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. 2016 Dec;6(1):62.
doi: 10.1186/s13568-016-0228-6. Epub 2016 Aug 31.

Degeneration of aflatoxin gene clusters in Aspergillus flavus from Africa and North America

Bishwo N Adhikari  1 , Ranajit Bandyopadhyay  2 , Peter J Cotty  3
Affiliations

Degeneration of aflatoxin gene clusters in Aspergillus flavus from Africa and North America

Bishwo N Adhikari et al. AMB Express. 2016 Dec.

Abstract

Aspergillus flavus is the most common causal agent of aflatoxin contamination of food and feed. However, aflatoxin-producing potential varies widely among A. flavus genotypes with many producing no aflatoxins. Some non-aflatoxigenic genotypes are used as biocontrol agents to prevent contamination. Aflatoxin biosynthesis genes are tightly clustered in a highly conserved order. Gene deletions and presence of single nucleotide polymorphisms (SNPs) in aflatoxin biosynthesis genes are often associated with A. flavus inability to produce aflatoxins. In order to identify mechanisms of non-aflatoxigenicity in non-aflatoxigenic genotypes of value in aflatoxin biocontrol, complete cluster sequences of 35 A. flavus genotypes from Africa and North America were analyzed. Inability of some genotypes to produce aflatoxin resulted from deletion of biosynthesis genes. In other genotypes, non-aflatoxigenicity originated from SNP formation. The process of degeneration differed across the gene cluster; genes involved in early biosynthesis stages were more likely to be deleted while genes involved in later stages displayed high frequencies of SNPs. Comparative analyses of aflatoxin gene clusters provides insight into the diversity of mechanisms of non-aflatoxigenicity in A. flavus genotypes used as biological control agents. The sequences provide resources for both diagnosis of non-aflatoxigenicity and monitoring of biocontrol genotypes during biopesticide manufacture and in the environment.

Keywords: Aflatoxin gene cluster; Aspergillus flavus; Biocontrol; Cluster degeneration; Evolution; Non-aflatoxigenic.

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Figures

Fig. 1
Fig. 1
Schematic of the aflatoxin biosynthesis (dark grey) and sugar clusters (light grey) from 35 non-aflatoxigenic A. flavus genotypes. The bottom figure shows the aflatoxin gene cluster from A. flavus AF13 (Ehrlich et al. 2005) and genes putatively involved in sugar translocation. Genotypes are grouped based on presence or absence of genes. Absence of genes towards the telomeric end of the cluster indicates gene deletion. Sequences bordering deletions are indicated in grey shaded boxes. Letters on the right indicate groups of genotypes with similar clusters. Group A includes AF36, BA16-F, BY18-A, C6-E, DO107-L, EC69-E, GO67-10, Ka16127, La3279, La3304, M011-8, M102-11, M109-2, M129-5, M2-7, Ms14-19, Ss19-14; group B includes E63-I and R7-H; group C includes C8-F and DO38-B; group D includes AT5-B, DO114-A, GO18-2 and M092-15; group E includes M21-11; group F includes AT21-A, AT4-C, BA35-C, BY19-D, DO46-G, EC19-B, M110-7, NRRL21882, and Og0222
Fig. 2
Fig. 2
Neighbor-Net network of 35 non-aflatoxigenic A. flavus genotypes based on 17 SSR loci. Network was generated by the split decomposition algorithm with the distance matrix calculated by START2 from allelic profile data using SplitsTree 4 (Huson and Bryant 2006). Nodes are colored orange (complete cluster), purple and teal (partial deletion) and green (complete deletion) based on completeness of the aflatoxin gene cluster. The letters correspond with the grouping of genotypes based on aflatoxin gene cluster sequence alignment and detailed under the “Variation in the aflatoxin gene cluster” section of the results
Fig. 3
Fig. 3
Heat map of SNP density (SNPs/kb of gene) in aflatoxin gene cluster of 17 non-aflatoxigenic and 3 toxigenic A. flavus genotypes. SNPs were called in reference to A. flavus AF13. Genotypes with a common letter along the bottom and genes with a common letter along the side do not differ significantly in mean SNP density by Tukey’s HSD test (P < 0.05). Only genotypes having all the genes in the aflatoxin biosynthesis cluster are included. Genotype names in bold are toxigenic. Schematic representation of the aflatoxin biosynthesis cluster is presented at the top. Left is the telomeric end of the cluster
Fig. 4
Fig. 4
Plot of Ka/Ks values on Y-axis based on pairwise comparisons of orthologous genes from non-aflatoxigenic genotypes with A. flavus AF13. The length of coding sequence used to calculate Ka/Ks are as follow: cypA (390 bp), aflR (334 bp), estA (345 bp), ver-1 (389 bp), vbs (332 bp), and cypX (327 bp). Since not all genes are present in all genotypes, no values are present for the aflR and estA genes in DO38-B and for the cypA and aflR genes in GO18-2. Genotypes BA16-F, EC69-E and La3304 have complete sets of genes while genotypes DO38-B and GO18-2 have partially deleted aflatoxin gene clusters
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