The linear mitochondrial genome of the quarantine pest Synchytrium endobioticum ; Wart disease workshop 26-28 June 2019 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
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 2
Characterization of isolates ▪ As pathotypes based on their virulence on a reference set of potato cultivars ▪ SSR markers ▪ TaqMan assay MC Gagnon, TAJ van der Lee, PJM Bonants , DS Smith… - Phytopathology, 2016 PJM Bonants, MPE van Gent- Pelzer… - European journal of plant pathology, 2015 3
Migration of S. endobioticum Can we gain insights into the evolution and recent history of 4 introductions of this plant pathogen using the mitochondrial genome?
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 5
Why the mitochondrial genome? ▪ 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), 6
Why the mitochondrial genome? ▪ 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). 7
Assembly of the mitochondrial genome “Next generation sequencing (NGS) and new analysis methods have resolved this issue by dispensing with the need for PCR amplification for extracting mitochondrial sequences” 8
Assembly of the mitochondrial genome ▪ 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. 9
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 10
Primer design for assembly verification 11
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 12
Verification of linearity of the S. endobioticum mtDNA 13
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
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
Linearization of the S. endobioticum mitogenome is a recent event ▪ S. microbalum : conserved organization and orientation; circular mapping, no endonucleases or dpoB 16
mtDNA haplotypes reveal 4 major groups • Build on 141 informative sites (SNPs) from the entire mtDNA 17
More than one haplotype per sample ▪ 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 18
mtDNA haplotype diversity 19
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 20
Acknowledgements Biointeractions and Plant Health Other ▪ Margriet Boerma (HLB, NL) Bart van de Vossenberg, Marga van Gent- ▪ Bart van de Vossenberg, Gerard van Pelzer, Balazs Brankovics, Peter Bonants, Theo van der Lee Leeuwen, Patricia van Rijswick, Annebeth Kloosterman, Nico Mentink (NVWA, NL) ▪ Hai Nguyen, Kasia Dadej (AAFC, Canada) Bioinformatics ▪ André Levesque, Donna Smith (CFIA, Sven Warris, Henri van de Geest, Linda Bakker Canada) ▪ Jarek Przetakiewicz (IHAR, Poland) ▪ Jan Kreuze (CIP, Peru) Plant Breeding Jack Vossen, Gert van Arkel, Marjan Funding TKI wart project Averis seeds BV, Bergervoet, Charlotte Prodhomme Boehm-Nordkartoffel Agrarproduktion GmbH & Co. OHG, HZPC Holland, SaKa Pflanzenzucht Promotor Richard Visser GmbH & Co. KG, Taegasc, Meijer Potato, LKF Vandel, HLB BV and NVWA 21
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