Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates

Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria

“…In the present study, atoxigenic A. flavus isolates from Nigeria were found that did not generate PCR products for several regions within the aflatoxin gene cluster, indicating the presence of large deletions. Deletions within the aflatoxin gene cluster were found previously in isolates of both A. flavus and the closely related A. oryzae (Kusumoto et al 2000;Chang et al 2005). Kusumoto et al (2000) classified strains of A. oryzae into three groups based on the deletion pattern.…”

“…Primers for the genes norB, pksA, afIR, norA, ver-1, verB, omtB, pecA, and taka amylase were designed using DNAMAN version 6 (Lynnon Biosoft, Vandereuil, Canada). Primers used to PCR-amplify the genes C2, aflT, fasA(hexA), aflJ, estA, omtA, verA, avnA, avfA, vbs, cypX, ordB, hypA, and glcA were previously described by Chang et al (2005) and norB-cypA by . PCR used 5 ng genomic DNA, 50 pmol of forward and reverse oligonucleotides, and the HotMaster PCR kit (Eppendorf, Westbury, NY, USA) in a 50 ml final volume.…”

“…PCR used 5 ng genomic DNA, 50 pmol of forward and reverse oligonucleotides, and the HotMaster PCR kit (Eppendorf, Westbury, NY, USA) in a 50 ml final volume. Annealing temperatures (Table 2) were optimized for each primer set (Chang et al 2005;Ehrlich et al 2005). PCR reactions were performed with a MyCycler thermocycler (Bio-Rad Laboratories, Richmond, CA, USA) under the following conditions: 5 min at 95 C followed by 38 cycles of 95 C for 30 s, locus-specific annealing temperature for 20 s, 72 C for 30 s, and 10 min at 72 C. Amplicons were visualized with SYBR Gold after 1.2% agarose gel electrophoresis.…”

“…The aflatoxin biosynthesis genes in A. oryzae contain deletions, frame-shift mutations, and base pair substitutions that explain the lack of aflatoxin production . Deletion of portions of the aflatoxin biosynthetic gene cluster within atoxigenic A flavus isolates is not rare (Chang et al 2005) and strains of A. flavus with large deletions in the aflatoxin gene cluster have been used to study the genetics of aflatoxin biosynthesis for over a decade (Prieto et al 1996). A single nucleotide polymorphism (SNP) in a polyketide synthase gene results in atoxigenicity in the biocontrol strain AF36 Ehrlich et al 2007).…”

“…First, sequences from 14 genes from the aflatoxin gene cluster were generated to assess nucleotide polymorphism. Second, eight additional cluster genes were polymerase chain reaction (PCR)-amplified to determine the presence/ absence of the genes (Chang et al 2005). To assess the diversity of atoxigenic strains available for biocontrol in Nigeria, relationships among the examined isolates were assessed with phylogenetic analyses that included two additional protein coding loci outside the aflatoxin gene cluster, pecA and taka amylase.…”