2.2.1 RADseq Library Preparation and Sequencing
RADseq libraries were prepared from the isolated DNA as described
following (Grewe et al., 2017). In summary, DNA isolations were pooled
with sequence adapters (Rubin and Moreau, 2016), digested with the
restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA) and
ligated using T4 ligase (New England Biolabs). All samples with
compatible barcodes were pooled and selected for fragment sizes between
300 and 500 bp using the BluePippin DNA size selection system (Sage
Science, Beverly, MA, USA). The pooled libraries were amplified using
the REDTaq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) prior to
sequencing on an Illumina MiSeq using the MiSeq Reagent Kit v3 for 150
cycles (Illumina, San Diego, CA, USA) to produce single-end sequences
with a length of 150 bp.