Illumina HiSeq 2500 paired end sequencing
A 17 mm tissue disc from the meristematic region of the sporophyte blade was dried in silica gel for storage. 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 10 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 60�C. The integrity of genomic DNA integrity 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 60 �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 150 and 600 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 a total of five lanes of an Illumina HiSeq 2500 (chemistry V4) platform to produce 966 million raw reads. After filtering and trimming, 660 million reads were retained giving an average of 3.5 million reads per sample and a mean coverage per sample of 16x.