Q5 hot start high fidelity 2 master mix dna polymerase

For each species, a single cell was isolated from the original sample and washed five times with 0.22 μm filtered in situ water to remove potential contaminants. Genomic DNA was extracted from the cleaned cells using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions but modified by using 1/4 of the suggested volume for each solution. Q5^® Hot Start High-Fidelity 2× Master Mix DNA polymerase (New England BioLabs) was used to amplify the 18S and ITS-5.8S rDNA using universal eukaryotic primers 82F (5′-GAAACTGCGAATGGCTC-3′) and ITS-R (5′-TACTGATATGCTTAAGTTCAGCGG-3′) (Sogin, 1989 (link); Chi et al., 2021 (link)). PCR amplifications were performed according to the following procedure: initial denaturation at 98°C for 30 s, followed by 18 cycles of amplification (98°C, 10 s; 69–51°C touchdown, 30 s; 72°C, 1 min), and another 18 cycles (98°C, 10 s; 51°C, 30 s; 72°C, 1 min), with a final extension of 72°C for 5 min (Lian et al., 2020 (link)). PCR products were sequenced bidirectionally in Tsingke Biological Technology Company (Qingdao, China) and assembled by SeqMan (DNAStar).

Three cells of each species were isolated for DNA extraction. All cells were washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination (Liu et al. 2022 (link)). Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Germany) following the optimized manufacturer’s protocol, with 25% of the suggested volume used for each solution. The primers 82-F (5′-GAA ACT GCG AAT GGC TC-3′) and ITS-R (5′-TAC TGA TAT GCT TAA GTT CAG CGG-3′) were used for PCR amplifications of the 18S rRNA gene and ITS1-5.8S-ITS2 region (Gao et al. 2012 (link); Jerome et al. 1996 (link)). To minimize the possibility of PCR amplification errors, Q5^® Hot Start High-Fidelity 2 × Master Mix DNA Polymerase (New England BioLabs, USA) was used (Li et al. 2023 (link)). The thermal cycler program used was that described by Li et al. (2022 (link)). The quality of the amplified DNA was checked by 1% agarose gel electrophoresis. PCR products were purified using the EasyPure R Quick Gel Extraction Kit (TransGen Biotech Co., Ltd., Beijing, China), and then sequenced on an ABI-PRISM 3730 automatic sequencer (Applied Biosystems, Tsingke Biological Technology Company, Qingdao, China).