2.4. DNA Library Preparation for Genotyping by ddRADseq

The ddRADseq DNA library was prepared according to the method of Peterson et al. [41]. The composition of the restriction mix of each P. cactorum isolate was: 300 ng of sample DNA, 5 U of MspI and 7 U of Sau3AI endonucleases and 1 × Tango buffer (all components from Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA), added to nuclease-free water to 30 µL. The reaction was performed in a thermocycler at 37 °C for three hours. Single-stranded oligos for the adapter were synthesised by the MilliporeSigma(Burlington, Massachusetts, USA) according to a template proposed by Peterson et al. [41], of which ends were redesigned to meet the MspI and Sau3AI restriction sites’ characteristics. The double-stranded adapters (one for MspI and twelve for Sau3AI) were prepared according to Peterson et al. [41]. The adapters’ ligation onto DNA fragments prepared in a restriction reaction was carried out in a mixture containing 40 ng of fragmented DNA, with both adapter stocks in a final concentration of 0.075 pmol/µL of Sau3AI and 0.041 pmol/µL of MspI adapters, 1 × ligation buffer (Thermo Fisher Scientific, Inc.) and 5 U of T4 DNA ligase (Thermo Fisher Scientific, Inc.) added to nuclease-free water to 40 µL. The reaction conditions were: 23 °C/60 min, 65 °C/10 min, and then the mixture was cooled by 0.6 °C/min until the temperature reached 0 °C. The length selection of DNA fragments in twelve partial DNA libraries was carried out using Pippin prep (Sage Science, Inc., New Castel upon Tyne, United Kingdom), and the selection window was set to 170–370 bp. Where required by protocol, the samples were purified using AMPure XP beads (Beckman Coulter, Brea, California, USA). The DNA concentration in partial libraries was increased by PCR using primers complementary to adapter sequences (one for the Sau3AI and twelve for the MspI adapter). Using the combination of twelve adapters and twelve primers, each sample was characterised by a unique combination of barcodes on both ends of the DNA fragments. The PCR master mix included 20 ng of DNA, 0.5 µM of both primers, 20 µM of dNTP mix, 1 U of Phusion DNA polymerase and 1 × final of Phusion HF buffer (New England BioLabs, Inc., Ipswitch, Massachusetss, USA). The PCR conditions were: 98 °C/45 s, 10 cycles consisting of 98 °C/10 s, 58 °C/10 and 72 °C/15 s and 72 °C/5 min as a final extension. The content of DNA fragments and their length distribution in each partial DNA library was evaluated by Agilent TapeStation (Agilent Technologies, Inc., Santa Clara, California, USA), and the concentration was fluorimetrically measured. The total library was finalised by an equimolar mixture of all twelve partial libraries.

The sequencing on an Illumina MiSeq device was processed by the Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (Vestec, Czech Republic) using a MiSeq Reagent Kit v3 (Illumina, Inc., San Diego, CA, USA).