Spin column protocol

DNA extraction for tissue samples was done using the DNeasy Blood & Tissue Kit (Qiagen) following the protocol for purification of total DNA from animal tissues (Spin‐Column Protocol, Qiagen). Pieces of 25 mg consisting of muscle, tendon, bone, or some hair were cut off from the samples and stored in 2 ml tubes at −20°C until further analysis. DNA concentration was assessed using quantus (QuantiFluor ONE System, Promega). The quality of the DNA samples was estimated with nanodrop 2000 (Thermo Fisher Scientific), based on the 260/280 nm ratio and concentration of the DNA, and quantitatively by the spectrum observed. Additionally, DNA quality was checked for all samples through electrophoresis using EZ‐Vision Blue Light Dye (Amresco, LLC) on a 1% agarose gel.
Based on the concentrations assessed using quantus (Promega), DNA samples were diluted to 2.5 ng/μl. Genotyping of tissue samples followed the protocol described above for NiG samples.

DNA from 34 P. liolepis lizards from the studied populations was extracted by complete digestion of a piece of tail muscle using the DNeasy Blood and Tissue kit following the manufacturer’s recommended procedures (Spin-Column Protocol, Qiagen). In order to assign individuals to a mitochondrial DNA lineage, we sequenced the ND4 gene in all analyzed individuals. Polymerase chain reaction (PCR) amplifications were performed in a 10 μl reaction volume containing 1 μl DNA solution, 5 μl of Quiagen Multiplex Kit and 1 μl of primers at 2 μM. PCRs started with an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 90 s and extension at 72°C for 1 min. All reactions were finished with a final extension at 72°C for 7 min. Successful PCRs were selected on ethidium-bromide stained agarose gels.
All sequences were obtained for both the reverse and forward primers. Alignment was performed manually using CodonCode Aligner 3.9 (Codon Code Corporation) and automatic base calls were checked along the sequence, both where the two sequences were in disagreement and elsewhere. Genetic structure based on the ND4 mitochondrial marker was estimated with Maximum-likelihood based on the Tamura-Nei model [90 (link)] in MEGA, version 4.1 [91 (link)].

We studied the genomic DNA integrity of the liver of all the studied rabbits. In accordance with the purification protocol of total DNA from rabbits^, tissues, DNA extraction was processed using (Spin-Column Protocol, QIAGEN Group). Agarose gel (2% agarose gel in 1x TAE buffer) was prepared according to Raj Kumar [24 ].