RAD-Seq of backcross between Aphelinus rhamni control and selection lines
Genomic DNA was extracted from pools of wasps from each population using Qiagen DNeasy Blood and Tissue Kits (Qiagen, Valencia, CA), following the standard protocol. The resulting DNA was digested with restriction endonucleases using one rare cutter (NgoMIV with a 6 bp recognition site) and one frequent cutter (CviQI with a 4 bp recognition site) (New England Biolabs, Inc., Ipswich, MA), which together determined the number of unique locations of fragments across the genome and the lengths of these fragments. Custom adaptors, with barcodes for each population that also served to register clusters on the Illumina HiSeq during sequencing, were ligated onto the fragments using T4 ligase (New England Biolabs, Inc., Ipswich, MA). The ligates were pooled and purified using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN). The purified ligate was separated into 10 aliquots that were amplified in separate PCR reactions to both increase copy number at each locus and add more adaptor sequence for sequencing. The adaptors were designed so that the only fragments that amplify would have the rare-common combination of cut sites. After PCR, the products were pooled and then size-selected (300-350 bp) using the BluePippin system (Sage Science, Beverly, MA). After quantification with qPCR, the resulting fragments were sequenced for ~100 nucleotides in single-end reads an Illumina HiSeq 2500 (Illumina, San Diego, CA) at the Delaware Biotechnology Institute.