Abstract
BackgroundAspergillus spp. comprises a very diverse group of lower eukaryotes with a high relevance for industrial applications and clinical implications. These multinucleate species are often cultured for many generations in the laboratory, which can unknowingly propagate hidden genetic mutations. To assess the likelihood of such events, we studied the genome stability of aspergilli by using a combination of mutation accumulation (MA) lines and whole genome sequencing.ResultsWe sequenced the whole genomes of 30 asexual and 10 sexual MA lines of three Aspergillus species (A. flavus, A. fumigatus and A. nidulans) and estimated that each MA line accumulated mutations for over 4000 mitoses during asexual cycles. We estimated mutation rates of 4.2 × 10−11 (A. flavus), 1.1 × 10−11 (A. fumigatus) and 4.1 × 10−11 (A. nidulans) per site per mitosis, suggesting that the genomes are very robust. Unexpectedly, we found a very high rate of GC → TA transversions only in A. flavus. In parallel, 30 asexual lines of the non-homologous end-joining (NHEJ) mutants of the three species were also allowed to accumulate mutations for the same number of mitoses. Sequencing of these NHEJ MA lines gave an estimated mutation rate of 5.1 × 10−11 (A. flavus), 2.2 × 10−11 (A. fumigatus) and 4.5 × 10−11 (A. nidulans) per base per mitosis, which is slightly higher than in the wild-type strains and some ~ 5–6 times lower than in the yeasts. Additionally, in A. nidulans, we found a NHEJ-dependent interference of the sexual cycle that is independent of the accumulation of mutations.ConclusionsWe present for the first time direct counts of the mutation rate of filamentous fungal species and find that Aspergillus genomes are very robust. Deletion of the NHEJ machinery results in a slight increase in the mutation rate, but at a rate we suggest is still safe to use for biotechnology purposes. Unexpectedly, we found GC→TA transversions predominated only in the species A. flavus, which could be generated by the hepatocarcinogen secondary metabolite aflatoxin. Lastly, a strong effect of the NHEJ mutation in self-crossing was observed and an increase in the mutations of the asexual lines was quantified.
Highlights
The taxon of filamentous fungi comprises organisms of great importance for human daily life, as friends or foes
We found that Aspergillus genomes are very robust and the deletion of the non-homologous endjoining (NHEJ) machinery results in a slight increase in the mutation rate, but at a rate, which we postulate, is still safe to use for biotechnology purposes
mutation accumulation (MA) lines, sequencing and identification of the mutations Three different aspergilli species have been selected based on their different characteristics and relevance in the aspergilli community: A. nidulans, A. flavus and A. fumigatus
Summary
The taxon of filamentous fungi comprises organisms of great importance for human daily life, as friends or foes. Within the filamentous fungi group, aspergilli are widely used for biotechnological applications in industry They have become model organisms to study different aspects of the bioenergy production, such as the production of cellulolytic enzymes useful for bioethanol production [4,5,6], the carbon-regulation of these enzymes [4, 7] and natural product synthesis as drug sources [3, 8, 9]. Some Aspergillus species are pathogens of humans, animals and/or plants, or appear as dangerous contaminants of crops before or after the harvest They are capable of producing an ample diversity of secondary metabolites, such as the carcinogen aflatoxin [13,14,15,16]. To assess the likelihood of such events, we studied the genome stability of aspergilli by using a combination of mutation accumulation (MA) lines and whole genome sequencing
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