Genome sequencing of evolved aspergilli populations reveals robust genomes, transversions in A. flavus, and sexual aberrancy in non-homologous end-joining mutants

RESEARCH ARTICLE Open Access Genome sequencing of evolved aspergilli populations reveals robust genomes, transversions in A. flavus, and sexual aberrancy in non-homologous end-joining mutants Isidro Álvarez-Escribano 1,2† , Christoph Sasse 3† , Jin Woo Bok 4† , Hyunsoo Na 5 , Mojgan Amirebrahimi 5 , Anna Lipzen 5 , Wendy Schackwitz 5 , Joel Martin 5 , Kerrie Barry 5 , Gabriel Gutiérrez 1 , Sara Cea-Sánchez 1 , Ana T. Marcos 1,6 , Igor V. Grigoriev 5,7 , Nancy P. Keller 4,8 , Gerhard H. Braus 3 and David Cánovas 1* Abstract Background: Aspergillus 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. Results: We 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 endjoining (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. Conclusions: We 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. Keywords: Aspergillus, Aflatoxin, Mutation accumulating lines, Genome stability, Non-homologous end-joining, ku70 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] † Isidro Álvarez-Escribano, Christoph Sasse and Jin Woo Bok contributed equally to this work. 1 Department of Genetics, Faculty of Biology, University of Seville, Seville, Spain Full list of author information is available at the end of the article Álvarez-Escribano et al. BMC Biology (2019) 17:88 https://doi.org/10.1186/s12915-019-0702-0 Introduction The taxon of filamentous fungi comprises organisms of great importance for human daily life, as friends or foes. As friends, we take advantage of the capabilities of filamentous fungi to produce enzymes and compounds of interest to humans, such as pharmaceuticals. Some of these enzymes have proved to be highly useful in the production of bio-energy [1,2] and fungal natural products impactful in many applications such as the antifungal echinocandins [3]. Within the filamentous fungi group, aspergilli are widely used for biotechnological applications in industry. In particular, they have become model organisms to study different aspects of the bioenergy production, such as the production of cellulolytic enzymes useful for bioethanol production [4–6], the carbon-regulation of these enzymes [4,7] and natural product synthesis as drug sources [3,8,9]. These studies rely on two important aspects of the biology of aspergilli: their fantastic features for fermentation purposes and the genetic amenability of the genus. The availibilty of many genome sequences of Aspergillus species makes this group of fungi very useful for comparative genomics [10–12]. As foes, 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 [13–16]. However, the evolution of aspergilli is still understudied. One of the main problems studying the evolution of fitness in filamentous fungi is the concept of an individual [17] (see Fig. 1a for a diagram for the morphological structure of a filamentous fungus). Thus, most evolutionary studies in filamentous fungi involved the comparison between different species or extremely laborious phenotypic analysis in combination with genetic techniques, e.g. RFLP analysis [18]. Some previous studies have shown that filamentous fungal evolution can be assessed experimentally under laboratory conditions [19, 20]. Using mutation accumulating (MA) lines for 40 generations, the base pair mutation rate in Aspergillus nidulans was estimated to be 2.26 × 10 −10 using the classical Bateman-Mukai method [21]. The recent availability of the genome sequences can boost the study of filamentous fungal evolution in a laboratory experiment to a level never experienced before. So far, evolution experiments in filamentous fungi have considered growth rates as a measurement of fitness. However, variations in growth rate could be the result of a physiological adaptation to the established growth conditions rather than the consequence of genome evolution. The stability of genomes highly depends on the lifestyle and the complexity of each organism. Whereas viruses have very dynamic genomes, eukaryotic genomes are highly stable and eukaryotes do not cope well with insults to the genome. Yet cells are continuously exposed to DNA damaging agents. Therefore, the stability of the genome relies on a complex molecular machinery that repairs the damage. One such example of DNA repair machinery is the non-homologous end-joining (NHEJ), which joins two DNA molecules that had suffered double-strand breaks (DSBs) [22–24]. Thus, a doubt arose with the discovery of the NHEJ genes in filamentous fungi and the use of the NHEJ deletions as genetic tools [25]. The underlying fundamental question behind the doubt is what is the role of the NHEJ system in the stability of an eukaryotic genome. Although this is a fundamental question in biology, the knowledge has direct consequences on biotechnology with the use of NHEJ mutants to generate new fungal strains with desired genetic modifications. The fungal homologues of the KU70 and KU80 required for NHEJ were first identified in Neurospora crassa [25]. Following the spectacular results of N. crassa Δku70 strains as genetic tools, NHEJ mutants have been constructed in many other fungal species (for some examples, see [26–31]). N. crassa NHEJ mutants are more sensitive to bleomycin, a compound producing double-strand DNA breaks [25], although an A. nidulans NHEJ mutant did not appear to show increase sensitivity to this drug [32]. However, there is an additional body of data generated in fungi that suggests that the KU70/KU80 heterodimer plays additional roles. For example, N. crassa and Aspergillus fumigatus mutants, but not A. nidulans, are more sensitive to agents producing point mutations [25,33,34], which were presumed to get repaired using different mechanisms. They are telomere-associated proteins contributing to the formation of heterochromatin in the telomeres [35]. In A. nidulans,NkuA KU70 appears to regulate telomere position effects [36]. Both ku70 and ku80 homologues were found to be overexpressed during asexual reproduction (conidiation) in a histone acetyltransferase mutant, suggesting that they are involved in the maintenance of genome stability in conidia, DNA repair or the responses to DNA stress [37]. In haploid cells, as Aspergillus species normally propagate, DNA repair by homologous recombination (HR) of doublestrand breaks is only possible in S and G2 phases of the cell cycle, when a sister chromatid is available. Therefore, the repair of double-strand breaks is expected to be more error-prone in a haploid because of the higher likelihood of using NHEJ repair. As these additional roles of the KU70/KU80 heterodimer on fungal biology and potentially DNA stability have emerged, it is prudent for the academic community and industry to determine if the NHEJ mutants display concerning genome instability properties. A stable genome is especially important for biotechnology applications, where Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 2 of 17 Fig. 1 (See legend on next page.) Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 3 of 17 much time and money is spent to optimize processes and a small change in yield can have large financial consequences. In addition to insights on Aspergillus genome evolution and stability, we expect our results will be widely applicable to other fungi where the use of NHEJ mutants is expanding [28,38,39]. Therefore, and considering that strains lacking KU70/KU80 are commonplace in biotechnology, we decided to test the global consequences of such mutants for the stability of the genome. We studied the genome stability of aspergilli by using a combination of MA lines and whole genome sequencing (WGS). We found that Aspergillus genomes are very robust and the deletion of the 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. Results 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. All species were already sequenced before this work started, and they have genome sizes ranging between 29.4 and 36.8 Mb [11,40]. Ten independent MA lines for each strain (wild type and Δku70 of each species) were set up by picking ten individual isolated colonies as described in the “Materials and methods” section, and the strains were allowed to accumulate mutations by growing in complete media for 60 passages (Fig. 1b). Based on the nuclear division time in A. nidulans and previous calculations [19,41] we estimated that nuclei underwent 4032 mitotic divisions. This strategy allowed the 10 populations of each strain to accumulate mutations independently from each other along the passages (Fig. 1b). Samples were taken at time 0 (controls) and at the end of the MA experiment for WGS. The starting strains before the MA lines were established, together with 2 of the 10 independent MA lines set at the beginning and all the 10 independent MA lines after the 60 growth passages were employed for DNA isolation. There were several reasons for sequencing the populations at passage 0. First, the Δku70 deletion strain of A. fumigatus is in the strain D141, a mat1-1 isolate (rather than in the mat1-2 strain Af293, which was the sequenced one). Sequencing of the controls at time 0 solved this issue and also provided the sequence of D141 strain, which was largely syntenic to the Af293 genome. In the case of A. nidulans, the strains employed in this work are derived from the sequenced wild-type strain FGSC4. The DNA from all the populations was sequenced with 100× coverage on average. This coverage was selected to allow the identification of mutations that were present at low frequency and not fixed in the (nuclear) populations yet. Previous reports using average coverages of 44× and 62×, which are lower than ours, calculated a false-positive error close to zero by Sanger sequencing [42,43]. Using this strategy, we were able to identify mutations with a frequency as low as 10% that were unique to one population (Fig. 2a). Mutation rate Globally in the MA lines, we identified a total of 122 single nucleotide variants (SNVs) and 17 insertions/deletions (indels) in A. flavus, 27 SNVs and 10 indels in A. fumigatus and 75 SNVs and 29 indels in A. nidulans. Out of those mutations, the wild-type MA lines accumulated 58 SNVs and 5 indel in A. flavus, 10 SNVs and 2 indels in A. fumigatus and 32 SNVs and 18 indels in A. nidulans.Aspergillus species like other filamentous fungi are multicellular and multinucleated organisms, i.e. the hyphal filament consists of connected cell compartments, each containing several nuclei (Fig. 1a). Consequently, for the estimation of the mutation rate, we need to consider nuclear divisions (mitoses) rather than cell divisions as previously employed for unicellular eukaryotes [42–47]. As mentioned above, the estimation of the number of mitoses is complicated in the case of filamentous fungi. Our calculations of the number of mitoses are based on previous cell cycle studies performed by Bergen and Morris on liquid media [41], which did not differ substantially from studies by Kaminskyj and Hamer [48] on solid media. The number of nuclear divisions occurring during conidiophore development was not taken into account because the division times and nuclear dynamics in the stalk and vesicle cells are not well characterized. This is partially compensated by the fact that the samples for the cultures passages were taken from the conidiation edge of the colony, which is (See figure on previous page.) Fig. 1 Diagram depicting the design of the evolution experiment in laboratory. aDiagram depicting the morphological characteristics of filamentous fungi. Growth occurred by apical extension at the tip. The older parts of the filament are separated in cell compartments by incomplete septa. The cell compartments are multinucleated. After the correct signals are perceived, the stalk arises from the filament. A morphogenetic program will control the development of the conidiophore, which harbours the uninucleated conidia. bA spore prep of each strain was diluted and plated to obtain isolated colonies. Ten random colonies showing normal wild-type characteristics were selected to establish each MA line and inoculated in an individual plate containing complete media. Fungal colonies were allowed to grow for 3 days at 37 °C (30 °C for A. flavus). One plug was taken from the conidiating edge of each plate, conidia were resuspended in 1000 μl of tween buffer and 10 μl were used to inoculate a new fresh plate. The procedure was repeated alternatively every 3 and 4 days (twice a week) 60 times to reach approximately 4032 mitosis Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 4 of 17 far behind from the real growing colony front. This strategy to estimate the number of mitoses during the growth passages has been previously carried out by Schoustra et al. [19]. This approach gave a total mutation rate of 4.2 × 10 −11 ±5.6×10 −12 (A. flavus), 1.1 × 10 −11 ± 4.1 × 10 −12 (A. fumigatus) and 4.1 × 10 −11 ±6.4×10 −12 (A. nidulans) per base per mitosis and an average mutation rate for all three species of 3.1 × 10 −11 ±5.4×10 −12 per base per mitosis revealing that indeed Aspergillus genomes are very robust in comparison to yeasts and higher eukaryotes. The distribution of mutations across the MA lines was consistent with a uniform distribution (Kolmogorov-Smirnov test (K-S test); P= 0.93, 0.31 and 0.91 for A. flavus,A. fumigatus and A. nidulans, respectively). The Δku70 deletion mutants of the three species accumulated a slightly higher number of mutations: 64 SNVs and 12 indels in A. flavus,17SNVsand8indelsinA. fumigatus and 43 SNVs and 11 indels in A. nidulans (Fig. 2b), which gives an estimate of a total mutation rate of 5.1 × 10 −11 ±4.7×10 −12 ,2.2×10 −11 ±3.8×10 −12 and 4.5 × 10 −11 ±8.1×10 −12 per base per mitosis for A. flavus,A. fumigatus and A. nidulans, respectively, and an average mutation rate for all three species of 3.9 × 10 −11 ±5.5× 10 −12 per base per mitosis. The distribution of mutations across the MA lines of the Δku70 mutants was also consistent with a uniform distribution (K-S test; P= 1.00, 0.77 and 0.97 for A. flavus,A. fumigatus and A. nidulans, respectively). The differences between the wild-type and the Δku70 mutant strains for each species are not statistically significant (two-way ANOVA with Tukey’s post hoc test, P= 0.675 for each pair of strains; two-tailed ttest, P= 0.33, P=0.14 and P=0.67 for A. flavus,A. fumigatus and A. nidulans, respectively). However, if we did not consider the different species, and we take into account only the genetic background combining the data from the three wild-type strains and the data from the three Δku70 strains, then the differences between the wild-type and the mutant backgrounds are statistically significant (two-way ANOVA with Tukey’s post hoc test, P= 0.025), suggesting that the NHEJ has a role in the genome stability of aspergilli. Distribution of mutations The concept of an individual in filamentous fungi is a matter of debate and the definition of a physical individual a Sites examined Real sites A. flavus 1,327 139 A. fumigatus 144 37 A. nidulans 427 104 b WT ku70 0 1 2 3 4 5 6 7 8 9 A. flavus A. fumigatus A. nidulans Average number of mutations / MA line Fig. 2 Number of mutations accumulated in the MA lines after 4032 mitosis. aTable of the total number of mutations found in the three species before and after filtering. bAverage number of mutations per MA line in the wild-type and the Δku70 mutant strains in all three Aspergillus species. Lighter colours represent wild-type strains (WT), and darker colours represent the Δku70 mutants of each species. Error bars depict the standard error of the mean Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 5 of 17 has been even considered a “hopeless task”[17]. Therefore, we considered our MA lines as populations of nuclei. Thanks to the high coverage of the sequencing, it was possible to detect mutations present in as low as 10% of the nuclei. A boxplot representation of the frequency of all the mutations in the six strains showed that there were a larger proportion of mutations that were not fixed in the Δku70 mutants than in the wild-type strain, suggesting that the mutations in the Δku70 mutants keep appearing at a slightly faster rate than in the wild-type strain (Additional file 1: Figure S1). The distribution of single nucleotide mutations in the eight chromosomes appeared to be random and not statistically different from the expected number according to their sizes (K-S test; P= 0.92 and 0.92 for A. fumigatus wild-type and Δku70 mutant, and 0.92 and 1.00 for A. nidulans wild-type and Δku70 mutant, respectively) (Additional file 2: Figure S2). In the particular case of A. flavus, this analysis was complicated because the 138 contigs are not assigned to specific chromosomes. We tried to map these contigs to the chromosomes of the closely related species A. oryzae with little success. Therefore, we performed the analysis asking the question whether the mutations were uniformly distributed in the 138 contigs using the K-S test. In this case, we found that P< 0.001, meaning that the mutations were not uniformly distributed. Further analysis revealed that all the mutations were located in 20 contigs, which account for 99.25% of the total length of the genome, while the other 118 contigs only provide the remaining 0.75% of the genome, and consequently represent a minor portion of the genome. We repeated the test using only the 20 big contigs accounting for 99.25% of the genome and containing the mutations. In this case, P= 0.68 suggests that the distribution of mutations is random in these 20 contigs and in the genome of A. flavus. Single nucleotide mutations and spectrum Out of the 280 total number of mutations, we identified a total of 224 substitutions after filtering and confirmation by visual inspection, of which 58, 10 and 32 were found in wild-type A. flavus,A. fumigatus and A. nidulans, respectively, and 64, 17 and 43 were found in the Δku70 mutants of A. flavus,A. fumigatus and A. nidulans, respectively. That gives a base-substitution mutation rate of 3.9 × 10 −11 ± 4.6 × 10 −12 (A. flavus WT), 8.9 × 10 −12 ± 3.2 × 10 −12 (A. fumigatus WT) and 2.6 × 10 −11 ± 4.7 × 10 −12 (A. nidulans WT) per base per mitosis, which is one to two orders of magnitude lower than in ascomycete and basidiomycete yeasts [42,43,46, 47,49]. The base-substitution rate for the Δku70 mutants was slightly higher: 4.3 × 10 −11 ± 3.5 × 10 −12 (A. flavus Δku70), 1.5 × 10 −11 ± 3.3 × 10 −12 (A. fumigatus Δku70) and 3.5 × 10 −11 ± 7.9 × 10 −12 (A. nidulans Δku70) per base per mitosis, but still lower than in the yeasts (Fig. 3). We found the spectrum of base-substitution mutations varied enormously between the species. The transition/ transversion (ts/tv) ratio was extremely low in A. flavus (0.3) and higher in A. fumigatus (2.3) and A. nidulans (1.9). However, the number of base-substitution mutations in A. fumigatus was very low. The number of transitions and transversions and the ts/tv ratio did not change significantly between the wild-type and the Δku70 mutant strains (Fig. 4a). The major changes found in the Δku70 mutants with respect to the wildtype strains were that the difference between the number of transitions and transversions was statistically significant in A. fumigatus Δku70 (two-tailed ttest; P=0.027) but not in the wild type (two-tailed ttest; P=0.36). In A. nidulans, the number of transversions (22) increased to reach nearly the number of transitions (21) in the Δku70, and consequently, the ts/tv ratio was close to 1. Considering the very low ts/tv ratio in A. flavus,we note that this species produces the toxic and mutagenic secondary metabolite aflatoxin, well known for its induction of G→TandC→A transversions in human p53 gene [50,51]. Following this fact, it was interesting to observe a strong G/C to A/T bias in A. flavus (Fig. 5a). Specifically, there were 5.8 times more G/C to A/T mutations than A/T to G/C mutations. This strong bias was not observed in A. fumigatus (1.0×) or A. nidulans (2.4×). This mutation bias towards A/T seemed to have been observed previously in many other species ([44]). However, it is only found in A. flavus and not in the other two Aspergillus species, which do not produce aflatoxin, although A. nidulans produces the aflatoxin precursor sterigmatocystin. In A. flavus MA lines, the GC→TA transversions are particularly enriched (χ 2 ;P< 0.001; df = 5), while in A. nidulans MA lines the GC→AT transitions are the most frequent substitutions (χ 2 ;P< 0.001; df = 5). The most frequent SNPs were 31 times C→A and 27 times G→TinA. flavus, 6 times A→G, 5 times G→A and T→CinA. fumigatus and 22 times C→T and 17 times G→AinA. nidulans (Fig. 5b). According to these estimates, the genomes of A. flavus and A. nidulans have not reached a nucleotide-content equilibrium. The expected G + C% would be 15% in A. flavus and 29% in A. nidulans based solely on the basesubstitution mutation rate. However, the G + C% content is 50.5% and 52.2% in the genic regions and 45.1% and 47.4% in the intergenic regions of A. flavus and A. nidulans, respectively. Since the differences between the expected and the observed G + C were very different, we further looked at the frequency of G + C in the third position in the triplet of the fourfold degenerate codons. A plot of the G + C% of each gene vs the G + C% of the synonymous sites revealed that Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 6 of 17 the genome of A. flavus shows a higher dispersion ofthedatawithalowercoefficient of determination (Additional file 3:FigureS3).Theregression coefficient (slope) in A. flavus is also the lowest of the three, suggesting that A. flavus has the lowest bias towards G + C of the three species, which is in agreement with the observed data. The analysis of the trinucleotide context can provide information about the causes of the mutations. Therefore, we also analysed the trinucleotide context of the base-substitution mutations, considering the nucleotides adjacent immediately before and after to the mutated nucleotide. This gives 64 possible combinations that can be reduced to 32 possibilities combining strand orientation nucleotides [46]. The mutated nucleotide together with the neighbouring positions can be assigned to one of these 32 trinucleotides. From this, it was clear that G/ C were mutated on average 6.6 and 2.4 times more than A/T in A. flavus and A. nidulans, respectively (Fig. 6). Although in A. flavus the trinucleotide that was most frequently mutated was ACA, the CpG environment accounted for 28% of the base-substitution mutations, while NCA accounted for 25%. In A. nidulans however, the most frequent trinucleotide was TCT, and NCT accounted for 24%, which was far higher than the 14% of mutations observed in CpG. Most of the mutations in all the species were either non-synonymous (missense) or intergenic (Fig. 7, Additional file 4: Figure S4). In Additional file 5:TableS1, Additional file 6: Table S2 and Additional file 7:TableS3, there is a list with the genes found to contain mutations in our MA experiment. When we compared the total number of mutations in the wild-type strains with the Δku70 strains for each species, we found only a clear trend in the intergenic mutations (23 in the wild type vs 29 in the Δku70,2 vs 10 and 22 vs 29 in A. flavus,A. fumigatus and A. nidulans, respectively). The ratio of synonymous to nonsynonymous mutations was 0.21, 0.29 and 0.14 in the wildtype strains of A. flavus,A. fumigatus and A. nidulans,respectively, and 0.28, 0.09 and 0.41 in the Δku70 mutants of A. flavus,A. fumigatus and A. nidulans, respectively. Indel mutations Out of the 280 total number of mutations, 56 indel mutations were identified in the six strains of Aspergillus, 19 were deletions and 37 were insertions, which on average accounts for 1.9 times more insertions than deletions (Fig. 4b). The distribution varied between species, being A. nidulans the species accumulating more indel mutations (7 deletions and 11 insertions in the wild-type strain). This is 1.8 times lower than the number of basesubstitution mutations. The low proportion of indel Rhodotorula toruloides Saccharomyces cerevisiae Aspergillus WT (pooled species) Chlamydomonas reinhardtii Paramecium tetraurelia Tetrahymena thermophila Caenorhabditis elegans Schizosaccharomyces pombe Drosophila melanogaster Arabidopsis thaliana germline A spergillus ku70 (pooled species) Base-substitution mutation rate (per site per generation) 10-12 10-11 10-10 10-9 10-8 Fig. 3 The spontaneous mutation rate in Aspergillus species compared to other eukaryotes. The data of Aspergillus species is presented as an average of the three species under study for the wild-type and the Δku70 mutant strains. The data from the other eukaryotes were obtained from [42–47,49,79–83]. Data shown refers to base-substitution mutations Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 7 of 17 mutations found in aspergilli (ranging from 0.08 to 0.36) is consistent with other organisms. In general, indels were observed to be increased in Δku70 mutants compared to the corresponding wild-type strain. However, insertions in the Δku70 were only significantly increased in A. flavus (one-tailed ttest; P= 0.038) and deletions in A. fumigatus (one-tailed ttest; P= 0.033). In A. nidulans, the number of indel mutations was even higher in the wild type (18 indel mutations) than in the Δku70 mutant (11 indel mutations), with a particular but not significant increase in the number of deletions (7 in the wild type vs 2intheΔku70 mutant strain) (one-tailed ttest; P=0.09). Fig. 4 The spectrum of mutation types differs between the three Aspergillus species. aAverage number of base-substitution mutations in the wild-type (WT) and Δku70 mutant (KU) of each species. In A. flavus, the number of transversions is higher than the number of transitions, opposite to what was found in A. fumigatus.InA. nidulans, the number of transitions is higher than the number of transversions in the wild-type strain but not in the Δku70 mutant. bAverage number of indel mutations in the wild-type and Δku70 mutant of each species. The number of indels is higher in A. nidulans than in the other two aspergilli, but in this case, the Δku70 mutation seems to protect the genome against indels opposite to what happens in the other two aspergilli. Error bars show the standard error of the mean. Statistically significant differences are shown with *P< 0.05 and **P< 0.01 (one-tailed ttest) Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 8 of 17 Based on these data, we estimated an indel mutation rate of 3.4 × 10 −12 ±1.5×10 −13 (A. flavus), 1.8 × 10 −12 ±1.2× 10 −13 (A. fumigatus) and 1.5 × 10 −11 ±4.4×10 −13 (A. nidulans) per base per mitosis for the wild-type strains and 8.1 × 10 −12 ±2.2×10 −13 (A. flavus), 7.1 × 10 −12 ±2.2× 10 −13 (A. fumigatus) and 9.1 × 10 −12 ±1.5×10 −13 (A. nidulans) per base per mitosis for the Δku70 mutant strains. Cessation of cleistothecia development in the sexual MA lines of the A. nidulans NHEJ mutants A. nidulans is the only homothallic species of these three. Self-crossing provides a framework in which any mutations or changes in genome sequence or structure that arise during the meiotic divisions are not due to the genetic heterogeneity of two parental strains. Taking advantage of this characteristic, a similar procedure was carried out with A. nidulans during sexual reproduction. We employed the same 10 independent MA lines established for the asexual passages. This scenario combined mitotic with meiotic divisions. As it can be observed in Fig. 8, all the Δku70 MA lines stopped producing cleistothecia after 5–9 passages. On the other hand, most of the wild-type MA lines (7 out of 10) still produced cleistothecia after 10 passages. We can assume that the cessation of cleistothecia production was not a consequence of the accumulation of mutations, as only 15 mutations were identified in 9 populations of the Δku70 strain after 5 passages and 5 mutations were found in the wild-type MA lines, and none of them seem to play an essential role (Additional file 8: Table S4). Therefore, the causes of cessation in the production of cleistothecia could be either epigenetics, or big chromosomal re-arrangements, or shortening of the telomeres. Although null mutations in histone modification genes associated with defects in 0 10 20 30 40 50 60 70 AT->GC GC->AT AT->CG AT->TA GC->CG GC->TA Number of mutations A. flavus A. fumigatus A. nidulans 0 5 10 15 20 25 30 35 Number of mutations A. flavus A. fumigatus A. nidulans a b Fig. 5 The spectrum of base-substitution mutations differs between the three Aspergillus species. aThe total number of mutations for the six possible transitions and transversions during the evolution of the six strains of aspergilli.bAll possible 12 substitutions in the six strains. Changes of C or G are particularly elevated in A. flavus and A. nidulans Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 9 of 17 17. Pringle A, Taylor J. The fitness of filamentous fungi. Trends Microbiol. 2002; 10(10):474–81. 18. Xu J. Analysis of inbreeding depression in Agaricus bisporus. Genetics. 1995; 141(1):137–45. 19. Schoustra SE, Debets AJ, Slakhorst M, Hoekstra RF. Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulans. J Evol Biol. 2006;19(4):1115–27. 20. Schoustra SE, Slakhorst M, Debets AJ, Hoekstra RF. Comparing artificial and natural selection in rate of adaptation to genetic stress in Aspergillus nidulans. J Evol Biol. 2005;18(4):771–8. 21. Bruggeman J, Debets AJ, Wijngaarden PJ, deVisser JA, Hoekstra RF. Sex slows down the accumulation of deleterious mutations in the homothallic fungus Aspergillus nidulans. Genetics. 2003;164(2):479–85. 22. Davis AJ, Chen DJ. DNA double strand break repair via non-homologous end-joining. Transl Cancer Res. 2013;2(3):130–43. 23. Dudasova Z, Dudas A, Chovanec M. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol Rev. 2004;28(5):581–601. 24. Walker JR, Corpina RA, Goldberg J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature. 2001; 412(6847):607–14. 25. Ninomiya Y, Suzuki K, Ishii C, Inoue H. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A. 2004;101(33):12248–53. 26. El-Khoury R, Sellem CH, Coppin E, Boivin A, Maas MF, Debuchy R, Sainsard- Chanet A. Gene deletion and allelic replacement in the filamentous fungus Podospora anserina. Curr Genet. 2008;53(4):249–58. 27. Nakazawa T, Honda Y. Absence of a gene encoding cytosine deaminase in the genome of the agaricomycete Coprinopsis cinerea enables simple marker recycling through 5-fluorocytosine counterselection. FEMS Microbiol Lett. 2015;362(15):fnv123. 28. Gandia M, Xu S, Font C, Marcos JF. Disruption of ku70 involved in nonhomologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum. Fungal biology. 2016;120(3):317–23. 29. Verbeke J, Beopoulos A, Nicaud JM. Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol Lett. 2013;35(4):571–6. 30. Choquer M, Robin G, Le Pecheur P, Giraud C, Levis C, Viaud M. Ku70 or Ku80 deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to knock out in a wild-type context. FEMS Microbiol Lett. 2008;289(2):225–32. 31. Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang T, Seidl V, Seiboth B. Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol. 2009;139(2):146–51. 32. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA, Oakley BR. A versatile and efficient gene targeting system for Aspergillus nidulans. Genetics. 2006;172(3):1557–66. 33. da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, Brakhage AA, Goldman GH. The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell. 2006;5(1):207–11. 34. Nayak T, Edgerton-Morgan H, Horio T, Xiong Y, De Souza CP, Osmani SA, Oakley BR. Gamma-tubulin regulates the anaphase-promoting complex/ cyclosome during interphase. J Cell Biol. 2010;190(3):317–30. 35. Nandakumar J, Cech TR. Finding the end: recruitment of telomerase to telomeres. Nat Rev Mol Cell Biol. 2013;14(2):69–82. 36. Palmer JM, Mallaredy S, Perry DW, Sanchez JF, Theisen JM, Szewczyk E, Oakley BR, Wang CC, Keller NP, Mirabito PM. Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans. Microbiology. 2010;156(Pt 12):3522–31. 37. Canovas D, Marcos AT, Gacek A, Ramos MS, Gutierrez G, Reyes-Dominguez Y, Strauss J. The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development. Genetics. 2014;197(4):1175–89. 38. Catalano V, Vergara M, Hauzenberger JR, Seiboth B, Sarrocco S, Vannacci G, Kubicek CP, Seidl-Seiboth V. Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation. Curr Genet. 2011;57(1):13–23. 39. Liu H, Wang G, Li W, Liu X, Li E, Yin WB. A highly efficient genetic system for the identification of a harzianum B biosynthetic gene cluster in Trichoderma hypoxylon. Microbiology. 2018;164:769–78. 40. Nierman WC, Yu J, Fedorova-Abrams ND, Losada L, Cleveland TE, Bhatnagar D, Bennett JW, Dean R, Payne GA. Genome sequence of Aspergillus flavus NRRL 3357, a strain that causes aflatoxin contamination of food and feed. Genome announcements. 2015;3(2):e00168–15. 41. Bergen LG, Morris NR. Kinetics of the nuclear division cycle of Aspergillus nidulans. J Bacteriol. 1983;156(1):155–60. 42. Farlow A, Long H, Arnoux S, Sung W, Doak TG, Nordborg M, Lynch M. The spontaneous mutation rate in the fission yeast Schizosaccharomyces pombe. Genetics. 2015;201(2):737–44. 43. Behringer MG, Hall DW. Genome-wide estimates of mutation rates and spectrum in Schizosaccharomyces pombe indicate CpG sites are highly mutagenic despite the absence of DNA methylation. G3. 2015;6(1):149–60. 44. Sung W, Tucker AE, Doak TG, Choi E, Thomas WK, Lynch M. Extraordinary genome stability in the ciliate Paramecium tetraurelia. Proc Natl Acad Sci U S A. 2012;109(47):19339–44. 45. Long H, Winter DJ, Chang AY, Sung W, Wu SH, Balboa M, Azevedo RBR, Cartwright RA, Lynch M, Zufall RA. Low base-substitution mutation rate in the germline genome of the ciliate Tetrahymena thermophil. Genome Biol Evol. 2016;8(12):3629–39. 46. Zhu YO, Siegal ML, Hall DW, Petrov DA. Precise estimates of mutation rate and spectrum in yeast. Proc Natl Acad Sci U S A. 2014;111(22):E2310–8. 47. Long H, Behringer MG, Williams E, Te R, Lynch M. Similar mutation rates but highly diverse mutation spectra in ascomycete and basidiomycete yeasts. Genome Biol Evol. 2016;8(12):3815–21. 48. Kaminskyj SG, Hamer JE. hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans. Genetics. 1998;148(2):669–80. 49. Lynch M, Sung W, Morris K, Coffey N, Landry CR, Dopman EB, Dickinson WJ, Okamoto K, Kulkarni S, Hartl DL, et al. A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc Natl Acad Sci U S A. 2008;105(27): 9272–7. 50. Lasky T, Magder L. Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure? Environ Health Perspect. 1997;105(4):392–7. 51. Zhang W, He H, Zang M, Wu Q, Zhao H, Lu LL, Ma P, Zheng H, Wang N, Zhang Y, et al. Genetic features of aflatoxin-associated hepatocellular carcinoma. Gastroenterology. 2017;153(1):249–62 e242. 52. Lee DW, Freitag M, Selker EU, Aramayo R. A cytosine methyltransferase homologue is essential for sexual development in Aspergillus nidulans. PLoS One. 2008;3(6):e2531. 53. Carvalho ND, Arentshorst M, Jin Kwon M, Meyer V, Ram AF. Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl Microbiol Biotechnol. 2010;87(4):1463–73. 54. Chang PK, Scharfenstein LL, Wei Q, Bhatnagar D. Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus.J Microbiol Methods. 2010;81(3):240–6. 55. Krappmann S, Sasse C, Braus GH. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous endjoining- deficient genetic background. Eukaryot Cell. 2006;5(1):212–5. 56. Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF. Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 2007;128(4):770–5. 57. Nakazawa T, Ando Y, Kitaaki K, Nakahori K, Kamada T. Efficient gene targeting in ΔCc.ku70 or ΔCc.lig4 mutants of the agaricomycete Coprinopsis cinerea. Fungal Genet Biol. 2011;48(10):939–46. 58. Zhang J, Mao Z, Xue W, Li Y, Tang G, Wang A, Zhang Y, Wang H. Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger. Curr Microbiol. 2011;62(4):1342–6. 59. de Boer P, Bastiaans J, Touw H, Kerkman R, Bronkhof J, van den Berg M, Offringa R. Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants. Fungal Genet Biol. 2010; 47(10):839–46. 60. Bugeja HE, Boyce KJ, Weerasinghe H, Beard S, Jeziorowski A, Pasricha S, Payne M, Schreider L, Andrianopoulos A. Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei. Fungal Genet Biol. 2012;49(10):772–8. 61. Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, Morin D, Bruel C, Beffa R, Lebrun MH. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genet Biol. 2008;45(1):68–75. 62. Lynch M. Evolution of the mutation rate. Trends Genet. 2010;26(8):345–52. Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 16 of 17 63. Huertas P. DNA resection in eukaryotes: deciding how to fix the break. Nat Struct Mol Biol. 2010;17(1):11–6. 64. Hopfner KP, Putnam CD, Tainer JA. DNA double-strand break repair from head to tail. Curr Opin Struct Biol. 2002;12(1):115–22. 65. Aguilera A, Gomez-Gonzalez B. Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet. 2008;9(3):204–17. 66. Antequera F, Tamame M, Villanueva JR, Santos T. DNA methylation in the fungi. J Biol Chem. 1984;259(13):8033–6. 67. Liu SY, Lin JQ, Wu HL, Wang CC, Huang SJ, Luo YF, Sun JH, Zhou JX, Yan SJ, He JG, et al. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation. PLoS One. 2012;7(1):e30349. 68. Tamame M, Antequera F, Villanueva JR, Santos T. High-frequency conversion to a “fluffy”developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene. Mol Cell Biol. 1983;3(12):2287–97. 69. Smela ME, Currier SS, Bailey EA, Essigmann JM. The chemistry and biology of aflatoxin B(1): from mutational spectrometry to carcinogenesis. Carcinogenesis. 2001;22(4):535–45. 70. Kuczuk MH, Benson PM, Heath H, Hayes AW. Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae. Mutat Res. 1978;53(1):11–20. 71. Kale SP, Milde L, Trapp MK, Frisvad JC, Keller NP, Bok JW. Requirement of LaeA for secondary metabolism and sclerotial production in Aspergillus flavus. Fungal Genet Biol. 2008;45(10):1422–9. 72. Pfannenstiel BT, Greco C, Sukowaty AT, Keller NP. The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus. Fungal Genet Biol. 2018;120:9–18. 73. Lan H, Wu L, Fan K, Sun R, Yang G, Zhang F, Yang K, Lin X, Chen Y, Tian J, et al. Set3 is required for asexual development, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus. Front Microbiol. 2019;10:530. 74. Lan H, Wu L, Sun R, Keller NP, Yang K, Ye L, He S, Zhang F, Wang S. The HosA histone deacetylase regulates aflatoxin biosynthesis through direct regulation of aflatoxin cluster genes. Mol Plant Microbe Interact. 2019;32(9): 1210–28. 75. Cove DJ. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta. 1966;113(1):51–6. 76. Schoustra SE, Bataillon T, Gifford DR, Kassen R. The properties of adaptive walks in evolving populations of fungus. PLoS Biol. 2009;7(11):e1000250. 77. Li H, Durbin R. Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics. 2009;25(14):1754–60. 78. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–6. 79. Denver DR, Wilhelm LJ, Howe DK, Gafner K, Dolan PC, Baer CF. Variation in base-substitution mutation in experimental and natural lineages of Caenorhabditis nematodes. Genome Biol Evol. 2012;4(4):513–22. 80. Drost JB, Lee WR. Biological basis of germline mutation: comparisons of spontaneous germline mutation rates among drosophila, mouse, and human. Environ Mol Mutagen. 1995;25(Suppl 26):48–64. 81. Ness RW, Morgan AD, Vasanthakrishnan RB, Colegrave N, Keightley PD. Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii. Genome Res. 2015;25(11):1739–49. 82. Ossowski S, Schneeberger K, Lucas-Lledo JI, Warthmann N, Clark RM, Shaw RG, Weigel D, Lynch M. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science. 2010;327(5961):92–4. 83. Saxer G, Havlak P, Fox SA, Quance MA, Gupta S, Fofanov Y, Strassmann JE, Queller DC. Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum. PLoS One. 2012;7(10):e46759. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Álvarez-Escribano et al. BMC Biology (2019) 17:88 Page 17 of 17