Single ascospore or conidium isolates were prepared and grown on 2 % malt extract agar (MEA), or on 2 % corn meal agar plus 2 % w/v dextrose (CMD).
Growth of liquid culture and extraction of genomic DNA was performed as reported previously (Voglmayr & Jaklitsch 2011 , Jaklitsch et al. 2012 (link)) using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany) or the modified CTAB method of Riethmüller et al. (2002) (link).
The following loci were amplified and sequenced: the complete internal transcribed spacer region (ITS1-5.8S-ITS2) and a c. 900 bp fragment of the large subunit nuclear ribosomal DNA (nuLSU rDNA), amplified and sequenced as a single fragment with primers V9G (De Hoog & Gerrits van den Ende 1998 (link)) and LR5 (Vilgalys & Hester 1990 (link)); a 450–454 bp fragment of the calmodulin (cal) gene with primers CAL-228F and CAL-737R (Carbone & Kohn 1999 ); a 441–445 bp fragment of the histone H3 (his) gene with primers CYLH3F (Crous et al. 2004 ) and H3-1b (Glass & Donaldson 1995 (link)); a c. 1 kb fragment of the guanine nucleotide-binding protein subunit beta (ms204) gene with primers MS-E1F1 and MS-E5R1 (Walker et al. 2012 (link)); a 711 bp fragment of the RNA polymerase II subunit 1 (rpb1) gene with primers RPB1-Af and RPB1-Cr (Stiller & Hall 1997 (link)); a c. 1.2 kb fragment of the RNA polymerase II subunit 2 (rpb2) gene with primers fRPB2-5f and fRPB2-7cr (Liu et al. 1999 (link)) or dRPB2-5f and dRPB2-7cr (Voglmayr et al. 2016 (link)); a c. 1.3 kb fragment of the translation elongation factor 1-alpha (tef1) gene containing introns 4 and 5 and part of the exon with primers EF1-728F (Carbone & Kohn 1999 ) and TEF1LLErev (Jaklitsch et al. 2005 (link)); and a 441–445 bp fragment of the β-tubulin (tub2) gene with primers T1 (O’Donnell & Cigelnik 1997 (link)) and the newly designed BtHV2r (5’ CATCATRCGRTCNGGGAACTC 3’). PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994 (link)) as described in Voglmayr & Jaklitsch (2008) (link). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington, UK) and the PCR primers; in addition, primers ITS4 (White et al. 1990 ) and LR3 (Vilgalys & Hester 1990 (link)) were used as internal sequencing primers for the ITS-LSU rDNA region. Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyzer, Applied Biosystems).
Growth of liquid culture and extraction of genomic DNA was performed as reported previously (Voglmayr & Jaklitsch 2011 , Jaklitsch et al. 2012 (link)) using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany) or the modified CTAB method of Riethmüller et al. (2002) (link).
The following loci were amplified and sequenced: the complete internal transcribed spacer region (ITS1-5.8S-ITS2) and a c. 900 bp fragment of the large subunit nuclear ribosomal DNA (nuLSU rDNA), amplified and sequenced as a single fragment with primers V9G (De Hoog & Gerrits van den Ende 1998 (link)) and LR5 (Vilgalys & Hester 1990 (link)); a 450–454 bp fragment of the calmodulin (cal) gene with primers CAL-228F and CAL-737R (Carbone & Kohn 1999 ); a 441–445 bp fragment of the histone H3 (his) gene with primers CYLH3F (Crous et al. 2004 ) and H3-1b (Glass & Donaldson 1995 (link)); a c. 1 kb fragment of the guanine nucleotide-binding protein subunit beta (ms204) gene with primers MS-E1F1 and MS-E5R1 (Walker et al. 2012 (link)); a 711 bp fragment of the RNA polymerase II subunit 1 (rpb1) gene with primers RPB1-Af and RPB1-Cr (Stiller & Hall 1997 (link)); a c. 1.2 kb fragment of the RNA polymerase II subunit 2 (rpb2) gene with primers fRPB2-5f and fRPB2-7cr (Liu et al. 1999 (link)) or dRPB2-5f and dRPB2-7cr (Voglmayr et al. 2016 (link)); a c. 1.3 kb fragment of the translation elongation factor 1-alpha (tef1) gene containing introns 4 and 5 and part of the exon with primers EF1-728F (Carbone & Kohn 1999 ) and TEF1LLErev (Jaklitsch et al. 2005 (link)); and a 441–445 bp fragment of the β-tubulin (tub2) gene with primers T1 (O’Donnell & Cigelnik 1997 (link)) and the newly designed BtHV2r (5’ CATCATRCGRTCNGGGAACTC 3’). PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994 (link)) as described in Voglmayr & Jaklitsch (2008) (link). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington, UK) and the PCR primers; in addition, primers ITS4 (White et al. 1990 ) and LR3 (Vilgalys & Hester 1990 (link)) were used as internal sequencing primers for the ITS-LSU rDNA region. Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyzer, Applied Biosystems).
