Dna extraction, , PCR

Zhao, Haijun, Mapook, Ausana, Hyde, Kevin D., Doilom, Mingkwan, Al- Otibi, Fatimah O., Saichana, Natsaran & Dong, Wei, 2025, Xylolentia bambusae (Rhamphoriaceae, Rhamphoriales), a novel species associated with dead bamboo in Guangdong Province, China, Phytotaxa 681 (2), pp. 211-222 : 212

DNA extraction, PCR amplification and sequencing

A single germinated spore was cultivated on potato dextrose agar (PDA) medium at 25–28 °C for one month. Fungal genomic DNA was extracted from fresh mycelia, followed by a Maglso plant DNA isolation kit (Magen, China) manufacture instructions. DNA amplification was performed utilizing the polymerase chain reaction (PCR) technique, employing a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Three partial gene portions and two protein coding genes were used in this study: internal transcribed spacer (ITS), the large subunits of the nuclear ribosomal RNA genes (LSU), the small subunits of the nuclear ribosomal RNA (SSU), the translation elongation factor-1 alpha (tef1-α), and the second largest subunit of RNA polymerase II (rpb2). The primer pairs used were ITS5/ ITS4 ( White et al. 1990), LR0R/LR5 ( Vilgalys & Hester 1990), NS1/NS4 ( White et al. 1990), EF1-983F/EF1-2218R ( Rehner & Buckley 2005), and fRPB2-5F/fRPB2-7cR ( Liu et al. 1999), respectively. The amplifications were carried out in a 25 μL reaction volume containing 9.5 μL ddH 2 O, 12.5 μL 2 × FastTaq PCR Master Mix (Vazyme Co., People’s Republic of China), 1 μL of DNA template, and 1 μL of each primer (10 μM). The PCR thermal cycles program for the amplification of ITS, LSU, SSU, and tef1-α was commenced with an initial denaturation step at 94 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, elongation at 72 °C for 60 s, and a final extension step at 72 °C for 10 min. For rpb2 amplification, the annealing temperature was adjusted to 56 °C and the times of cycles was increased to 40. PCR products were detected on 1% agarose electrophoresis gels stained with ethidium bromide. PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red. The products with bright bands were sent to Tianyi Hui-yuan Biotechnology Co., (Guangzhou, China) for sequencing.