2.2. DNA Extraction and Procedures

Total genomic DNA was extracted from 0.5 mg of mycelia obtained from seven-day-old Fusarium oxysporum f.sp. apii monosporic, cultured on ¼ PDA using the standard cetyltrimethylammonium bromide (CTAB) protocol [19]. The DNA pellet was dissolved in 50 μL of TE buffer. The DNA concentration and purity were determined using a NanoDrop One C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The following loci were amplified using polymerase chain reaction (PCR): the 700-bp partial CDS of the translation elongation factor 1-alpha (EF-1) gene (TEF-1α) [20] and a fragment of 970-bp including the partial sequence of the 28S ribosomal RNA gene, the complete sequence of the intergenic spacer 28S-18S ribosomal RNA and the partial sequence of the 18S ribosomal RNA gene (IGS rDNA) [16,21] (Table S1). The PCR reaction was performed in a 25 μL mixture with 0.25 μL DreamTaq DNA Polymerase [5 U/μL] (Thermo Sientific, Waltham, MA, USA, Ref. EP0702), 2.5 μL 10× Dream Taq Buffer (included [20 mM] MgCl₂, Thermo Scientific), 2.5 μL dNTP Mix [each 2 mM] (Thermo Scientific Ref. R0241), 1.25 μL of each forward and reverse primers [10 nM], 1 μL of the template DNA [10–30 ng] and 16.25 μL sterile ultrapure water. PCR program consisted of an initial denaturing step of 95 °C for 120 s, followed by 35 cycles of 96 °C for 60 s, 52 °C (TEF-1α) and 57 °C (IGS) for 60 s, and 72 °C for 60 s, and a final extension at 72 °C for 10 min on a Mastercycler Pro Thermal (Eppendorf, Hamburg, Germany).

The amplified products were purified with the Exonuclease I (ExoSapI) (Thermo Scientific, Waltham, MA, USA, ref. EN0582) and sequenced at Macrogen Inc., Gangnam-gu, Seoul, Republic of Korea. All sequences were manually checked for quality, trimmed, and aligned with either ClustalW2 or Muscle. All polymorphisms were checked manually. NCBI GenBank accession numbers for genetic variable isolates are {"type":"entrez-nucleotide-range","attrs":{"text":"OR640020 to OR640041","start_term":"OR640020","end_term":"OR640041","start_term_id":"2599448646","end_term_id":"2599448688"}}OR640020 to OR640041 for the TEF-1α and {"type":"entrez-nucleotide-range","attrs":{"text":"OR626669 to OR626690","start_term":"OR626669","end_term":"OR626690","start_term_id":"2599448586","end_term_id":"2599448607"}}OR626669 to OR626690 for IGS rDNA.