Comparative Genomic DNA Extraction and Labeling

Whole genomic DNAs (gDNAs) from P. ridibundus and P. lessonae were extracted from muscle tissue using the conventional phenol-chloroform-isoamylalcohol method [13 (link)]. Probes prepared from both parental species were differentially labelled either with biotin-16-dUTP (2’-Deoxyuridine, 5’-Triphosphate, Roche, Mannheim, Germany) or digoxigenin-11-dUTP (Roche) using Nick Translation Mix (Abbott Molecular, Illinois, USA or Roche Diagnostics, Mannheim, Germany). For each slide, 1 μg of P. ridibundus gDNA, 1 μg of P. lessonae gDNA and 50 μg of sonicated salmon sperm DNA (Sigma-Aldrich) were added and the resulting probe was precipitated in 96 % ethanol, washed in 70 % ethanol, air-dried and re-dissolved in 25 μl of hybridization buffer (50 % formamide, 10 % dextran sulphate, 2× SSC (Standard saline buffer), 0.04 M NaPO₄ (Sodium Phosphate) buffer, 0.1 % SDS, Denhardt’s reagent, see [29 ]). In some experiments, the final probe also included 15–30 μg of unlabelled species-specific competitive DNA prepared from P. esculentus gDNA using a Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Buckinghamshire, UK), followed by sonication of the amplified product (40 cycles, 10 pulses, 100 % power) to approximate fragment size of 100–200 bp using the ultrasonic homogenizer Sonopuls HD 2070 (Bandelin Electric, Berlin, Germany).