Dna extraction

DNA extraction , amplification, and sequencing

Total genomic DNA was extracted from dried materials using a CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Company, Limited, Beijing, China) according to the manufacturer’s instructions, with some modifications ( Wu et al. 2022). The internal transcribed spacer (ITS) and large subunit nuclear ribosomal RNA gene (nLSU) were amplified using the primer pairs ITS5/ITS4 and LR0R/LR7 ( White et al. 1990) (https://sites.duke. edu/vilgalyslab/rdna_primers_for_fungi/). Part of tef 1-α was amplified with primer pairs EF1-983F and EF1-1567R ( Rehner & Buckley 2005).

The PCR procedure for ITS and tef 1-α was as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for ITS and 54 °C for tef 1-α for 45 s and 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 34 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. The PCR products were purified and sequenced using the same primers at the Beijing Genomics Institute in China. DNA sequencing was performed at the Beijing Genomics Institute, and the newly generated sequences were deposited in the GenBank database. All sequences analyzed in this study are listed in Table 1. Sequences generated from this study were aligned with additional sequences downloaded from GenBank using BioEdit ( Hall 1999) and ClustalX ( Thompson et al. 1997). The final ITS, nLSU, and tef 1-α datasets were subsequently aligned using MAFFT v.7 under the E-INS-i strategy, with no cost for opening gaps and equal cost for transformations (command line: mafft—genafpair—maxiterate 1000) ( Katoh & Standley 2013). The alignments were then visualized in BioEdit ( Hall 1999).