Dna extraction, , PCR

Gao, Ying, Tibpromma, Saowaluck, Mckenzie, Eric H. C., Eungwanichayapant, Prapassorn D., Gautam, Ajay Kumar, Lu, Li, Jayawardena, Ruvishika S., Hyde, Kevin D., Xu, Jian-Chu & Gui, Heng, 2025, Two new species of Puccinia (Pucciniaceae, Pucciniales) from herbaceous plants in Yunnan Province, China, Phytotaxa 694 (1), pp. 25-45 : 26-27

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from fresh fruiting bodies using the E.Z.N.A.® Forensic DNA Kit-D3591 (Omega Bio- Tek, Georgia, USA) following the manufacturer’s protocol. Polymerase chain reactions (PCR) were carried out for two genetic markers, the internal transcribed spacer region (ITS) with primers ITS5/ITS4 ( White et al. 1990), and 28S large subunit nuclear ribosomal DNA (LSU) with primers LR0R/LR5 ( Vilgalys & Hester 1990). The PCR thermal cycle programs for LSU and ITS were as follows: initial heat treatment for 5 min at 94 °C, 30 cycles with a denaturation step at 94 °C for 30 s, annealing at 52 °C for 45 s, and an elongation step of 90 s at 72 °C, and a final elongation period of 7 min at 72 °C. The total volume of PCR mixtures for amplification was 25 μL containing 8.5 μL ddH 2 O, 12.5 μL 2xF8FastLong PCR MasterMix (Beijing Aidlab Biotechnologie), 2 μL of DNA template, and 1 μL of each forward and reverse primer (stock of 10 pM). The PCR products were sent for sequencing to Shanghai Sangon Biological Engineering Technology and Service, China.

the sequences. The new taxa are in bold. Ex-type strains are indicated with superscript “T”, “NA” sequence is unavailable.