Computational protocol: Species diversity of Trichoderma in Poland

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Protocol publication

[…] Primary identification was based on the sequencing of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster. In cases where ITS1 and ITS2 did not provide unambiguous identification, a fragment of the translation-elongation factor 1-alpha (tef1) gene was sequenced. The ITS region of the rDNA of 170 isolates was amplified using primers ITS4, ITS5 (White et al. ). A fragment of tef1 gene containing the 4th and 5th introns was amplified using the primers Ef728M (Carbone and Kohn ) and tef1R (Kullnig-Gradinger et al. ). The PCR reaction was carried out in a 25-μl reaction mixture containing the following: 1 μl 50 ng/μl of DNA, 2.5 μl 10 × PCR buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.8, 0.1% Triton X-100), 1.5 μl 10 mM dNTP (GH Healthcare), 0.2 μl 100 mM of each primer, 19.35 μl MQ H2O, 0.25 μl (2 U/μl) DyNAzymeTM II DNA Polymerase (Finnzymes). Amplifications were performed in either a PTC-200 or PTC-100 thermocycler (MJ Research, USA) under the following conditions: initial denaturation 5 min at 94°C, 35 cycles of 45 s at 94°C, 45 s at 58°C (for the ITS region), or 63°C (for the tef1 fragment), 1 min at 72°C, with the final extension of 10 min at 72°C. Amplification products were separated on 1.5% agarose gel (Invitrogen) in 1 × TBE buffer (0.178 M Tris-borate, 0.178 M boric acid, 0.004 M EDTA) and stained with ethidium bromide. The 10-μl PCR products were combined with 2 μl of loading buffer (0.25% bromophenol blue, 30% glycerol). A 100-bp DNA Ladder Plus (Fermentas) was used as a size standard. PCR products were electrophoresed at 3 Vcm−1 for about 2 h, visualized under UV light, and photographed (Syngene UV visualizer). The 3-μl PCR products were purified with exonuclease I and shrimp alkaline phosphatase according to Chełkowski et al. (). Sequencing reactions were prepared using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit in 5 μl volume (Applied Biosystems, Switzerland). DNA sequencing was performed on an ABI PRISM 310 Genetic Analyzer (USA). Sequences were edited and assembled using Chromas v.1.43 (Applied Biosystems). CLUSTAL W (Thompson et al. ) and MUSCLE (Edgar ) were used to align the sequences; the resulting alignments were inspected and refined manually. [...] For species identification, ITS1 and ITS 2 sequences were submitted to the BLAST interface in TrichOKEY (; Druzhinina et al. ; Druzhinina and Kubicek ). In ambiguous cases, the result was re-checked using the TrichoBLAST program based on tef1 gene sequences (Druzhinina and Kopchinskiy , ). All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were performed in MEGA4 (Tamura et al. ). Both ITS1, ITS2 and tef1 gene sequences were analyzed using the maximum parsimony (Eck and Dayhoff ) approach of close-neighbor-interchange algorithm with search level 3 (Nei and Kumar ), in which the initial trees were obtained with the random addition of sequences (10,000 replicates). In total, there were 48 parsimony informative positions retained from an initial alignment of 368 for the ITS1, ITS2 sequences and 491 positions in the final dataset, of which 118 were parsimony informative for tef1 gene sequences. In both cases, to infer the consensus, phylogenetic trees bootstrapping with 10,000 data replicates was conducted (Felsenstein ). […]

Pipeline specifications

Software tools Clustal W, MUSCLE, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Trichoderma virens, Trichoderma gamsii