Genemapper 3.5 program

A total of 24 microsatellite primer pairs were used for genotyping in a multiplex amplification system (Table 2). The selection of these markers was based on their discriminative power, simple amplification pattern, and for having independent segregation (Borba et al., 2009) (link). The reactions were prepared with Multiplex PCR Kit (Qiagen) at a final volume of 5 µL, containing 3 ng DNA, 1X Master mix, Q-solution 0.5X various primer pairs concentrations (forward and reverse) according to Borba et al. (2009) (link), and RNase-free water. The amplification reactions were conducted in a thermocycler 9700 (Life Technologies) with the following schedule: initial denaturation at 95°C for 15 min, followed by 40 amplification cycles, with each cycle consisting of denaturation at 94°C for 30 s, annealing at 56°C for 90 s, and extension at 72°C for 90 s; and, finally, a final extension at 72°C for 10 min. The amplification products were separated by capillary electrophoresis on an ABI 3100 DNA sequencer (Life Technologies). Locus genotyping (in bp) was performed through the GeneMapper 3.5 program (Applied Biosystems), using the ROX 500 internal molecular weight marker (Life Technologies).