ074135 to JQ074170).Neighbor-joining phylograms of aligned mitochondrial sequences were assembled in MEGA version 4 [] with an Alviniconcha sp. 2 as an outgroup. Statistical-parsimony networks were assembled in TCS version 1.21 (default settings; []). Arlequin version 3.11, [] was used to estimate haplotype (H), nucleotide diversity (π), Fu's Fs, and pairwise φST. [...] Nine microsatellite markers (Ifr040, Ifr043, Ifr052, Ifr068, Ifr078, Ifr086, Ifr093, Ifr094, and Ifr103) were amplified from Manus, North Fiji, and Lau Basin samples following methods reported in Thaler et al. []. To assess marker quality, allelic richness and divergence from Hardy-Weinberg Equilibrium (HWE) were calculated in GENEPOP (version 4.0, []). Permutation tests to determine significant variation in allelic richness were conducted in F-stat (version 2.9.3.2; []). Departures from HWE toward heterozygote excess or deficiency were assessed for each locus using GENEPOP exact tests. Loci were screened using LOSITAN to test for the potential influence of selection (25,000 simulations; []). Microsatellite markers that showed deviations from HWE expectations or found to be under the influence of selection were excluded from subsequent analyses. Identity tests (PID and PSIB) were used to indicate whether a given set of microsatellites contains sufficient information to be useful for assessing population structure [,]. PID and PSIB were calculated for all useful sets of microsatellite markers (Gimlet version 1.3.3; []). […]

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