Genomic DNA was extracted using the Ultraclean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. Parts of the following loci were amplified and sequenced for the species listed in Table 1 : 1. RPB1, RNA polymerase II largest subunit (regions E and F; according Matheny et al. 2002 (link)), 2. RPB2, RNA polymerase II second largest subunit (regions 5–7), 3. Cct8, subunit of the cytosolic chaperonin Cct ring complex, related to Tcp1p and required for the assembly of actin and tubulins in vivo (Stoldt et al. 1996 (link), Kim et al. 1994 (link)), 4. Tsr1, protein required for processing of 20S pre-rRNA in the cytoplasm (Gelperin et al. 2001 (link), Léger-Silvestre et al. 2004 (link)). Partial RPB2 data was obtained for the majority of species listed in Table S1. Exceptions are strains used in the study of Houbraken et al. (2011c (link)); in that case, published partial β-tubulin sequences were used.
The RPB1 fragment was amplified using the primer pair RPB1-F1843 and R3096, and RPB1-R2623 was occasionally used as an internal primer for sequencing. A part of the RPB2 locus was amplified using the primer pair RPB2-5F and RPB2-7CR (Liu et al. 1999 (link)) or the primer pair RPB2-5F_Eur and RPB2-7CR_Eur. The internal sequencing primers RPB2-F311 and RPB2-R310 were occasionally used when poor results were obtained with the regular forward and reverse primers. Amplification of a part of the Cct8 gene was performed using the primer pair Cct8-F660 and Cct8-R1595. No amplicons could be obtained in the case of 5–10 % of the analysed strains. In those cases, amplicons were generated using the primer pair Cct8-R1595 and Cct8-F94. A part of the Tsr1 gene was amplified using the forward primers Tsr1-F1526Pc or Tsr1-F1526 in combination with Tsr1-R2434. Annealing temperatures and primers used for amplification and sequencing are shown inTable 2 .
The PCR reactions were performed in 25 μL reaction mixtures containing 1 μL genomic DNA 2.5 μL PCR buffer, 0.75 μL MgCl2 (50 mM), 16.55 μL demineralised sterile water, 1.85 μL dNTP (1 mM), 0.50 μL of each primer (100 mM) and 0.1 μL Taq polymerase (5 U/μL, BioTaq, Bioline). The PCR program typically was: 5 cycles of 30 s denaturation at 94 °C, followed by primer annealing for 30 s at 51 °C, and extension for 1 min at 72 °C; followed by 5 cycles with an annealing temperature at 49 °C and 30 cycles at 47 °C, finalised with an extension for final 10 min at 72 °C. Excess primers and dNTP's were removed from the PCR product using the QIAQuick PCR purification kit (Qiagen). Purified PCR fragments were resuspended in 30–50 μL of water. PCR products were sequenced directly in both directions with the same primers and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience, Roosendaal, The Netherlands). The cycle sequencing reaction mixture had a total reaction volume of 10 μL, and contained 1 μL of template DNA, 0.85 μL BigDye reagent, 3 μL buffer, 4.75 μL demineralised water and 0.4 μL primer (10 mM).
Sequencing products were purified according to the manufacturers' recommendations with Sephadex G-50 superfine columns (Amersham Bioscience, Roosendaal, The Netherlands) in a multiscreen HV plate (Millipore, Amsterdam, The Netherlands) and with MicroAmp Optical 96-well reaction plate (AB Applied Biosystems, Nieuwerkerk a/d Yssel, The Netherlands). Contigs were assembled using the forward and reverse sequences with the programme SeqMan from the LaserGene package (DNAStar Inc., Madison, WI).
The RPB1 fragment was amplified using the primer pair RPB1-F1843 and R3096, and RPB1-R2623 was occasionally used as an internal primer for sequencing. A part of the RPB2 locus was amplified using the primer pair RPB2-5F and RPB2-7CR (Liu et al. 1999 (link)) or the primer pair RPB2-5F_Eur and RPB2-7CR_Eur. The internal sequencing primers RPB2-F311 and RPB2-R310 were occasionally used when poor results were obtained with the regular forward and reverse primers. Amplification of a part of the Cct8 gene was performed using the primer pair Cct8-F660 and Cct8-R1595. No amplicons could be obtained in the case of 5–10 % of the analysed strains. In those cases, amplicons were generated using the primer pair Cct8-R1595 and Cct8-F94. A part of the Tsr1 gene was amplified using the forward primers Tsr1-F1526Pc or Tsr1-F1526 in combination with Tsr1-R2434. Annealing temperatures and primers used for amplification and sequencing are shown in
The PCR reactions were performed in 25 μL reaction mixtures containing 1 μL genomic DNA 2.5 μL PCR buffer, 0.75 μL MgCl2 (50 mM), 16.55 μL demineralised sterile water, 1.85 μL dNTP (1 mM), 0.50 μL of each primer (100 mM) and 0.1 μL Taq polymerase (5 U/μL, BioTaq, Bioline). The PCR program typically was: 5 cycles of 30 s denaturation at 94 °C, followed by primer annealing for 30 s at 51 °C, and extension for 1 min at 72 °C; followed by 5 cycles with an annealing temperature at 49 °C and 30 cycles at 47 °C, finalised with an extension for final 10 min at 72 °C. Excess primers and dNTP's were removed from the PCR product using the QIAQuick PCR purification kit (Qiagen). Purified PCR fragments were resuspended in 30–50 μL of water. PCR products were sequenced directly in both directions with the same primers and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience, Roosendaal, The Netherlands). The cycle sequencing reaction mixture had a total reaction volume of 10 μL, and contained 1 μL of template DNA, 0.85 μL BigDye reagent, 3 μL buffer, 4.75 μL demineralised water and 0.4 μL primer (10 mM).
Sequencing products were purified according to the manufacturers' recommendations with Sephadex G-50 superfine columns (Amersham Bioscience, Roosendaal, The Netherlands) in a multiscreen HV plate (Millipore, Amsterdam, The Netherlands) and with MicroAmp Optical 96-well reaction plate (AB Applied Biosystems, Nieuwerkerk a/d Yssel, The Netherlands). Contigs were assembled using the forward and reverse sequences with the programme SeqMan from the LaserGene package (DNAStar Inc., Madison, WI).
