Multiplex PCR was used to simultaneously amplify the 16 investigated markers, as shown in Supplementary Table 1 . The amplification was performed on ABI Verity thermocycler (Life Technologies, Foster City, CA, United States). A single multiplex reaction used Master Mix QIAGEN Multiplex PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The samples were incubated at 95 °C for 15 min, followed by 35 cycles at 94 °C for 45 s, 60 °C for 90 s, and 72 °C for 1 min, with a final extension at 70 °C for 30 min.
For fragment analysis, we used capillary electrophoresis on the ABI 3130 Genetic Analyzer instrument (Life Technologies). 1.0 μL of PCR product was added to 8.5 μL of HI-DI deionized formamide (Life Technologies) and 0.5 μL of GeneScan 500 LIZ pattern size standard (Life Technologies). After data collection, samples were analyzed on the GeneMapper ID v.3.7 software (Life Technologies).
For fragment analysis, we used capillary electrophoresis on the ABI 3130 Genetic Analyzer instrument (Life Technologies). 1.0 μL of PCR product was added to 8.5 μL of HI-DI deionized formamide (Life Technologies) and 0.5 μL of GeneScan 500 LIZ pattern size standard (Life Technologies). After data collection, samples were analyzed on the GeneMapper ID v.3.7 software (Life Technologies).