Dna extraction, , PCR

He, Yong-Ze, 2025, Neopyrenochaetopsis machili sp. nov. on Machilus nanmu from Shanxi Province, China, Phytotaxa 696 (3), pp. 224-234 : 225

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol a kit method (OGPLF-400, GeneOnBio Corporation, Changchun, China) take for sanger sequencing ( Guo et al. 2000; Zhao et al. 2023). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), the partial large subunit (LSU) nrRNA, part of the beta-tubulin gene region (tub), and partial RNA polymerase II second largest subunit (rpb2) genes were amplified and sequenced by using primers pairs ITS5/ITS4 ( White et al. 1990), LR0R/LR5 ( Rehner & Samuels 1994, Vilgalys & Hester 1990), Bt2a/Bt2b ( Glass & Donaldson 1995), fRPB2- 5F/fRPB2-7cR ( Liu et al. 1999, Sung et al. 2007).

The PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume, which contained 12.5 μL Green Taq Mix ( Vazyme , Nanjing , China), 1 μL of each forward and reverse primer (10 μM) ( TsingKe , Qingdao , China), and 1 μL template genomic DNA in amplifier, adjusted with distilled deionized water to a total volume of 25 μL The PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 50 s, extension at 72 °C for 1 min and a final elongation step at 72°C for 10 min. Annealing temperature for each gene were 55 °C for ITS, 52 °C for LSU, 53 °C for tub and 56 °C for rpb2. The PCR products were separated with the 1 % agarose gel, and add GelRed and use UV light to visualize the fragments. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 ( Kumar et al. 2016). New sequences generated in this study were deposited at NCBI’s GenBank (www.ncbi.nlm.nih.gov; Table 1) .