Illumina NovaSeq X Plus paired end sequencing
Whole gametophyte tissue, grown in culture, was harvested and frozen at -80�C. The tissue was disaggregated in a microtube using a micro-pestle and DNA was extracted using the NucleoSpin 96 Plant II kit (Macherey-Nagel GmbH & Co. KG, Germany). The lysis step was carried out for 30 min at room temperature with strong agitation to resuspend all the disrupted tissue. Extraction was carried out according to the manufacturer�s instructions except that one additional wash with buffer PW2 was carried out to more effectively remove PCR inhibitors and polysaccharides. DNA was eluted in 60 �L of PE Buffer pre-heated to 70�C. DNA concentration was measured using PicoGreen (Fisher Scientific) and DNA integrity was evaluated on a 1.5% agarose gel. Aliquots of 40 �L of genomic DNA were then digested with PstI and MseI (New England Biolabs, https://www.neb.com). These enzymes had been chosen based on in silico digestion simulations carried out with the R package SimRAD. DNA was 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. The concentration of each library was checked on a 1.5% agarose gel. The libraries were pooled into three pools (low, medium and high concentration) and concentrated with AMPure XP beads (Beckman Coulter Genomics). The concentration of each pool was measured using the Qubit DNA HS kit (Invitrogen) to allow equimolar pooling. Fragments of between 480 and 980 bp were then selected with a Pippin-Prep kit (Sage Science, Beverly, MA, USA). An Agilent 2100 Bioanalyzer (Agilent Technologies) was used to evaluate the insert size range of the final library and the library was quantified using the Qubit DNA HS kit (Invitrogen). The pooled ddRAD library was sequenced (150 bp paired-end reads) on a partial lane of and Illumina NovaSeq X Plus Series platform. The ddRAD protocol is described here: https://dx.doi.org/10.17504/protocols.io.rm7vzjo68lx1/v3. INDEX_01 ( ATCACG ), barcode: ACGCATT