Dimethyl sulfoxide (dmso)

For the primers inferred from the complete genome through the approach by Robert et al. (2011) , total genomic DNA was extracted from living cultures, cells preserved in liquid nitrogen or in lyophilisation and from dried fungarium specimens (Agaricomycotina only) using different variations of one-by-one or high-throughput 96-well plate DNA extraction techniques, routinely used by the respective collaborating laboratories (Ferrer et al. 2001 (link), Ivanova et al. 2006 , Yurkov et al. 2012 (link), 2015 , Feng et al. 2013 (link), Verkley et al. 2014 (link)). DNA extraction and PCR protocols differed between participating laboratories.
Primers and amplification conditions used varied between laboratories, an example of the ones used at the CBS is provided. PCR reactions for amplification of the ITS barcode employed primers ITS5/ITS1/ITS1F and ITS4 were performed under standard or semi-nested conditions in 12.5 μL reactions (the CBS-KNAW barcoding lab protocol) containing 2.5 μL purified DNA, 1.25 μL PCR buffer (Takara, Japan, incl. 2.5 mM MgCl₂), 1 μL dNTPs (1 mM stock; Takara, Japan), 0.6 μL v/v DMSO (Sigma, Netherlands), forward-reverse primer 0.25 μL each (10 mM stock), 0.06 μL (5 U) Takara HS Taq polymerase, 7.19 μL MilliQ water (White et al. 1990 , Stielow et al. 2012 , Yurkov et al. 2012 (link)). PCR conditions for amplifying partial LSU rDNA using primers LR0R and LR5 differed only by their annealing temperature (55 °C instead of 60 °C) and increased cycle extension time (90 s per cycle). Amplification of partial γ-actin (ACT), covering the more variable 5’-end including two small introns, and partial β -tubulin 2 (TUB2), covering the variable 5’-end with up to four small introns, followed the protocol of Aveskamp et al. (2009) (link) and Carbone & Kohn (1999) using the primers Btub2Fd and Btub4Rd, and ACT-512F, ACT-783R, respectively. TEF1α and RPB2 were amplified following the protocols of Rehner & Buckley (2005) (link) and Liu et al. (1999) (link), respectively (Table 2). PCR products were directly purified using FastAP thermosensitive alkaline phosphatase and shrimp alkaline phosphatase (Fermentas, Thermo Fisher Scientific). Cycle-sequencing reactions were set up using ABI BigDye Terminator v. 3.1 Cycle Sequencing kit (Thermo Fisher Scientific), with the manufacturers’ protocol modified by using a quarter of the recommended volumes, followed by bidirectional sequencing with a 3730xl DNA Analyser (Thermo Fisher Scientific). Sequences were archived, bidirectional reads assembled and manually corrected for sequencing artefacts using BioloMICS software v. 8.0 (www.bio-aware.com) (Vu et al. 2012 (link)). Edited sequences were exported to and aligned with MAFFT v. 7.0 (Katoh et al. 2005 (link)) and further corrected for indels and SNPs (single nucleotide polymorphisms) by replacing respective positions with ambiguity code letters.
For the primers inferred from the complete proteome through the Pfam approach by Lewis et al. (2011) , genomic DNA from fungal cultures was extracted using the OmniPrep™ Genomic DNA Extraction Kit (G-Biosciences, St. Louis, Missouri). Fungal tissue was ground into a fine powder in a mortar and pestle with liquid nitrogen and stored at -80 °C. Approximately 50 mg of ground tissue was placed in a 1.7 mL microfuge tube, resuspended in 250 μL of lysis buffer and vortexed for several seconds. An additional 250 μL of lysis buffer was added to the resuspension and the tube was incubated for 15 min at 55–60 °C without the addition of Proteinase K. The samples were cooled to room temperature and 200 μL of chloroform was added to the tube. The tube was mixed by inversion several times and then centrifuged for 10 min at 14 000 ×g. The upper aqueous phase was removed to a new microfuge tube and 100 μL of precipitation solution was added. If no white precipitate was produced an additional 50 μL of precipitation solution was added to the tube. The white precipitate was pelleted by centrifugation and the supernatant was moved to a fresh tube. The genomic DNA was precipitated by the addition of 500 μL of isopropanol to the supernatant and inversion of the tube several times. The genomic DNA was pelleted by centrifugation and washed with 700 μL of 70 % ethanol. The ethanol was decanted and the pellet was air dried for 15 s prior to resuspension in 50 μL of TE Buffer. DNA concentration was determined using the Qubit® 2.0 Fluorometer (Life Technologies, Burlington, Canada) and working solutions were prepared at a concentration of 0.05 ng/μL. PCR was carried out on 0.1 ng of genomic DNA in a total volume of 20 μL using 0.2 mM dNTPs, 0.5 μM of each primer, 1× of Titanium Taq Buffer and Titanium Taq DNA Polymerase (Clontech) using an Eppendorf GradientS thermal cycler. For the ITS primers, an initial denaturation at 95 °C for 3 min was followed by 40 cycles at the following conditions: 30 s at 95 °C, 45 s at 58 °C and 2 min at 72 °C. A final extension at 72 °C for 8 min completed the PCR. For the remaining primers, Touchdown PCR was performed where an initial denaturation at 95 °C for 5 min was followed by 10 cycles of 45 s at 95 °C, 45 s starting at 68 °C and dropping by 1 °C per cycle until a temperature of 58 °C was reached and a 1 min extension at 72 °C. The initial 10 cycles were then followed by 35 cycles of 45 s at 95 °C, 45 s at 58 °C and 1 min at 72 °C. A final extension at 72 °C for 5 min completed the PCR.