The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen

Abstract

Background: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen.

Results: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence.

Conclusions: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Keywords: Chytridiomycota; Fungal communities; Mitochondrial haplotypes; Pathotype formation; Pest introduction; Population dynamics.

Fig. 1

Assembly and annotation of the linear S. endobioticum mtDNA genome. Tracks ❶ to ❸ represent putative mtDNA scaffolds from three different assemblies (respectively putative mtDNA scaffolds from the S. endobioticum MB42 genome assembly, reference guided assembly with GRAbB using S. endobioticum MB42 HiSeq data, and a reference guided assembly using S. endobioticum MB42 PacBio CCS) mapped to the linear mtDNA genome. Darker shades are used to indicate regions where scaffolds from the same assembly overlap. The narrow line in track 2 indicates a gap relative to the linear mtDNA genome. ❹ G + C content determined with a 100 bp sliding window in which orange ≤40% G + C, light green 40–50% G + C, green 50–60% G + C, and dark green > 60% G + C. ❺ mtDNA annotation track showing genes (green), rRNA (red), tRNA (purple) and the terminal inverted repeats (orange). All genes, rRNAs and tRNAs are orientated in the 5′ to 3’direction. ❻ the 72,865 bp linear mitochondrial genome of S. endobioticum

Fig. 2

Verification of linearity of the S. endobioticum mtDNA. a Design of the verification experiments using a graphical representation of the 5′ and 3’ TIRs (orange) and forward and reverse primer sites (green and light green). Three PCR reactions, performed after TdT tailing, are displayed: ❶ M13F_polyG/mtDNA_00787-fw to verify linear conformation of the mtDNA. This reaction takes place at both telomeric ends since the TIRs are inverse orientated. ❷ mtDNA_0007-fw/mtDNA_03691-rv to verify presence of the TIR with its specific flanking sequence on the 5′ end of the mtDNA. ❸ mtDNA_0007-fw/mtDNA_69209-fw to verify presence of the TIR with its specific flanking sequence on the 3′ end of the mtDNA. b Gel images corresponding with reactions described under A. The 1 kb-plus size marker (M) was used for amplicon size estimation. c Sanger sequence data mapped to the S. endobioticum mtDNA corresponding with the reactions described under A. The mtDNA is used as reference, and bases similar to the reference are shown in grey, and differences to the reference are highlighted (black). Phred scores for the individual peaks are shown as blue bars, and low quality data (Phred < 30) is annotated in pink. For reaction 1, the first 800 bases of the 5′ end TIR are shown, whereas for reactions 2 and 3 the full TIR including the specific flanking sequences are shown (~ 4 kb)

Fig. 3

Bayesian tree (GTR model, G + I distributed sites) based on a concatenated alignment of atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6 of 43 chytrid isolates with A. macrogynus (Blastocladiomycota) serving as outgroup. Bayesian posterior probabilities are displayed at branch nodes. Highlighted in bold is the S. endobioticum mtDNA reference isolate MB42. Species with linear mtDNA genomes are indicted with an asterisk “*”

Fig. 4

Median Joining haplotype network based on 141 polymorphic sites on the mitochondrial genome of S. endobioticum, of which 122 were parsimony-informative. Nodes in the network are coloured based on pathotype identity. Colours are used for pathotypes of major importance in Europe and Canada, whereas a greyscale is used for pathotypes of lesser importance [9]. Black nodes represent hypothetical ancestors. Marks on the branches indicate the number of mutations, and the numbers are shown on branches with > 5 mutations. No signatures for multiple mitochondrial haplotypes were detected for isolates with an asterisk “*”