Table 1.
Species included in the phylogenetic analysis.
Table 2.
Mapping results for mitochondrial sequence for 6 NGS data set used for assembly.
Fig 1.
Genetic map of the Verticillium nonalfalfae mtDNA.
The concentric circles from the outside inwards represent different tracks. The outermost track represents the coding features of the V. nonalfalfae mitochondrial genome. The direction of the highlights (inward, outward) represents the strand in which the feature is present. The next track represents read counts of RNA-Seq mapping from 2 different pathotypes (mild-blue, lethal-red) of V. nonalfalfae. The tracks are made of Bowtie2 [56] mapped RNA-Seq reads and shown as counts per 100bp bins. The counts were normalized with the DESeq method [57]. Because of rRNA counts overwhelming the remaining expression profiles, the count number on the graph was capped below 17.5% of the top count profiles (a non-capped graph would show only rRNA expressed), in order to enable visualization of low-expressed regions. Following this track are GC-skew and GC-content tracks, respectively. GC-skew [(G-C)/(G+C)] reflects the relative number of cytosine to guanine and is often used to describe the strand-specific bias of a nucleotide composition. The GC-skew track is shown as a histogram of 250bp sliding windows with calculated gc-skew coefficients. Green regions represent windows for which the coefficient is larger than 0 and red regions windows for which the coefficient is smaller than 0. The neighbouring grayscale heatmap of the GC-content track represents 100bp sliding windows with calculated gc-contents. Regions in the GC content heatmap are shaded in gray, where darker gray represents higher gc-content and lighter gray represents lower gc-content. The two tracks show a similar pattern to other Sordariomycetes and were also used to scan for anomalies in GC content, which could indicate the introduction of heterologous DNA. No anomalies indicating such an event were detected. The cumulative GC skew analysis was also used to try and find the origins of replication and termination of replication loci (data not shown) but we could not determine them with this analysis.
Table 3.
Gene features of the Verticillium nonalfalfae mitochondrial genome.
Table 4.
rRNA and orf414 features of the Verticillium nonalfalfae mitochondrial genome.
Fig 2.
Column diagram of the Verticillium nonalfalfae mtDNA codon usage.
The diagram represents the codons (x-axis) and percentages of their occurrence (y-axis) in the V. nonalfalfae mitochondrial genome.
Table 5.
tRNA features of the Verticillium nonalfalfae mitochondrial genome.
Fig 3.
Expression analysis of the potential long non-coding RNA orf414 in two different strains of V. nonalfalfae (Rec = Slovenian mild strain and T2 = Slovenian lethal strain). Expression values were normalized with ubiquitin (ubq) as a housekeeping reference. Bars indicate standard errors of three biological replicates.
Fig 4.
Maximum likelihood phylogenetic tree of 20 mitochondrial genomes, based on a conserved set of proteins.
The tree was inferred from an alignment of amino-acid sequences of conserved mitochondrial proteins with 3093 distinct alignment positions and 100 rapid bootstrap inferences. The gamma model of rate heterogeneity and a maximum likelihood estimate of the alpha-parameter were used to prepare the final tree. Numbers above tree nodes represent the bootstrap support values. Next to the tree is a graphical presentation of an alignment of mitochondrial protein-coding genes and their order in the represented species. The conserved set of 14 protein-encoding genes is present in most of the species, with the exception of S. cerevisiae and S. pombe yeasts, which lack the NADH dehydrogenase family of genes, C. lindemuthianum, which lacks the nad2 gene, N. crassa, which has two copies of the nad2 gene and R. orthosporum and P. anserina, which lack the atp9 gene. The phylogenetic tree shows V. nonalfalfae joined with V. dahliae and C. lindemuthianum in a cluster corresponding to the established group of fungi called Glomerellales. C. lindemuthianum can be seen to have a very different gene order compared to the other two members of its group. The Hypocreales and Sordariales groups of the Sordariomycetes can also be seen in the tree. A. chrysogenum of the Hypocreales group contains a translocation of the cox2 gene, which distinguishes it from the rest of the members of its group. The Glomerellales and Hypocreales groups show a high degree of synteny within their respective groups and differ only by a translocation of the nad2-nad3 gene cluster.