Microsatellite genotyping
DNA was extracted from tissue and blood using the Sherlock AX Kit (A&A
Biotechnology, Gdynia, Poland), following the manufacturer’s protocol.
The extracts were quantified with a NanoDrop 2000 spectrophotometer
(Thermo Scientific, Wilmington, DE, USA). All individuals were genotyped
with a panel of 16 polymorphic microsatellite loci (S090, SW72, S155,
S026, S355, S215, SW951, SW857, SW24, SW122, IGF1, SW461, SW1492,
SW2021, SW2496, SW2532), which had been successfully used to study
relatedness and genetic variation in wild boar populations from the
study area (Podgórski et al., 2014a; Podgórski et al., 2014b). The 16
autosomal STR loci were amplified in two independent multiplexed mixes
using Qiagen Master Mix (Qiagen Inc, Hilden, Germany) reagents and
fluorescently label primers. For the first panel, the fluorochromes were
used: 6-FAM for the loci SW72, SW857; VIC for SW1492, IGF1; NED for
S026, S215; PET for SW2021, SW2532 and for second panel 6-FAM for SW122,
S0355 ; VIC for SW461, SW2496; NED for W24, S0155 and PET for SW951 and
S0090. PCR was performed on Veriti® Thermal Cycler amplifier (Applied
Biosystems, Foster City, CA, USA) using the following thermal profile: 5
min. of initial DNA denaturation at 95°C, followed by 35 cycles of
denaturation at 95°C for 30 s, annealing at 56°C for 90 s, elongation of
starters at 72°C for 30 s and final elongation of starters at 60°C for
30 min. Analysis of the obtained PCR products was performed using an ABI
3130xl capillary sequencer ( Applied Biosystems, Foster
City, CA, USA) at the Institute of National Research Institute of Animal
Production (Cracow, Poland). The amplified DNA fragments were subjected
to electrophoresis in 7% denaturing POP-7 polyacrylamide gel in the
presence of a standard length of 500 Liz and a reference sample. The
results of the electrophoretic separation were analysed automatically
using the GeneMapper ® Software 4.0 ( Applied Biosystems, Foster
City, CA, USA).