Supplementary Material, Conservation Genetics Resources

Development and characterization of thirteen microsatellite markers for the longbill spearfish (Tetrapturus pfluegeri)

Supplementary Material, Conservation Genetics Resources

Bernard, A.M.1, Hilsdorf, A.W.S.2, Amorim, A.F.3, Domingues, R.R.2, Shivji, M.S.1

1 Guy Harvey Research Institute, Oceanographic Center, Nova Southeastern University, 8000 North Ocean Drive,FL, USA 33004

2University of Mogi das Cruzes, Unit of Biotechnology, PO Box 411, 08701-970 Mogi das Cruzes, SP, Brazil.

3Fisheries Institute, Av. Bartolomeu de Gusmão, 192, 11030-906 Santos, SP, Brazil (Agência Paulista de Tecnologia dos Agronegócios).

Corresponding authors:

Mahmood S. Shivji () phone: (954) 262-3657; fax: (954) 262-3600

Alexandre W.S. Hilsdorf () phone: (55)(11)4798-7210; fax: (55)(11)4798-7106

Supplementary Materials

For each microsatellite locus, the polymerase chain reaction (PCR) was carried out in 25μL reaction volumes, containing: 1μL of unquantified genomic DNA, 1X PCR buffer (containing 1.5mM MgCl2), 0.2mM of each dNTP, 0.33-0.4mM MgCl2, 0.5U of HotStar Taq DNA Polymerase (QIAGEN Inc.), and locus-specific concentrations of fluorescently labeled universal M13 primer (0.08-0.2µM) (5’-TGTAAAACGACGGCCAGT-3’) (Schuelke 2000), locus-specific Forward primer with the associated M13 tail on its 5’ end (0.2-0.4µM), and locus-specific Reverse primer (0.4µM). The amplification thermal profile for all markers was: initial denaturation at 95⁰C for 15 minute (min), followed by 35 cycles of 1min at 95⁰C, 1min at the primer set annealing temperature (Table 1), 1min at 72⁰C, and a final extension of 20min at 72⁰C. Electrophoresis was performed on an AB3130 (Applied Biosystems) genetic analyzer. Fragments were scored using LIZ 600 (Applied Biosystems) and the software GENEMAPPER 3.7 (Applied Biosystems).

Diversity statistics (HO and HE) for all 13 microsatellite loci were estimated utilizing the Excel Microsatellite Toolkit (Park 2001) and GENEPOP on the web (Raymond and Rousset 1995; Rousset 2008). Tests for both Hardy-Weinberg and linkage disequilibrium were performed using GENEPOP, implementing the Markov chain (MC) algorithm of Guo and Thompson (1992) to estimate significance (1000 dememorizations, 100 batches, and 1000 iterations per batch). The program FreeNA (Chapuis and Estoup 2007) was utilized to estimate the frequency of null alleles across all loci.

References for Supplementary Materials

Chapuis M-P, Estoup A (2007) Microsatellite null alleles and estimation of population differentiation. Mol Biol Evol 24: 621-631

Guo SW, Thompson EA (1992) Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361-372

Park SDE (2001) Trypanotolerance in West African Cattle and the Population Genetic Effects of Selection. Dissertation, University of Dublin

Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249

Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225

Rousset F (2008) GENEPOP ‘007: a complete re-implementation of the GENEPOP software for Windows and Linux. Mol Ecol Resour 8:103–106

Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233–234

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