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Sep 19, 2023 193 likes •422 views

Comparative genome analyses of Synchytrium endobioticum representing six different pathotypes reveal pathotype-specific genes and SNPs useful for molecular diagnostics Hai Nguyen, Ph.D. Research Scientist How Canada became involved with potato

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Comparative genome analyses of Synchytrium endobioticum representing six different pathotypes reveal pathotype-specific genes and SNPs useful for molecular diagnostics

Hai Nguyen, Ph.D. Research Scientist

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SLIDE 2
  • In North America, the disease

was first detected in Newfoundland in 1909.

  • In 1912, Ottawa forbade the

movement of Newfoundland soil into Canada.

  • In 2000, potato wart was found

in Prince Edward Island. US prohibited the movement of seed and table stock potatoes from Prince Edward Island.

  • New requirements are now in

place for export of potato from PEI to the United States

  • AAFC/CFIA (only 2 scientists)

with focused program on potato wart

How Canada became involved with potato wart?

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SLIDE 3

ladder water control PEI contaminated soil Triparticalcar arcticum Spizellomyces plurigibbosus Gaertneriomyces semiglobiferus Kochiomyces dichotomus Powellomyces sp Entophlyctis conferrae-glomeratae Podochytrium sp Blastocladiella britannica Rhizoclosmatium globosum Chytriomyces hyalinus Rhizophydium patellarium Spizellomyces acuminatus Rhizophydium haynaldii

  • S. endobioticum (1944)
  • S. endobioticum (1903)
  • S. plantagineum
  • S. aureum
  • S. collapsum
  • S. perforans
  • S. papillatum

PCR test: Presence/absence Real time PCR tests: quantification of spores, viability tests

CA, PEI (2 samples), P6 CA, PEI, P6 CA, NFL, P? CA, NFL (3 samples), P? CA, PEI, P6 NL, P1 NL, P2 DE, P2 DE, P1 DE, P2 NL, P1 NL, P6 DE, P6 UK, P? DE, P18 NL, P18 UK, P1 UK, P1 UK, P?

  • Microsatellites. Each group creates a

unique pattern and strains could be differentiated from each other

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SLIDE 4

Pathotypes of S. endobioticum

  • Isolates of S. endobioticum grouped into pathotypes

based on interaction with a differential set of resistant potato varieties

  • 39 pathotypes (and maybe more)
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SLIDE 5

Pathotypes of S. endobioticum

  • Pathotypes manifest

themselves as a result of different R genes present in potato varieties and their cognate Avr genes in S. endobioticum

– Presence of Sen1 gene in potato gives it resistance to pathotype 1 isolates – Sen1 recognizes the effector AvrSen1 present only in S. endobioticum pathotype 1 isolates and it triggers HR in the potato, which gives the potato resistance to S. endobioticum pathotype 1

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SLIDE 6

None of the tests that exist so far can tell difference between various pathotypes of S. endobioticum The benefit of knowing (or predicting) the pathotype gives producers a tool to mitigate economic risk

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SLIDE 7
  • In 2018, we published a paper on the

mitochondrial genome of S. endobioticum

  • NGS data generated for 30 strains of S.

endobioticum

  • Phylogeny of concatenated mito genes

does not show any distinction between

  • S. endobioticum strains
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SLIDE 8

Haplotype network based on 141 polymorphic sites shows some groupings corresponding perhaps to certain pathotypes

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SLIDE 9

Would differences in the nuclear genome allow us to separate pathotypes of S. endobioticum?

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Would differences in the nuclear genome allow us to differentiate between pathotypes?

Technically yes because the pathotype is a manifestation of the pathogen’s genotype and its interaction with the host’s genotype, and there is more (relevant) information in the nuclear genome compared to the mitochondrial genome 3 STRATEGIES

1 6 8 2

Core genome / pathotype- specific genome Presence/absence of genes

Pathotype phylogeny represented by genes in the core genome Site specific variation in the genome (SNPs, indels, struct. variants)

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SLIDE 11
  • 1. Read mapping to reference: building a pan

genome

LEV6574 (Canadian) MB42 (Dutch) Pan genome Pan genome

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SLIDE 12
  • 1. Read mapping to reference: only some of the

30 datasets have enough coverage to proceed for in depth comparative genome analyses

Strain Coverage Reference Location 1D1_MB42 NA van de Vossenberg et al. (2018) Langenboom, the Netherlands 2G1_MB08 9.9 van de Vossenberg et al. (2018) Mussel, the Netherlands 2G1_SE4 20.4 van de Vossenberg et al. (2018) Germany 6O1_LEV6574 NA van de Vossenberg et al. (2018)

  • St. Eleanor’s, Prince Edward Island, Canada

6O1_LEV6602 19.5 van de Vossenberg et al. (2018) Augustine Cove, Prince Edward Island, Canada 6O1_LEV6687 40.4 van de Vossenberg et al. (2018) New Annan, Prince Edward Island, Canada 6O1_CHY1000 41.7 new Carmanville, Newfoundland, Canada 6O1_CHY1001 55.1 new

  • St. Philips, Newfoundland, Canada

6O1_SE5 63.9 van de Vossenberg et al. (2018) the Netherlands 6O1_SE6 93.7 van de Vossenberg et al. (2018) Germany 8F1_DEN01 32.7 van de Vossenberg et al. (2018) Jylland, Denmark 8F1_CHY1002 79.1 new Avondale, Newfoundland, Canada 18T1_SE7 48.6 van de Vossenberg et al. (2018) Borgercompagnie, the Netherlands 38Nevsehir_MB56 12.2 van de Vossenberg et al. (2018) Nevsehir, Turkey

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  • 2. De novo genome assembly

786 3500 3325 3275 1285 3079 3654 3102 3226 3105 3367 3238 3341 3077 3434 3372

1 D 1 _ M B 4 2 _ r e f e r e n c e 1 D 1 _ M B 4 2 _ r e

  • a

s s e m b l e d 2 G 1 _ M B 8 2 G 1 _ S E 4 6 O 1 _ L E V 6 5 7 4 _ r e f e r e n c e 6 O 1 _ L E V 6 5 7 4 _ r e

  • …

6 O 1 _ L E V 6 6 2 6 O 1 _ L E V 6 6 8 7 6 O 1 _ C H Y 1 6 O 1 _ C H Y 1 1 6 O 1 _ S E 5 6 O 1 _ S E 6 8 F 1 _ D E N 1 8 F 1 _ C H Y 1 2 1 8 T 1 _ S E 7 3 8 N e v s e h i r _ M B 5 6

Number of contigs (>= 1000 bp)

21483073210010962119336621383118 23209196 21492804 20608936 211960742104956021249904 20174921 214316142127949521316832 20008646 21296695

1 D 1 _ M B 4 2 _ r e f e r e n c e 1 D 1 _ M B 4 2 _ r e

  • a

s s e m b l e d 2 G 1 _ M B 8 2 G 1 _ S E 4 6 O 1 _ L E V 6 5 7 4 _ r e f e r e n c e 6 O 1 _ L E V 6 5 7 4 _ r e

  • …

6 O 1 _ L E V 6 6 2 6 O 1 _ L E V 6 6 8 7 6 O 1 _ C H Y 1 6 O 1 _ C H Y 1 1 6 O 1 _ S E 5 6 O 1 _ S E 6 8 F 1 _ D E N 1 8 F 1 _ C H Y 1 2 1 8 T 1 _ S E 7 3 8 N e v s e h i r _ M B 5 6

Total length of assembly (>= 1000 bp)

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SLIDE 14
  • 2. Genome annotation

85.1 82 81.7 82.8 84.2 83.8 82.1 83.1 82.1 83.4 82.7 84.1 82 84.5 81.7 82.8

1 D 1 _ M B 4 2 _ r e f e r e n c e 1 D 1 _ M B 4 2 _ r e

  • a

s s e m b l e d 2 G 1 _ M B 8 2 G 1 _ S E 4 6 O 1 _ L E V 6 5 7 4 _ r e f e r e n c e 6 O 1 _ L E V 6 5 7 4 _ r e

  • …

6 O 1 _ L E V 6 6 2 6 O 1 _ L E V 6 6 8 7 6 O 1 _ C H Y 1 6 O 1 _ C H Y 1 1 6 O 1 _ S E 5 6 O 1 _ S E 6 8 F 1 _ D E N 1 8 F 1 _ C H Y 1 2 1 8 T 1 _ S E 7 3 8 N e v s e h i r _ M B 5 6

Complete BUSCO (%)

8031 6675 6696 6668 8671 6562 6688 6579 6642 6585 6558 6624 6646 7342 6584 6712

1 D 1 _ M B 4 2 _ r e f e r e n c e 1 D 1 _ M B 4 2 _ r e

  • a

s s e m b l e d 2 G 1 _ M B 8 2 G 1 _ S E 4 6 O 1 _ L E V 6 5 7 4 _ r e f e r e n c e 6 O 1 _ L E V 6 5 7 4 _ r e

  • …

6 O 1 _ L E V 6 6 2 6 O 1 _ L E V 6 6 8 7 6 O 1 _ C H Y 1 6 O 1 _ C H Y 1 1 6 O 1 _ S E 5 6 O 1 _ S E 6 8 F 1 _ D E N 1 8 F 1 _ C H Y 1 2 1 8 T 1 _ S E 7 3 8 N e v s e h i r _ M B 5 6

Number of validated gene models

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Orthogroup analysis

  • Validated gene models were pooled and clustered into

gene families (orthogroups) with orthofinder2

  • 8012 orthogroups were found in total (pan genome)
  • 4625 orthogroups were considered single copy (large

subset of the core genome)

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Orthogroup Se_MB42.1D1 Se_MB08.2G1 Se_SE4.2G1 Se_LEV6574.6O1 Se_LEV6602.6O1 Se_LEV6687.6O1 Se_CHY1000.6O1 Se_CHY1001.6O1 Se_SE OG0007119 1 1 OG0007125 1 1 OG0007127 1 1 OG0007134 1 1

  • Orthogroups of interest are those that are present in a given pathotype but absent

in all other pathotypes analysed

  • Pathotypes (* represented by a single strain)
  • 1(D1)* – 47 orthogroups
  • 2(G1) – 4 orthogroups
  • 8(F1) – 2 orthogroups
  • 18(T1)* – 60 orthogroups
  • 38(Nevsehir)* – 24 orthogroups
  • 6(O1) – 5 orthogroups
  • Caveat: not all strains positive for all 5, they usually have 3 out of the 5
  • Note: Some pathotype specific orthogroups are going to be strain specific because

there was only 1 representative genome for that pathotype

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SLIDE 17

4625 single copy gene neighbour joining phylogeny (amino acid level)

(Newfoundland, Canada) (PEI, Canada) (PEI, Canada) (Newfoundland, Canada) (PEI, Canada) (Langeboom, The Netherlands) (Mussel, The Netherlands) (Germany) (Germany) (The Netherlands) (Borgercompagnie, The Netherlands) (Nevsehir, Turkey) (Jylland, Denmark) (Newfoundland, Canada)

Alignment Length = 2.4M columns Variable sites = 1.6% (38.4k columns)

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Look for genes that have similar relationships as our hypothesis of the pathotyping system

  • Search for relationships in single copy genes that resemble the pathotyping

groupings

  • Calculate the symmetric difference between two trees (Robinson-Foulds)
  • No good candidates were found, but might need more technical development
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SNP analysis Map read fragments to ref. genome, detect variation (SNPs)

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Unique and shared homozygous SNPs in 3 pathotypes 226 184 584 471 9 146 Pathotype 1 (1 strain) Pathotype 2 (2 strains) Pathotype 6 (3 LEV strains) 66 NOTE – the reference strain is LEV6574 (pathotype 6)

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Summary & Conclusions

  • Objective: Comparative genomic analyses were performed in an

attempt to find pathotype specific genes/sequences in S. endobioticum useful for the design of a molecular diagnostic test for pathotyping

  • Results:
  • Orthogroup specific to pathotypes were found but more representative

genomes of certain pathotypes are required to strengthen the analysis

  • The relationships of genes analysed did not correspond well to pathotype

relationships/groupings

  • Pathotype specific SNPs were found but additional representative genomes

required

  • Future
  • Need additional S. endobioticum genomes of other pathotypes
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Acknowledgements

Kasia Dadej (AAFC) NGS Sequencing Donna Smith (CFIA) Isolation and DNA extraction

  • f S. endobioticum

Wendy Findlay (AAFC) SNP analyses Funding – Project J-0001799 - NGS genotyping tool for potato wart