The linear mitochondrial genome of the quarantine pest ... · Synchytrium endobioticum Soil-borne,...
Transcript of The linear mitochondrial genome of the quarantine pest ... · Synchytrium endobioticum Soil-borne,...
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The linear mitochondrial genome of the
quarantine pest Synchytrium endobioticum;
Bart T. L. H. van de Vossenberg, Balázs Brankovics, Hai D. T. Nguyen,
Marga P. E. van Gent-Pelzer, Donna Smith, Kasia Dadej, Jarosław
Przetakiewicz, Jan F. Kreuze, Margriet Boerma, Gerard C. M. van
Leeuwen, C. André Lévesque and Theo A. J. van der Lee
Wart disease workshop 26-28 June 2019
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Synchytrium endobioticum
▪ Soil-borne, obligate biotrophic (non-
culturable) fungus on potato causing wart
disease
▪ Wart formation on tubers and shoots
▪ Can result up to 100% yield loss
▪ Production of robust resting spores
(infectious > 40 years)
▪ World-wide quarantine status and on the
USA bioterrorism list
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Characterization of isolates
▪ As pathotypes based on their virulence on a reference set of potato
cultivars
▪ SSR markers
▪ TaqMan assay
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PJM Bonants, MPE van Gent-Pelzer… - European journal of plant pathology, 2015
MC Gagnon, TAJ van der Lee, PJM Bonants, DS Smith… - Phytopathology, 2016
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Migration of S. endobioticum
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Can we gain insights into the evolution and recent history of introductions of this plant pathogen using the mitochondrial genome?
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Why the mitochondrial genome?
Mitogenomes:
• do not mix or recombine with the
nuclear genome
• have a mutation rate about ten times
higher than nucDNA
• are relatively small, ranging up to
240 kb in fungi
• form a single haplotype/haplogroup
per organism
• have many copies (hundreds) per cell
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▪ One of the most ideal markers for monitoring the distribution and spread of populations is the mitochondrial genome (Harrison, 1989; Taylor, 1986).
▪ Mitochondrial genomes are relatively small and, therefore, can be studied in their entirety.
▪ Due to its high copy number within individual cells, the mitochondrial genome is easy to access.
▪ Simple organization that makes homologous regions easy to identify.
▪ Finally, in many fungal groups mitogenomes are inherited maternally (Basse, 2010),
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Why the mitochondrial genome?
https://doi.org/10.1016/0169-5347(89)90006-2https://doi.org/10.1016/0147-5975(86)90011-3https://doi.org/10.1016/j.mib.2010.09.003
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▪ Mitochondrial sequences have been used for resolving phylogenetic and evolutionary relationships
between fungi at all taxonomic levels (Liu et al., 2009; Avila-Adame et al., 2006; Fourie et al., 2013).
▪ In 2003, the DNA barcoding initiative started, aiming at using a single marker for taxon identification.
The marker that was selected was a mitochondrial gene, cytochrome c oxidase I—COI or cox1 (Hebert
et al., 2003).
▪ The use of cox1 was abandoned as a barcoding region, because the frequent presence of introns in the
gene made this region impractical for PCR amplification (Gilmore et al., 2009).
▪ Next generation sequencing (NGS) and new analysis methods have resolved this issue by dispensing
with the need for PCR amplification for extracting mitochondrial sequences (Brankovics et al., 2016).
▪ In addition, de novo assembly of mitochondrial sequences from NGS data is not confounded by the
presence of nuclear mitochondrial DNA segments (NUMTs), while NUMTs are known to cause problems in
PCR-based barcoding (Song et al., 2008).
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Why the mitochondrial genome?
https://doi.org/10.1186/1471-2148-9-272https://doi.org/10.1007/s00294-005-0016-3https://doi.org/10.1186/1471-2164-14-605https://doi.org/10.1098/rspb.2002.2218https://doi.org/10.1111/j.1755-0998.2009.02636.xhttps://doi.org/10.1371/journal.pcbi.1004753https://doi.org/10.1073/pnas.0803076105
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“Next generation sequencing (NGS) and new analysis methods have resolved this issue by dispensing with the need for PCR amplification for extracting mitochondrial sequences”
Assembly of the mitochondrial genome
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▪ Individual assemblies were aligned to create a consensus mtDNA assembly
▪ The consensus mtDNA assembly was annotated using the online Mfannot tool
Mfannot: University of Montreal; http://megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannotInterface.pl.
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Assembly of the mitochondrial genome
http://megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannotInterface.pl
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The consensus mtDNA
▪ Assembly size: 72.8 kb
▪ No circular confirmation found
▪ Drop in read coverage at 5’ and 3’ ends: consistent with linear
mtDNA hypothesis
▪ Inverted repeats at 5’ and 3’ ends (~3 kb)
▪ All 14 “core” genes for fungi, 5 tRNAs and 2 rRNAs predicted
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Primer design for assembly verification
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Improved procedures
▪ New DNA extraction protocol shows high molecular DNA extract
▪ WGA step to generate sufficient material for confirmation
experiments
▪ Fragmentation controls show amplification up to 4.3 kb
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Verification of linearity of the S. endobioticum mtDNA
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Assembly and Annotation of the linear
mtDNA▪ Three assemblies were
used to determine mtDNA
genome sequence
▪ “standard” fungal mtDNA
genes identified
▪ Reduced set of tRNAs
▪ GC-rich intergenic regions
(up to 68.5%)
▪ Codes for dpoB and intron
encoded endonucleases
▪ Linear with terminal
inverted repeats (TdT
tailing and sanger
sequence verified) 14
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Linear mtDNA genomes in Chytrid species
▪ Bayesian Inference of
phylogeny with high support
for all nodes
▪ Three linear mtDNA genomes
are known in Chytrid
species: Sendo, Bdend, and
Hcurv
● Independent events
▪ Splits the Chytridiales
▪ Little intraspecies variation
for Sendo (19
polymorphisms)15
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Linearization of the S. endobioticum
mitogenome is a recent event
▪ S. microbalum: conserved organization and orientation; circular
mapping, no endonucleases or dpoB
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mtDNA haplotypes reveal 4 major groups
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• Build on 141 informative sites (SNPs) from the entire mtDNA
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More than one haplotype per sample
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▪ mtDNA haplotype was build on 141
informative sites
▪ SNPs do not show a black and white
distribution
● Intermediate SNP frequencies
● >1 mtDNA haplotype is present in many samples
▪ SNP frequency distribution and haplotype
composition seems to be fairly conserved
for mtDNA groups
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mtDNA haplotype diversity
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Conclusions
▪ The S. endobioticum mtDNA genome is linear and shows that the
pest has been introduced in Europe at least three times
▪ Several pathotypes emerged more than once independently
▪ S. endobioticum isolates are populations and have more than one
haplotype per sample
▪ These findings have an impact on breeding for potato wart
resistance, as the diversity of S. endobioticum virulence is
underestimated
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Acknowledgements
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Biointeractions and Plant Health
Bart van de Vossenberg, Marga van Gent-
Pelzer, Balazs Brankovics, Peter Bonants,
Theo van der Lee
Bioinformatics
Sven Warris, Henri van de Geest, Linda
Bakker
Plant Breeding
Jack Vossen, Gert van Arkel, Marjan
Bergervoet, Charlotte Prodhomme
Promotor Richard Visser
Other
▪ Margriet Boerma (HLB, NL)
▪ Bart van de Vossenberg, Gerard van
Leeuwen, Patricia van Rijswick, Annebeth
Kloosterman, Nico Mentink (NVWA, NL)
▪ Hai Nguyen, Kasia Dadej (AAFC, Canada)
▪ André Levesque, Donna Smith (CFIA,
Canada)
▪ Jarek Przetakiewicz (IHAR, Poland)
▪ Jan Kreuze (CIP, Peru)
Funding TKI wart project Averis seeds BV,
Boehm-Nordkartoffel Agrarproduktion GmbH &
Co. OHG, HZPC Holland, SaKa Pflanzenzucht
GmbH & Co. KG, Taegasc, Meijer Potato, LKF
Vandel, HLB BV and NVWA