DNA extraction and PCR
DNA was extracted as described previously (Kratochwil & Rijli, 2014).
Briefly, we incubated fin or muscle tissue for 2h on a shaking incubator
at 55°C using 200 µg/ml proteinase K (Sigma) in 500 µl extraction buffer
(100 mM Tris–HCl pH 8.5, 200 mM NaCl, 5 mM EDTA, 0.2 % SDS) in 1.5ml
tubes. Tubes were centrifuged at 12,000–16,000 × g. 500 µl isopropanol
were added to the supernatant and centrifuged again. The supernatant was
removed, and the pellet was washed with 500 µl of 70% ethanol. The
pellet was air dried and diluted in 500 μl 10 mM Tris-Cl, pH 8.5. We did
not use EDTA as it might affect restriction enzyme activity. PCRs were
performed for eight samples (with the exception of a few cases where we
did not have enough individuals) of the target population (ingroup) and
5–8 samples that did not belong to the target population (outgroup).
For the outgroups, we tried to use representatives of each lake (for the
lake comparisons, i.e., population comparisons) or other species living
in sympatry (for the species comparisons). We did not include samples
that were part of the previously-generated genome re-sequencing dataset.
For PCRs, we used 2µl template and the standard protocol of DreamTaq DNA
Polymerases (Thermo Fisher) with a total volume of 20µl, an annealing
temperature of 58°C, 30 cycles and 30s extension time.