title: HiPlex pool-Seq of Lolium populations
design: 185 genomic regions of 80-140 bp positioned in, or around, 42 genes were resequenced using Highly Multiplex Amplicon Sequencing (HiPlex dataset). HiPlex primers were designed with Primer3 (Untergasser et al., 2012) using prior knowledge of allelic sequence variation (Veeckman et al., 2018). The primer pairs were then divided into two highly multiplex PCR-reactions according to their amplification efficiency (Supporting Information, Table S1). Per population, leaf material of around 300 individual seedlings were pooled. Genomic DNA was extracted using the CTAB method of Murray & Thompson (1980) and DNA concentration was measured using the Quantus double-stranded DNA assay (Promega, Madison, WI, USA). Per sample, the final DNA concentration was adjusted to 40 ng/L and the amplicons were PCR-amplified while adding sample-specific indices. Each DNA extract was amplified in 3 replicate PCRs. All PCR-products of all samples were pooled before final adapter ligation using the KAPA Hyper Prep PCR-free Kit according to manufacturer directions (Kapa Biosystems, USA). The libraries were sequenced with 2x150 PE on a HiSeq2500 instrument in rapid run mode. Forward and reverse reads are merged with PEAR (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933873/) and all read data of the three PCR replicates are merged per sample, and adapter sequences were removed.