Computational protocol: New and revisited species in Aspergillus section Nigri

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Protocol publication

[…] The cultures used for the molecular studies were grown on malt peptone (MP) broth using 10 % (v/v) of malt extract (Oxoid) and 0.1 % (w/v) bacto peptone (Difco), 2 mL of medium in 15 mL tubes. The cultures were incubated at 25 °C for 7 d. DNA was extracted from the cells using the Masterpure™ yeast DNA purification kit (Epicentre Biotechnol.) according to the instructions of the manufacturer. The ITS region and parts of the β-tubulin and calmodulin genes were amplified and sequenced as described previously (Varga et al. , , ).Part of the FUM8 gene was amplified using primers vnF1 and vnR3 as described by Susca et al. (). Primer sets were also designed to target part of the chloroperoxidase gene of black aspergilli presumably taking part in ochratoxin biosynthesis. Construction of the primers was carried out by using the homologous sequences identified in the genomic sequences of Aspegillus niger CBS 513.88 and Aspergillus carbonarius ITEM 5010 isolates. The designed chloroperoxidase specific PCR primers were BCPOF (5'-CTGGGCGACTGCATCCAC – 3') and BCPOR (5'-TTCATCGTACGGCAGACGCT - 3') which generated specific amplicons of about 250 base pairs. Amplifications were performed on a PTC-0148 Mini48 thermocycler (BioRad, USA), using the following amplification steps: 4 min of initial denaturation at 94 °C followed by 35 amplification cycles of 20 s at 94 °C, 15 s at 62 °C and 30 s at 2 °C and a final extension step for 1 min at 72 °C.DNA sequences were edited with the DNASTAR computer package and an alignment of the sequences and neighbour joining analyses were performed using the MEGA v. 4 software (). To determine the support for each clade, a bootstrap analysis was performed with 1 000 replications. Aspergillus flavus CBS 100927T was used as outgroup in these analyses.Phylogenetic analysis of sequence data was also performed using PAUP* v. 4.0b10 (). Alignment gaps were treated as fifth character state, parsimony uninformative characters were excluded and all characters were unordered and equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option. To assess the robustness of the topology, 1 000 bootstrap replicates were run by maximum parsimony (). Other measures including tree length, consistency index and retention index were also calculated. Sequences were deposited at GenBank under accession numbers listed in .UP-PCR analyses were carried out according to Bulat et al. (). DNA was isolated as described in the literature (). The primers used were L45, AS15inv, L15/AS19, AA2M2, L21, 3-2, AS4, AS15 (, ). The amplification process consisted of a predenaturation step for 1 min at 94 °C, followed by 35 cycles (30 s at 94 °C, 45 s at 55 °C, and 1 min at 72 °C), plus a final extension of 2 min at 72 °C. The amplification products were separated by electrophoresis in 1 % agarose gels, stained with ethidium bromide, and visualised under UV light. All amplifications were repeated at least two times. The faint bands which did not appear in all repeated experiments were not counted during cluster analysis.Altogether 88 fragments were noted and a binomial matrix was created so that presence and absence of DNA fragments were scored as 1 or 0, respectively. Cluster analysis was carried out by using PHYLIP v. 3.67 software package (). Phylogenetical tree was created by using neighbor-joining method (Saitou et al. 1987) with the program NEIGHBOR from the PHYLIP program package. […]

Pipeline specifications

Software tools MEGA-V, PAUP*, PHYLIP
Applications Phylogenetics, GWAS
Organisms Lactuca sativa, Guizotia abyssinica
Diseases Leishmaniasis, Visceral
Chemicals Carbon, Deoxyglucose