GBS pool-Seq of Lolium populations
Per population, leaf material of around 300 individual seedlings were pooled. Genomic DNA was extracted with a CTAB-based method. GBS libraries were created with a PstI single-enzyme GBS protocol modified from Elshire et al. (2011) and Byrne et al. (2013). 100 ng of genomic DNA was digested with PstI (New England Biolabs), and barcoded adapters were ligated with T4 ligase (New England Biolabs). Ligation products were purified with 1xAMPure magnetic beads (Beckman Coulter). Each ligation product was split and amplified in 3 replicate PCRs using indexed Illumina Flowcell binding site primers and high-fidelity Phusion DNA polymerase (New England Biolabs). Individual PCR replicates were purified using 1xAMPure beads and quantified using Qubit fluorescence spectrophotometry, then pooled equally for each replicate PCR set. GBS libraries were single-end sequenced on an Illumina Hiseq2000 instrument (IBERS), until a mimimum of 10M reads were obtained per sample. All read data of the PCR replicates are merged per sample, and quality trimmed (maximal length of 86 bp).