2.5. CRISPR amplification and sequencing

Complete CR1, CR2, and CR3 arrays were sequenced for the 36 E. amylovora isolates, using primers detailed in Table 2. The PCR cycling parameters were 5 min at 94°C, followed by 40 cycles of 94°C for 30 s, 58°C or 55°C (CR1/2, and CR3 respectively) for 30 s, 72°C for 4 min or 45 s (CR1/2, and CR3 respectively), and a final extension at 72°C for 7 min. PCR reactions were established in a final volume of 50 μL containing 1x DreamTaq Buffer with 2.0 mM MgCl₂ (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2 mM of dNTPs (GRiSP, Porto, Portugal), 0.4 μM of each primer, 1U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and 10 ng of DNA template. PCR amplicons were separated by electrophoresis in a 0.8% agarose gel stained with GreenSafe Premium (NZYTech, Lisbon, Portugal), at a constant voltage (90V) in 1x TE buffer, and purified using the Illustra GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Chicago, Illinois, USA) following manufacturer’s instructions. Sequencing of the three CRISPR arrays was outsourced to STAB Vida (Costa da Caparica, Lisbon, Portugal), and primer walking sequencing was employed to obtain the full sequence of the CR1/2 arrays.