Fungal DNA Extraction and Sequencing Protocol

Fungi were grown on PDA for 20–30 days at 25°C. A Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Hangzhou, China) was used to extract total genomic DNA from fresh mycelia according to the manufacturer’s instructions. DNA amplification was performed by polymerase chain reaction (PCR). LSU, SSU, ITS and RPB2 gene regions were amplified using the primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4 and RPB2-5F/RPB2-7cR, respectively (Vilgalys and Hester, 1990 (link); White et al., 1990 (link); Rehner and Samuels, 1994 (link); Liu et al., 1999 (link)). The amplifications were carried out in a 25 μL reaction volume containing 9.5 μL ddH₂O, 12.5 μL 2 × PCR Master Mix, 1 μL DNA template, 1 μL each primer (10 μM). The PCR thermal cycles for the amplification of the gene regions followed the methods in Jeewon et al. (2004) ; Réblová et al. (2011) (link), and Su et al. (2015) (link). PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red. The sequencing reactions were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.