Complete mitochondrial genome sequence of Aspergillus flavus SRRC1009: insight of intraspecific variations on A. flavus mitochondrial genomes.

The mitogenome of Aspergillus flavus SRRC1009 was sequenced to investigate intraspecific variations on mitochondrial genomes of A. flavus. It shows 29,202 bp with a typical configuration of Aspergillus mitogenome. Sixteen SNPs and 22 INDELs and 17 SNPs and 27 INDELs were identified against AflaGuard® and JQ355000, respectively. Phylogenetic trees present in the three A. flavus mitochondrial genomes were clustered with A. oryzae mitochondrial genome in one clade.

Aspergillus flavus produces aflatoxin, a notorious mycotoxin that can cause liver cancer (Squire 1981). To date many genome sequences of A. flavus were sequenced (Payne et al. 2006; Nierman et al. 2015; Weaver et al. 2017); however, only three mitogenomes are available (Joardar et al. 2012; Yan et al. 2016; Park et al., under review). To understand intraspecific variations on A. flavus mitogenomes, we completed the mitogenome of A. flavus SRRC1009.

DNA was extracted using the optimized protocol (Lee et al. 2017). Raw data generated by HiSeq2500 and de novo assembly were conducted by Velvet v1.2.10 (Zerbino and Birney 2008). Gap filling was done by SOAPGapCloser v1.12 (Zhao et al. 2011). Basepair confirmation was done by BWA v0.7.17 and SAMtools v1.9 (Li et al. 2009; Li 2013) under the environment of Genome Information System (GeIS; http://geis.infoboss.co.kr/). Geneious R11 11.0.5 (Biomatters Ltd, Auckland, New Zealand) was used to annotate its mitogenome with A. flavus (MT335777; Park et al., under review). Voucher sample isolated from barley in Denmark was deposited into South Regional Research Center of USDA (SRRC1009; Klich and Pitt 1988).

Aspergillu. flavus SRRC1009 mitogenome length (GenBank accession is MT354769) is 29,202 bp, the shortest mitogenomes among three A. flavus mitogenomes. Numbers of PCGs, tRNAs, and rRNAs are 17, 27, and 2, respectively. GC ratio is 26.2%, samilar to the two A. flavus mitogenomes.

The first mitogenome of A. flavus (Yan et al. 2016) presented different features in various aspects including length (31,602 bp) and GC ratio (25.2%). Based on our mitogenome, this might be another Aspergillus species together with the recent study (Park et al., under review) and phylogenetic tree (Figure 1(A)).

Figure 1.

(A) Maximum likelihood (bootstrap repeat is 10,000) and neighbor joining (bootstrap repeat is 10,000) phylogenetic trees of 12 Aspergillus, one Penicillium mitochondrial genomes, and Podospora mitochondrial genome as an outgroup: Aspergillus flavus (MT354769 in this study, MT335777, JQ355000, and NC_026920), Aspergillus oryzae (NC_018100), Aspergillus parasiticus (NC_041445), Aspergillus fischeri (JQ354995), Aspergillus fumigatus (NC_017016), Aspergillus pseudoglaucus (NC_041427), Aspergillus niger (NC_007445), Aspergillus kawachii (AP012272), Aspergillus luchuensis (NC_040166), Aspergillus nidulans (NC_017896), Penicillium digitatum (NC_015080), and Podospora anserina (NC_001329). Phylogenetic tree was drawn based on maximum likelihood phylogenetic tree. The numbers above or below branches indicate bootstrap support values of maximum likelihood and neighbor joining phylogenetic trees, respectively. Star-marked A. flavus TCM2014 is not placed in A. flavus/oryzae group, indicating that it may be misidentified. (B) Summary of intraspecific variation of three A. flavus strains.

Sixteen SNPs and 22 INDELs were identified in comparison to AflaGuard® mitogenome (MT335777; Figure 1(B)). Five non-synonymous SNPs (nsSNPs) were identified in three genes, NAD1 (two nsSNPs), the hypothetical protein (two nsSNPs), and NAD3 (one nsSNP). Four synonymous SNPs (sSNPs) were identified in COX3 (one sSNP), NAD5 (one sSNP), and COX1 (two sSNPs). In addition, 17 SNPs and 27 INDELs were found against JQ355000, one more sSNP was found in NAD5 and number of SNPs in genes is same to those of the previous comparison (Figure 1(B)). Number of variations found in this study occupied small proportion on mitogenomes in comparison to the other fungal species, including Fusarium oxysporum (Kwon et al. 2019) as well as insect (e.g. Nilaparvata lugens (Choi et al. 2019; Park, Kwon, et al. 2019), Laodelphax striatellus (Park, Jung, et al. 2019; Seo, Jung, et al. 2019), Spodoptera frugiperda (Seo, Lee, et al. 2019), and Aphis gossypii (Bee et al. 2020; Park, Xi, et al. 2019)) and plant species (Liriodendron tulipifera (Park, Kim, et al. 2019)).

Sequence alignments of 10 conserved genes from 13 Aspergillus, one Penicillium, and one Podospora mitogenome (outgroup) were calculated by MAFFT v7.450 (Katoh and Standley 2013) and concatenated. The bootstrapped neighbor-joining and maximum likelihood phylogenetic trees were constructed using MEGA X (Kumar et al. 2018). Phylogenetic trees showed that three A. flavus mitogenomes were clustered in one clade together with A. oryzae mitogenome (Figure 1(A)).