Five new species of Hydnellum (Bankeraceae, Thelephorales) from the Changbai Mountains and Dabie Mountains, China

Total genomic DNA was extracted from dried specimens using the NuClear Plant Genomic DNA Kit (CoWin Biotech Co., Ltd., Jiangsu, China, CW0531M) according to the manufacturer’s protocols. The internal transcribed spacer (ITS) region was amplified with primers ITS1F and ITS4, and the large subunit of the nuclear ribosomal RNA gene (nrLSU) was amplified with primers LROR and LR7. Polymerase chain reaction (PCR) was performed in a 15 μL reaction system containing the following components: 1.0 μL of genomic DNA template; 7.5 μL of SanTaq® PCR Master Mix (Sangon Biotech, Shanghai, China, B532081-0020); 1.0 μL of each forward and reverse primer (10 μM); and 4.5 μL of ddH₂O. The PCR cycling parameters for the ITS region were as follows: initial denaturation at 95 °C for 3.5 min; 35 cycles of denaturation at 95 °C for 40 s, annealing at 56 °C for 45 s, and extension at 72 °C for 90 s; and a final extension at 72 °C for 10 min. The cycling parameters for nrLSUwere: initial denaturation at 94 °C for 2 min; 35 cycles of 94 °C for 40 s, 56 °C for 60 s, and 72 °C for 1.5 min; followed by a final extension at 72 °C for 8 min. The PCR amplicons were purified using a DiaSpin Kit (Sangon Biotech, B110093-0100), sent to Comate Bioscience Co., Ltd. (Jilin, China) for sequencing, assembled into consensus sequences using SeqMan (DNASTAR, v7.1), and submitted to GenBank.