Taq pcr master mix with blue dye

The total genomic DNA was extracted from yeast strains using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer’s instructions (Sangon Biotech Co., Shanghai, China). Two nuclear loci, which include the ITS regions and the D1/D2 domains of the LSU rRNA gene, were amplified using ITS1/ITS4 (White et al. 1990 (link)) and NL1/NL4 (Kurtzman and Robnett 1998 (link)) primers, respectively. The amplifications were performed in a 25 µL reaction-volume tube containing 9.5 µL of ddH₂O, 12.5 µL of 2 × Taq PCR Master Mix with blue dye (Sangon Biotech Co., Shanghai, China), 1 µL of DNA template, and 1 µL of each primer. The following parameters were used to amplify the ITS and D1/D2 regions: an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C, 40 s at 72 °C, and a final extension of 10 min at 72 °C (Wang et al. 2014 (link)). The PCR products were purified and sequenced at Sangon Biotech Co., Ltd (Shanghai, China) with the same primers. We determined the identity and accuracy of the newly-obtained sequences by comparing them to sequences in GenBank and assembled them using BioEdit 7.1.3.0 (Hall 1999 ). All newly generated sequences were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), and the accession numbers are listed in Table 2.

Isolates were grown on PDA and/or MEA medium at 25 °C for one month. Fungal mycelium was scraped off and transferred to a 1.5-ml microcentrifuge tube using a sterilised lancet for genomic DNA extraction. Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China) was used to extract DNA following the manufacturer’s instructions. ITS, LSU and TEF1α gene regions were amplified using the primer pairs ITS5 or ITS1 with ITS4 (Vilgalys and Hester 1990 (link)), LROR with LR5 or LR7 (White et al. 1990 (link)) and EF1-983F with EF1-2218R (Rehner 2001 ). The amplifications were performed in a 25 μl reaction volume containing 9.5 μl ddH₂O, 12.5 μl 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, China), 1 μl of DNA template and 1 μl of each primer (10 μM). The amplification condition for ITS, LSU and TEF1α consisted of initial denaturation at 94 °C for 3 min; followed by 40 cycles of 45 s at 94 °C, 50 s at 56 °C and 1 min at 72 °C and a final extension period of 10 min at 72 °C. Purification and sequencing of PCR products were carried out using the above-mentioned PCR primers at Sangon Biotech (Shanghai) Co. Ltd. in China.

Pure cultures were grown on MEA/PDA media at 25 °C for one month. Fresh fungal mycelia were scraped off from the surface of the cultures and transferred to 1.5 mL microcentrifuge tubes. Meanwhile, fungal genomic DNA was extracted with the Biospin Fungus Genomic DNA Extraction Kit (Biospin Fungus Genomic DNA Extraction Kit, BioFlux^®, Shanghai, China) following the manufacturer’s instructions. LR0R and LR5 (Vilgalys and Hester 1990) and ITS5 and ITS4 (White et al. 1990) primers were used to amplify the large subunit of the ribosomal DNA (LSU) and the internal transcribed spacer (ITS) gene regions. The amplification reactions were performed in a 50 μL reaction volume, which contained 2 μL of DNA template, 2 μL of each forward and reverse primer (10 μM), 25 μL of 2× Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China) and 19 μL of distilled–deionized water. The following thermo-cycling parameters were used for the LSU and ITS region: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 50 s, elongation at 72 °C for 1 min and a final extension period for 10 min at 72 °C. The quality of the PCR products was checked on a 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were performed at Sangon Biotech (Shanghai, China) using the same primers.