2.2 DNA extraction, library preparation, and sequencing

Total genomic DNA was extracted from mesothorax muscle tissues of each beetle for library preparation and sequencing using the TruSeq DNA Sample Preparation kit (Illumina, USA)according to the manufacturer’s instructions. Sequencing libraries were generated using the Truseq Nano DNA HT Sample Preparation kit (Illumina, USA) following the manufacturer’s recommendations, and index codes were added to label sequences from each sample. For each individual, the DNA sample was sheared into fragments of 350 bp fragments by using a Covaris S2 (Covaris). The DNA fragments were end-polished, A-tailed, and ligated with a full-length adapter for Illumina sequencing with further PCR amplification. The PCR products were purified (AMPure XP system), and the libraries were analyzed for size distribution using an Agilent 2100 Bioanalyzer and quantified using real-time PCR. The DNA fragments were then processed and sequenced using the Illumina HiSeq PE150 platform. Quality control was achieved by removing low costs and low-quality paired reads (reads with ≥10% unidentified nucleotides; >10 nt aligned to the adaptor, allowing ≤10% mismatches; >50% of bases having phred quality <5; and putative PCR duplicates generated in the library construction process), which mainly resulted from base-calling duplicates and adaptor contamination.