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Return of the mitochondrial DNA

Authors:

Abstract

In the 1990s, the barcoding of life project was started, aiming to use a single sequence to identify animals, fungi and plants to the species level. The first marker that was proposed was a mitochondrial marker: the mitochondrially encoded cytochrome oxidase I (cox1 in filamentous fungi). However, amplifying the cox1 sequence proved problematic in many fungal groups, because the frequent insertions of introns into the region made universal primer design difficult. Hence, the mitochondrial marker was abandoned and the barcoding community chose the ITS region as official barcode for fungi. Unfortunately, the ITS barcode proved to have insufficient resolution in many closely related species. Thus, multi-locus analysis became the new standard, most of which included at least one mitochondrial marker. There has been no consensus on which mitochondrial loci to include. With next generation sequencing and new assembly tools it is possible to assemble the complete mitochondrial genome of isolates, which provide all the benefits that are associated with using mitochondrial markers. In addition, using the complete mitochondrial genome offers better resolution for phylogenetic analyses and with sufficient sampling it can be placed in the context of previous works done on mitochondrial barcoding markers.
Return of the Mitochondrial DNA
AFusarium oxysporum story
Bal´
azs Brankvics1,2, Peter van Dam3, Martijn Rep3, G. Sybren de Hoog1,2,
Theo A. J. van der Lee4, Cees Waalwijk4& Anne D. van Diepeningen1,4
1Westerdijk Fungal Biodiversity Institute (former CBS-KNAW), Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands; 2Institute of
Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; 3Swammerdam Institute for Life Sciences, University of Amsterdam,
Amsterdam, The Netherlands; 4Wageningen Plant Research,Wageningen University and Research Centre, Wageningen, The Netherlands
Introduction
In the 1990s, the barcoding of life project was started, aiming to use a single sequence to identify
animals, fungi and plants to the species level. The first marker that was proposed was a mitochondrial
marker: the mitochondrially encoded cytochrome oxidase I (cox1 in filamentous fungi).
However, amplifying the cox1 sequence proved problematic in many fungal groups, because the
frequent insertions of introns into the region made universal primer design difficult. Hence, the mi-
tochondrial marker was abandoned and the barcoding community chose the ITS region as official
barcode for fungi. Unfortunately, the ITS barcode proved to have insufficient resolution in many
closely related species. Thus, multi-locus analysis became the new standard, most of which included
at least one mitochondrial marker. There has been no consensus on which mitochondrial loci to
include.
With next generation sequencing and new assembly tools it is possible to assemble the complete
mitochondrial genome of isolates, which provide all the benefits that are associated with using mito-
chondrial markers. In addition, using the complete mitochondrial genome offers better resolution for
phylogenetic analyses and with sufficient sampling it can be placed in the context of previous works
done on mitochondrial barcoding markers.
Demonstration: How the mitochondrial genome revealed recombination[1]
Two parts of the mitochondrial genome
The mitochondrial genome of Fusarium spp. can be divided into two parts: the conserved part (shows
complete synteny between species) and the large variable (LV) region (Fig. 1). From NGS data, we
have assembled, annotated and compared the mitochondrial genomes of 61 strains of the Fusarium
oxysporum species complex (FOSC) together with 1 F. commune and 2 F. proliferatum strains as
outgroup.
Figure 1: Mitochondrial genome of Fusarium oxysporum f. sp. cumini strain F11. Green blocks: tRNA coding genes,
blue arrows: genes, yellow arrows: protein coding sequences, red arrows: rDNA coding sequence, purple arrows: intron
encoded homing endonuclease genes, gray segment: large variable (LV) region with orf2285 (LV-uORF).
Non-homologous variants of the LV region
Three different variants of the LV region (Fig. 2) were found within the FOSC. Variant 1 (Fig. 2a)
is homologous to the LV region found in other Fusarium spp. The three variants contain at least
13 tRNA genes, their order shows partial synteny. However, the variants have highly divergent se-
quences, which is demonstrated by the fact that BLASTN is unable to identify synteny blocks between
the variants.
Figure 2: The three variants of the large variable region. a) Variant 1 represented by F. oxysporum strain Fon015,
b) variant 2 represented by F. oxysporum strain FOSC3-a and c) variant 3 represented by F. oxysporum strain NRRL37622.
Green blocks: tRNA coding genes, blue arrows: ORFs, yellow arrows: ORFs that are not present in all representatives of
the given variant.
Both variants 1 & 2 are not clade specific
The strains of the FOSC used in this study could be grouped into three clades which were recognized
as phylogenetic species based on genealogical concordance. Variant 1 was present in all three clades,
variant 2 was present in clades 2 & 3 and variant 3 was restricted to clade 2. Possible hypotheses for
the distribution of variants 1 & 2 are as follows:
H1: Variant 2 emerged in either clade 1 or 2 and the complete mitochondrial genome was transferred
to a strain of the other clade, then the genome spread in the population without recombination.
H2: Variant 2 emerged in either clade 1 or 2 and the complete mitochondrial genome was transferred
to a strain of the other clade, then the genome spread in the population by recombination.
H3: Variant 2 emerged in the ancestor of clades 1 & 2 and was maintained within both lineages during
the separation of the two phylogenetic species without recombination.
H4: Variant 2 emerged in the ancestor of clades 1 & 2 and was maintained within both lineages during
the separation of the two phylogenetic species by recombination.
Co-evolution of the conserved part and the LV region variants
The conserved part of the mitochondrial genome of the strains supports the separation of the three
clades (Fig. 3: Conserved mt region). Thus, the conserved part of the mitogenomes of strains belong-
ing to the same clade are more similar irrespective of which variant the strains have, this rules out H1
& H3. Both variants 1 & 2 support the separation of the two clades (Fig. 3), this also rules out H1&
H3. If H2were correct, we would expect that one of the clades is paraphyletic both for the tree based
on variant 2 and the conserved part, but there is no sign of this in the data. Therefore, we conclude
that H4is correct: variant 2 emerged in the ancestor of clades 1 & 2 and was maintained within both
lineages during the separation of the two phylogenetic species by recombination.
Variant 2 Conserved mt region Variant 1
Figure 3: Tanglegram of the trees based on the LV region variant 2, the conserved part of the mitogenome and
the variant 1 of the LV region, respectively. The trees were constructed using MrBayes. Clades with high Bayesian
posterior probability (BPP) support are displayed with thicker branches. The support values are BPP values. The three
phylogenetic clades identified within the FOSC are highlighted in different shades of gray. The strains that contain variant
3 are highlighted by blue boxes.
Recombination within the FOSC
Variant 2 emerged in the common ancestor of clades 2 & 3 of the FOSC and was maintained within
both lineages during the separation of the two phylogenetic species. This shows that mitochondrial
recombination is going on within the FOSC.
Conclusions
Mitochondrial genomes can be efficiently assembled from NGS data
It is feasible to analyze the mitogenome of a large number of strains
Complete mitogenomes offer sufficient information to delineate even closely related species
A detailed analysis of the mitogenomes may offer new insights into the biology of the organism
Funding
The investigations are supported by the Division for Earth and Life Sciences (ALW) with financial aid from the Nether-
lands Organization for Scientific Research (NWO) under grant number 833.13.006. This work was further supported by
the Horizon programme (project 93512007) of the Netherlands Genomics Initiative (NGI) through a grant to Martijn Rep.
Reference
[1] Brankovics, B., van Dam, P., Rep, M., de Hoog, G. S., van der Lee, T. A. J., Waalwijk, C. and van Diepeningen, A. D.
Mitochondrial genomes reveal recombination in the presumed asexual Fusarium oxysporum species complex. (Under
review)
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Article
Full-text available
Background The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties. Results A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species. Conclusions The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.