Dna

DNA Extraction, PCR Amplification, and Sequencing

Fungal genomic DNA was extracted from 20-day-old mycelia using the Trelief™ Plant Genomic DNA Kit ( Beijing Qingke Biotech), following the manufacturer’s instructions.Polymerase chain reaction (PCR) was performed to amplify the internal transcribed spacer (ITS) region, the large subunit ribosomal RNA (LSU), the second largest subunit of RNA polymerase II ( rpb 2), and beta-tubulin ( tub 2) genes using the primer pairs ITS9mun/ITS4_KYo1 ( Toju et al. 2012), LR0R ( Hopple & Vilgalys 1999)/ LR5 ( Cubeta et al. 1991), fRPB2-7cR/fRPB2-5F ( Voglmayr et al. 2016), and TI/T22 (O'Donnel & Cigelnik 1997), respectively. The PCR reaction was set up at 30 μL volume, containing 15 μL of PCR Master Mix (CoWin Biosciences, Taizhou, China), 11 μL of ddH 2 O, 1 μL each of forward and reverse primers (10 μM), and 2 μL of DNA template. The PCR conditions were performed as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for a suitable time, extension at 72 °C for 2 min, and a final extension of 72 °C for 10 min. The annealing time of each gene was 50 s for ITS, LSU and tub 2; 2 min for rpb 2. The PCR products were analyzed by 1% agarose gel electrophoresis, and Sanger sequencing was carried out by Sangon Biotech Co., Ltd. Raw sequence data were processed using SeqMan v.7.1.0 software, which was used to assess chromatogram quality, trim low-quality bases at terminals, and assemble bidirectional sequencing reads.