Illumina HiSeq 2000 paired end sequencing
Genomic DNA was extracted from females and males separately in pools of 100 individuals using an insect-specific modification of Qiagen's Genomic Tip 20/G protocol (available at https://www.qiagen.com/gb/resources/resourcedetail?id=b45c3cc3-7f2b-4f4a-aa37-21d814ed3730&amp;lang=en). In brief, pools of individuals were homogenised twice for 30 seconds in 75 µl of PBS using a TissuelyserTM at a frequency of 25 Hz/s. Aliquots of 1.8 ml of lysis buffer (20 mM EDTA, 100 mM NaCl, 1% Triton X-100, 500 mM Guanidine-HCl, 10 mM Tris-HCl pH 7.9) with RNase A (final concentration 20 µg/ml) were added to the homogenised material and incubated at 37 ˚C for 30 minutes. Proteinase K (final concentration 0.8 mg/ml) was then added and the solution was further incubated at 50 ˚C for 2 hours mixing from time to time. The samples were centrifuged for 20 minutes at 11000 rpm to pellet the debris, and the supernatant (lysate) transferred to a Genomic Tip (20/G) previously equilibrated with 1 ml of QBT buffer. The lysate was allowed to flow through the column by gravity. The column was then washed 4 times with 1 ml of Buffer QC by gravity flow. The DNA was eluted with 0.8 ml of buffer QF (pre-warmed at 50 ˚C) by gravity flow and then precipitated with 0.7 volumes of isopropanol, followed by a 20 minutes' centrifugation at 4 ˚C at 15000g. The DNA pellet was washed with 70% ethanol, air-dried and resuspended in 25 μl of 0.1 x TE. DNA extracted from each sex was pooled and the concentration was measured using the Broad Range dsDNA assay in a Qubit 2.0 Fluorometer (Life Technologies). The integrity of the genomic DNA was visualised using a 5 μl sample on a 1% (w/v) agarose gel.