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Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale.

Moore GG, Elliott JL, Singh R, Horn BW, Dorner JW, Stone EA, Chulze SN, Barros GG, Naik MK, Wright GC, Hell K, Carbone I - PLoS Pathog. (2013)

Bottom Line: For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus.A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations.This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

View Article: PubMed Central - PubMed

Affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, Louisiana, United States of America.

ABSTRACT
Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

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Schematic diagram of the aflatoxin gene cluster in A. flavus and A. parasiticus.The shaded blocks indicate the regions sequenced and the numbers correspond to the LD blocks outlined in Figure 3. For A. parasiticus, LD block 2 spans three regions and block 5 spans two regions [24].
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ppat-1003574-g001: Schematic diagram of the aflatoxin gene cluster in A. flavus and A. parasiticus.The shaded blocks indicate the regions sequenced and the numbers correspond to the LD blocks outlined in Figure 3. For A. parasiticus, LD block 2 spans three regions and block 5 spans two regions [24].

Mentions: The influence of asexual and sexual reproduction on recombination in the aflatoxin cluster and overall toxin diversity was examined by reconstructing patterns of LD in the aflatoxin cluster for a subset of isolates representing distinct MLSTs in A. flavus L and S, A. parasiticus and A. minisclerotigenes. Previous population genetic studies showed that multilocus cluster haplotypes are identical within a VCG and that recombination in the aflatoxin cluster is detected only between VCGs [14], [18], [24]. The subset for LD analysis was therefore selected to maximize VCG (MLST) and toxin diversity. Moreover, recombination is nonrandom and species-specific such that LD blocks and recombination hotspots are conserved among geographically separated strains [31]. We therefore determined the LD block structure and rate of recombination in the aflatoxin cluster by focusing on the intergenic regions separating LD blocks identified in the United States populations of A. flavus and A. parasiticus[14], [24]. For A. flavus L and S and A. minisclerotigenes, the regions sequenced were aflE/aflM, aflM/aflN (hypE), aflN/aflG, aflG/aflL, aflL/aflI, and aflI/aflO, which define six distinct LD blocks [14]. For A. parasiticus, we sequenced aflB/aflR, aflS/aflH, aflH/aflJ, aflJ/aflE, aflE/aflM, aflG/aflL, and aflK/aflV, which define five LD blocks [24]. Figure 1 shows a schematic representation of the aflatoxin gene cluster and the regions that were sequenced for LD analysis. LD was examined by 1) combining all sequenced loci for each locality, species and morphotype using SNAP Combine [32] into a single concatenated sequence alignment, 2) collapsing the alignment to infer multi-locus haplotypes using SNAP Map [32] with the options of recoding indels (insertions/deletions) as binary characters and excluding infinite sites violations, and 3) generating an LD plot for all variable positions using the Clade and Matrix [33] programs implemented in SNAP Workbench [34]. LD was quantified using the coefficient of determination (r2) between the allelic states at pairs of sites and a two-sided Fisher's Exact test, as implemented in Tassel version 1.1.0 [35]. LD blocks were based on the number of contiguous pairs of sites that were both strongly correlated (0.8<r2<1) and significantly linked (P<0.01). Because highly divergent haplotypes sampled once or at a low frequency could be potential targets of balancing selection in aflatoxin gene clusters [14], [24], they were not excluded in the LD analyses and the strength of LD was assessed using both r2 and 2×2 contingency tests. All sequences have been deposited in GenBank under Accession numbers HM353147–HM355445 and HM745560–HM745901.


Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale.

Moore GG, Elliott JL, Singh R, Horn BW, Dorner JW, Stone EA, Chulze SN, Barros GG, Naik MK, Wright GC, Hell K, Carbone I - PLoS Pathog. (2013)

Schematic diagram of the aflatoxin gene cluster in A. flavus and A. parasiticus.The shaded blocks indicate the regions sequenced and the numbers correspond to the LD blocks outlined in Figure 3. For A. parasiticus, LD block 2 spans three regions and block 5 spans two regions [24].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757046&req=5

ppat-1003574-g001: Schematic diagram of the aflatoxin gene cluster in A. flavus and A. parasiticus.The shaded blocks indicate the regions sequenced and the numbers correspond to the LD blocks outlined in Figure 3. For A. parasiticus, LD block 2 spans three regions and block 5 spans two regions [24].
Mentions: The influence of asexual and sexual reproduction on recombination in the aflatoxin cluster and overall toxin diversity was examined by reconstructing patterns of LD in the aflatoxin cluster for a subset of isolates representing distinct MLSTs in A. flavus L and S, A. parasiticus and A. minisclerotigenes. Previous population genetic studies showed that multilocus cluster haplotypes are identical within a VCG and that recombination in the aflatoxin cluster is detected only between VCGs [14], [18], [24]. The subset for LD analysis was therefore selected to maximize VCG (MLST) and toxin diversity. Moreover, recombination is nonrandom and species-specific such that LD blocks and recombination hotspots are conserved among geographically separated strains [31]. We therefore determined the LD block structure and rate of recombination in the aflatoxin cluster by focusing on the intergenic regions separating LD blocks identified in the United States populations of A. flavus and A. parasiticus[14], [24]. For A. flavus L and S and A. minisclerotigenes, the regions sequenced were aflE/aflM, aflM/aflN (hypE), aflN/aflG, aflG/aflL, aflL/aflI, and aflI/aflO, which define six distinct LD blocks [14]. For A. parasiticus, we sequenced aflB/aflR, aflS/aflH, aflH/aflJ, aflJ/aflE, aflE/aflM, aflG/aflL, and aflK/aflV, which define five LD blocks [24]. Figure 1 shows a schematic representation of the aflatoxin gene cluster and the regions that were sequenced for LD analysis. LD was examined by 1) combining all sequenced loci for each locality, species and morphotype using SNAP Combine [32] into a single concatenated sequence alignment, 2) collapsing the alignment to infer multi-locus haplotypes using SNAP Map [32] with the options of recoding indels (insertions/deletions) as binary characters and excluding infinite sites violations, and 3) generating an LD plot for all variable positions using the Clade and Matrix [33] programs implemented in SNAP Workbench [34]. LD was quantified using the coefficient of determination (r2) between the allelic states at pairs of sites and a two-sided Fisher's Exact test, as implemented in Tassel version 1.1.0 [35]. LD blocks were based on the number of contiguous pairs of sites that were both strongly correlated (0.8<r2<1) and significantly linked (P<0.01). Because highly divergent haplotypes sampled once or at a low frequency could be potential targets of balancing selection in aflatoxin gene clusters [14], [24], they were not excluded in the LD analyses and the strength of LD was assessed using both r2 and 2×2 contingency tests. All sequences have been deposited in GenBank under Accession numbers HM353147–HM355445 and HM745560–HM745901.

Bottom Line: For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus.A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations.This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

View Article: PubMed Central - PubMed

Affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, Louisiana, United States of America.

ABSTRACT
Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

Show MeSH
Related in: MedlinePlus