Illumina HiSeq 2500 paired end sequencing
Whole gametophyte tissue, grown in culture, was harvested and frozen at -80�C. The tissue was disrupted using a TissuLyzer II (Qiagen) (four pulses of 45 sec) and DNA was extracted using the NucleoSpin� 96 Plant II kit (Macherey-Nagel GmbH & Co. KG, Germany). The lysis step was carried out for 15 min at 60�C with strong agitation to resuspend all the disrupted tissue. Extraction was carried out according to manufacturer�s instructions except that we carried out one additional wash with buffer PW1 and one additional wash with buffer PW2 to more effectively remove PCR inhibitors and polysaccharides. DNA was eluted twice with 100 �L of PE Buffer pre-heated to 70�C. The genomic DNA extracts were purified using the NucleoSpin gDNA Clean-up XS, Micro kit for DNA clean up and concentration (Macherey-Nagel GmbH & Co. KG, Germany). The purifications were performed according to the manufacturer's instructions with elution into 30 _L (2 x 15 �L) of the supplied elution buffer. The integrity of genomic DNA was assessed by electrophoresis on an agarose gel. DNA concentration was measured using PicoGreen (Fisher Scientific) and adjusted to 2.5 ng.�L-1. The genomic DNA was then digested with PstI and MseI (New England Biolabs, https://www.neb.com), which had been chosen based on in silico digestion simulations carried out with the R package SimRAD. Samples of 100 ng of DNA were digested overnight at 37�C with 10 U MseI and 10 U PstI in 1X CutSmart buffer (total volume of 50 �L). Digested DNA was purified with AMPure XP beads (Beckman Coulter Genomics). Adapters were added using T4 DNA ligase (New England Biolabs, https://www.neb.com/). Illumina flowcell annealing sequences, multiplexing indices and sequencing primer annealing regions were added to all fragments bearing adapters by PCR amplification using the Q5 Hot Start High-Fidelity DNA polymerase kit (New England Biolabs) so that each individual library could be identified with a unique combination of barcode and index. Aliquots (5 �L) of each library were pooled and the pools precipitated with ethanol (2.5 vol. ethanol, 0.1 vol. 3M sodium acetate pH 5.2). Fragments of between 480 and 930 bp were then selected with a Pippin-Prep kit (Sage Science, Beverly, MA, USA). Both an Agilent 2100 Bioanalyzer (Agilent Technologies) and qPCR were used to quantify the pooled library. The pooled ddRAD library was sequenced (125 bp paired-end reads) on an Illumina HiSeq 2500 (chemistry V4) platform. The ddRAD protocol is described here: https://dx.doi.org/10.17504/protocols.io.rm7vzjo68lx1/v3.