Computational protocol: The Genera of Fungi – G 4: Camarosporium and Dothiora

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Protocol publication

[…] Genomic DNA was extracted from fungal colonies growing on MEA using the Wizard® Genomic DNA purification kit (Promega, Madison, WI), according to the manufacturer’s protocol. The primers V9G (de Hoog & Gerrits van den Ende 1998) or ITS5 () and LR5 () were used to amplify for all isolates part of the nuclear rDNA operon (ITS) spanning the 3’ end of the 18S nrRNA gene, the first internal transcribed spacer (ITS1), the 5.8S nrRNA gene, the second ITS region (ITS2) and approximately 900 bp of the 5’ end of the 28S nrRNA gene. The primers ITS4 () and LR0R () were used as internal sequence primers to ensure good quality sequences over the entire length of the amplicon. Part of the beta-tubulin gene (tub2) was amplified and sequenced for selected isolates using T1 () or Bt-2a and Bt-2b (). In addition, part of the first or second half of the translation elongation factor 1-alpha gene (tef1) was amplified and sequenced for selected isolates using EF1-728F () and EF2 (), and EF1-983F and EF1-2218R (), respectively. Part of the 18S small subunit nrRNA gene (SSU) was amplified and sequenced for selected isolates using NS1 and NS4 (). Amplification conditions followed and . The programme SeqMan Pro v. 13.0.0 (DNASTAR, Madison, WI) was used to obtain consensus sequences of each isolate. Blast searches using ITS and LSU sequences were performed for each isolate and the closest matches were retrieved from GenBank and included in the phylogenetic analyses. The SSU, tef1 and tub2 sequences were not included in phylogenetic analyses but were used in blast searches to confirm the species identification where possible. Multiple sequence alignments for ITS and LSU were generated using the online version of MAFFT (http://mafft.cbrc.jp/alignment/software/) and subsequent phylogenetic analyses were conducted using parsimony in PAUP v. 4.0b10 () as described by . Sequence data were deposited in GenBank () and the alignments and trees in TreeBASE (http://www.treebase.org). […]

Pipeline specifications

Software tools DNASTAR Molecular Biology Suite, MAFFT, PAUP*
Databases TreeBASE
Application Phylogenetics