Dna extraction, , PCR

DNA extraction, PCR amplification, and sequencing

Fresh mycelia were scraped from living cultures, which grew and incubated at room temperature for 15 days. Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) was used to extract DNA , following the manufacturer’s protocol. The extracted DNA was maintained at –20 °C for long-term storage. The polymerase chain reactions (PCR) were performed for three genes: internal transcribed spacer (ITS), RNA polymerase II subunit 2 ( RPB2), and translation elongation factor 1-alpha ( TEF 1-α) gene, using the primers ITS5 and ITS4 ( White et al. 1990), fRPB2-5F and fPB2-7R ( Liu et al. 1999), and EF1 and EF2 ( O’Donnell et al. 1998), respectively. The PCR amplifications were carried out in a 25 µL reaction volume, containing 12.5 µL of 2 × Power Taq PCR Master Mix ( Beijing Bomaide Biotechnology Co., Ltd., Haidian District, Beijing, China), 8.5 µL distilled-deionized water (ddH 2 O), 2 µL of DNA template, and 1 µL of each forward and reverse primer ( Tibpromma et al. 2018).

The amplification condition for ITS and TEF 1-α consisted of initial denaturation at 94 °C for 2 min, followed by 35 cycles of 30 s denaturation at 95 °C, 50 s annealing at 55 °C, and 1.5 min extension at 72 °C, and a final extension period of 10 min at 72 °C. The amplification condition for RPB2 consisted of initial denaturation at 94 °C for 2 min, followed by 35 cycles of 45 s denaturation at 95 °C, 50 s annealing at 58 °C, and 1.5 min extension at 72 °C, and a final extension period of 10 min at 72 °C. The PCR products were detected by 1% agarose gel electrophoresis stained with TS-GelRed (TSJ002, Beijing Kinco Biotechnology Co., Ltd. Kunming Branch, P.R. China). Purification and sequencing of PCR products were performed at the Tsingke Biological Engineering Technology and Services Co., Ltd ( Yunnan, P.R. China).