RRL sequencing
Mallard DNA samples were prepared from ethanol preserved whole blood collected from nine individuals from three locations across Europe: two females and a male each from Doñana (Spain), Northern Netherlands and Ottenby (Sweden). Each of these individuals was either directly caught from the wild, or was a first generation descendant from local wild mallard parents. DNA extraction was performed using the Gentra Systems Puregene DNA purification Kit according to the manufacturer’s instructions. Briefly, ~200µl blood was digested with 9 µg Proteinase K (Sigma) in Cell Lysis Solution (Gentra Systems) at 55ºC over night. Proteins were subsequently precipitated with Protein Precipitation Solution (Gentra Systems) and spun down. DNA from the supernatant was precipitated with isopropanol and washed twice with 70% ethanol. DNA quantity and purity were measured using the Nanodrop ND1000. Possible degradation was inspected on an agarose gel and only high quality DNA samples were used to prepare the DNA pool. Equal amounts of DNA from the nine mallards were combined into two pools of 25 µg each. Aliquots of 5 µg for each pool were digested with either AluI or HhaI (10 units per reaction, Pharmacia). The digested pools in O’range loading dye (Fermentas) were size-fractionated on precast 10% polyacrylamide in 1xTBE with the Criterion™ Cell (BioRad). The gel was run 190 minutes at 100 volt and stained for 30 minutes in ethidium bromide solution. After staining, the target fragment size range between 110-130bp was sliced out of the gel. The gel slice was sheared by nesting a 0.5ml Eppendorf tube (with a hole in the bottom formed with a needle) containing the gel slice inside a 2ml Eppendorf tube, and centrifuged at 14000 rpm for 2 minutes. The sheared gel pieces were covered with 300µl DNA recovery buffer (8mM Tris pH 8.0, 0.08 mM EDTA, 1.25M ammonium acetate), vortexed, and eluted at 4°C overnight, followed by 15 minutes incubation at 65°C. The slurry was divided over two Montage DNA gel extraction devices (Millipore) and centrifuged at 5000g for 10 minutes to purify the eluted gel. DNA was precipitated by adding 1/10 volume 3M sodium acetate pH 5.2, 1 volume isopropanol and 1/500 volume glycogen, washed with ethanol and resuspended in DNA hydration solution (Gentra Systems). The genomic libraries were combined.