Computational protocol: Co-occurring species of Teratosphaeria on Eucalyptus

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Protocol publication

[…] Genomic DNA was extracted from mycelia taken from fungal colonies on MEA using the UltraClean TM Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc., Solana Beach, CA, USA). The Primers V9G () and LR5 () were used to amplify part of the nuclear rDNA operon spanning the 3’ end of the 18S rRNA gene (SSU), the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region (ITS2) and the 5’ end of the 28S rRNA gene (LSU). The primers ITS4 () and LR0R () were used as internal sequence primers to ensure high quality sequences over the entire length of the amplicon. The PCR amplifications were performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) in a total volume of 12.5 μL solution containing 10–20 ng of template DNA, 1 × PCR buffer, 2.5 mM MgCl 2 , 15 pmol for each primer, 60 μM of each dNTP and 0.75 U Taq DNA polymerase (Bioline GmbH, Luckenwalde, Germany). PCR amplification conditions were set as follows: an initial denaturation temperature of 94 °C for 5 min, followed by 40 cycles of denaturation temperature of 94 °C for 45 s, primer annealing at 48 °C for 30 s, primer extension at 72 °C for 90 s and a final extension step at 72 °C for 7 min. The resulting fragments were sequenced using the PCR primers together with a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA) and analysed on an ABI Prism 3100 DNA Sequencer (Perkin-Elmer, Norwalk, CN).Separate alignments were made for the ITS and LSU regions. The generated sequences were compared with other fungal DNA sequences from NCBI’s GenBank sequence database using a blastn search; sequences with high similarity were added to the alignments. Additional GenBank sequences were manually aligned using Sequence Alignment Editor v. 2.0a11 (). Phylogenetic analyses of the aligned sequence data were performed using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 () and consisted of neighbour-joining analyses with the uncorrected (‘p’), the Kimura 2-parameter and the HKY85 substitution models. Alignment gaps were treated as missing data and all characters were unordered and of equal weight. Any ties were broken randomly when encountered. For parsimony analyses, alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed using the heuristic search option with 100 random (ITS) or simple (LSU) taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1 000 bootstrap replications (). Tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) were calculated and the resulting trees were printed with TreeView v. 1.6.6 (). New sequences were lodged in GenBank and the alignments and phylogenetic trees in TreeBASE (www.treebase.org). […]

Pipeline specifications

Software tools BLASTN, PAUP*, TreeViewX
Databases TreeBASE
Application Phylogenetics
Organisms Eucalyptus cladocalyx, Encephalartos lehmannii