The six STRs markers (#022, #108, #138, #189, #278, and #279) were amplified separately as previously described (Gits-Muselli et al., 2015 (link)). Briefly, the forward primers were tagged with FAM, HEX or ATTO565 fluorophores. PCR reactions were performed on a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems) in a final volume of 20 μL. The reaction mixture was composed of 1 × AmpliTaq Gold buffer (Life technologies) with 0.25 μM of each primer, 2.5 mM of MgCl2, 0.8 μM of dNTPs, 0.25 UI of AmpliTaq Gold polymerase (Life technologies) and 2 μL of DNA. The PCR protocol was 10 min at 95°, followed by 35 cycles of 30 s at 95 °C (denaturation), 30 s at 56° (primer annealing) and 60 s at 72 °C (extension) followed by a final extension of 10 min at 72 °C. A calibration sample was used in each PCR run as an internal control. Then, 2 μL of the PCR product was prepared for fragment analysis by the addition of 18 μL of formamide (Life Technologies) and 1 μL of Genescan-500 LYZ Size Standard (Life Technologies). The lengths of the PCR fragments were determined by capillary electrophoresis (50 mm) on an ABI 3500 genetic analyser using denaturing polymer POP-7 (Life Technologies) at 60 °C. Analysis was performed with the ABI Gene mapper v4.1 software (Life Technologies).
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