Dna

Cai, Yuan-Ting, Zhang, Liang, Shen, Hong-Wei, Bao, Dan-Feng & Luo, Zong-Long, 2025, Setophoma aseptata sp. nov. and new record of Minutisphaera aspera from Yuanjiang River Basin, China, Phytotaxa 702 (3), pp. 287-299 : 288

DNA Extraction, PCR Amplification and Sequencing

Fungal mycelium was scraped from the surface of colonies grown on potato dextrose agar (PDA), transferred into a 1.5 mL centrifuge tube. The TreliefTM Plant Genomic DNA Kit (TSP101-50) (Beijing Tsingke Biological Engineering Technology and Services Co., Ltd, Beijing, P.R. China) was used to extract DNA from the ground mycelium according to the manufacturer’s instructions. Three gene regions: ITS, LSU, SSU were amplified using ITS5/ITS4, LR0R/LR5, NS1/NS4 ( Vilgalys, 1990, White et al. 1990, Liu et al. 1999). The amplification was performed in a 25 μL reaction volume containing 9.5 μL deionized water, 12.5 μL 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, China), 1 μL of DNA template and 1 μL of each primer (10 μM). The amplification condition for ITS, LSU, SSU were followed Luo et al. (2018). PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were sent for sequencing at Tsingke Biological Engineering Technology and Services Company, Yunnan, China.