Computational protocol: Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya

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Protocol publication

[…] PCR analysis of the internal transcribed spacers (ITS1–5.8S-ITS2 cluster) regions of the ribosomal DNA gene cluster, and parts of the B-tubulin and calmodulin genes was done for each isolate using the primer pairs shown in Table . PCR amplifications were performed on 25 μL of a reaction mixture containing MgCl2-free reaction buffer, 50 mM MgCl2, 10 mM dNTP mix, 10 μM of each primer, 5 U/μL Taq DNA polymerase and 5 ng/μL of template DNA. The AccuPower® Taq PCR PreMix (Bioneer, Korea) was preferred as the Taq in the premix is not a high fidelity enzyme.PCR was carried out as follows: (1) one step at 94°C for 3 min; (2) 30 cycles of the following three steps: 1 min at 94°C, 1 min at 57°C, 1 min at 72°C, and (3) one final 10-min step at 72°C. The PCR products were separated by 1.2% agarose gel electrophoresis in a Tris-base, acetic acid and EDTA buffer, and stained with ethidium bromide. PCR product purification was done using QIAquick PCR Purification Kit (Qiagen) and sequencing performed at Inqaba Ltd (Cape Town, Republic of South Africa). The obtained DNA sequences were assembled and edited using CLC Main Workbench 7.7.3 (https://www.qiagenbioinformatics.com/), and then compared with the sequences that are deposited in the GenBank (http://www.ncbi.nlm.nih.gov/). The sequences of each of the three genes were aligned separately with references in the NCBI database, and the SNPs/Indels were observed. Phylogenetic trees were constructed using maximum likelihood with 1000 bootstraps replicates using MEGA v 6. Sequences were deposited at GenBank under accession numbers indicated on Supplementary Tables –. […]

Pipeline specifications

Software tools CLC Main Workbench, MEGA-V
Applications Phylogenetics, GWAS