Computational protocol: Three new additions to the genus Talaromyces isolated from Atlantis sandveld fynbos soils

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Protocol publication

[…] DNA was extracted from strains grown on MEA for 7 d using the ZR Fungal/Bacterial DNA Kit (Zymo Research, California, USA). The subsequent PCR of the ITS1–5.8S–ITS2 rDNA region were prepared in 25 μL total volume reactions and consisted of 2.5 μL 10X Kapa Taq High Yield Buffer A, 2.5 U Kapa Taq (Kapa Biosystems, Woburn, USA), 250 μM dNTPs and 0.25 μM of primers ITS1 and ITS4, respectively (). The thermal cycle profile had an initial denaturing step at 94 °C for 5 min, followed by 36 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 60 s, followed by a final elongation step at 72 °C for 10 min. The β-tubulin gene was amplified using the same reagents as for the ITS PCR, except for primers Bt2a and Bt2b used (). The thermal cycle profile had an initial denaturing step at 94 °C for 3 min, followed by 36 cycles at 94 °C for 45 s, 52 °C for 45 s, 72 °C for 60 s, and was completed with a final elongation step at 72 °C for 10 min.PCR products were purified using the MSB®Spin PCRapace (Invitek, Berlin) kit. Sequencing reactions of the PCR products were set up using a Big Dye terminator cycle sequencing premix kit (Applied Biosystems, CA). The thermal cycle profile had an initial denaturing step at 94 °C for 5 min, followed by 25 cycles at 94 °C for 10 s, 55 °C for 10 s and 60 °C for 4 min. Sequence reactions were analysed with an ABI PRISM 310 genetic analyser, with the subsequent sequence contigs assembled in CodonCode Aligner (v. 3.7.1.1, Codon Code Corporation). The ITS and β-tubulin sequences of the fynbos soil strains were included in datasets containing sequences from GenBank of known Penicillium subg. Biverticillium and Talaromyces species (, , , , ).Datasets were aligned in MAFFT v. 6.850b () with the L-INS-i option selected. All of the aligned datasets can be obtained from TreeBASE (Submission ID 11467). Sequence analysis was done in PAUP v. 4.0b10 (Swofford 2000), using the BioNJ option for calculating a single tree for each dataset (). Confidence in nodes was calculated using a bootstrap analysis of 1 000 replicates. Talaromyces thermophilus was chosen as outgroup for the ITS phylogeny (). For β-tubulin, clade specific phylogenies were done which gave a better alignment than a total phylogeny. Penicillium lignorum and T. phialosporus were chosen as outgroups for the T. solicola clade phylogeny. For the T. chloroloma / T. ptychoconidium clade, synnemata-producing species T. calidi- canius, T. duclauxii, T. palmae and T. panamensis were chosen as outgroups. […]

Pipeline specifications

Software tools CodonCode Aligner, MAFFT, PAUP*, BIONJ
Databases TreeBASE
Application Phylogenetics