Fungal Genomic Amplification and Sequencing

The SSU region was amplified with the primers NS1 and NS4 (White et al. 1990 ), the LSU region with LSU1Fd (Crous et al. 2009b (link)) and LR5 (Vilgalys & Hester 1990 (link)), the ITS region with V9G (De Hoog & Gerrits van den Ende 1998 (link)) and ITS4 (White et al. 1990 ), the GAPDH region with gpd1 and gpd2 (Berbee et al. 1999 ), the RPB2 region with RPB2-5F2 (Sung et al. 2007 (link)) and fRPB2-7cR (Liu et al. 1999 (link)) and the TEF1 gene with the primers EF1-728F and EF1-986R (Carbone & Kohn 1999 ) or EF2 (O’Donnell et al. 1998 (link)). The PCRs were performed in a MyCycler™ Thermal Cycler (Bio-Rad Laboratories B.V., Veenendaal, The Netherlands) in a total volume of 12.5 μL. The SSU and LSU PCR mixtures consisted of 1 μL genomic DNA, 1′ GoTaq® Flexi buffer (Promega, Madison, WI, USA), 2 μM MgCl₂, 40 μM of each dNTP, 0.2 μM of each primer and 0.25 Unit GoTaq® Flexi DNA polymerase (Promega). The ITS and GAPDH PCR mixtures differed from the original mix by containing 1 μM MgCl₂, the RPB2 and TEF1 PCR mixtures differed from the original mix by containing 2 μL genomic DNA and the RPB2 mixture differed from the original mix by containing 0.5 U instead of 0.25 U GoTaq® Flexi DNA polymerase. Conditions for PCR amplification consisted of an initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C and 90 s at 72 °C for SSU, LSU, ITS and 40 cycles of 30 s at 94 °C, 30 s at 52 °C / 59 °C and 45 s at 72 °C for TEF1 using respectively EF2 or EF1-986R as reverse primer and a final elongation step of 7 min at 72 °C. The partial RPB2 gene was obtained by using a touchdown PCR protocol of 5 cycles of 45 s at 94 °C, 45 s at 60 °C and 2 min at 72 °C, followed by 5 cycles with a 58 °C annealing temperature and 30 cycles with a 54 °C annealing temperature. The PCR products were sequenced in both directions using the PCR primers and the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s recommendations, and analysed with an ABI Prism 3730XL Sequencer (Applied Biosystems) according to the manufacturer’s instructions. Consensus sequences were computed from forward and reverse sequences using the BioNumerics v. 4.61 software package (Applied Maths, St-Martens-Latem, Belgium). All generated sequences were deposited in GenBank (Table 1).