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2009 Fall, BG Journal Club2009 Fall, BG Journal ClubA multi-locus phylogeny for A multi-locus phylogeny for PhytophthoraPhytophthora utilizing markers derived from complete genome utilizing markers derived from complete genome sequencessequences
9/9/2009
Bongsoo Park,
Department of Plant Pathology
The Pennsylvania State University
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Irish Potato Famine (1845-Irish Potato Famine (1845-1852)1852)
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Courtesy D. Inglis, apsnet.org
Potato tuber with late blightPotato tuber with late blight
Grunwald - Blighted potato field Cochabamba
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Tomato leaf and step blightTomato leaf and step blight
Black/Brown lesions on leaves
Water-soaked or chlorotic bordersExpanding rapidly and necrotic!
www.phytophthoradb.org
www.phytophthoradb.org
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Disease cycle of late blight of Disease cycle of late blight of potato and tomato caused by P. potato and tomato caused by P. infestansinfestans
Plant Pathology, Agrios, 5ed
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A. Sporangiophore; B. Sporangiophore branch showing swellings at successive sites of sporangium formation; C. Sporangia germination by zoospore
Figures-Phytophthora Disease Worldwide, 1997, Donald C. Erwin and Olaf K. Ribeiro
Sporangiophore and zoosporesSporangiophore and zoospores
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Phytophthora genusPhytophthora genus Taxonomy
Eukaryota > Chromista (Kingdom)> Oomycota >
Oomycetes(Class)> Peronosporales > Pythiaceae > Phytophthora
Facultative parasitesSoil- and water-borne
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Father of Plant PathologyFather of Plant Pathology
Heinrich Anton de Bary (1831-1888)http://en.wikipedia.org/wiki/Heinrich_Anton_de_Bary
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Sudden Oak Death (SOD) in CA and OR
Phytophthora ramorumSymptoms include bleeding cankers on the tree’s trunk and dieback of the foliage.
Sudden Oak Death in Sudden Oak Death in CaliforniaCalifornia
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Tanoak cankerTip necrosis on Rhododendron leaves
Ivors, K. http://www.phytophthoradb.org/
Ivors, K. http://www.phytophthoradb.org/
Sudden Oak Death in Sudden Oak Death in CaliforniaCalifornia
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82 species(Phytophthora)82 species(Phytophthora)2 outgroup(Pythium)2 outgroup(Pythium)
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Why it is important?Why it is important?
1) Diagnostic and species identification
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Why it is important?Why it is important?
1) Diagnostic and species identification
2) Evolutionary history and hypothesis
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Why it is important?Why it is important?
1) Diagnostic and species identification
2) Evolutionary history and hypothesis
3) Utilizing available genome sequences
Phytophthora InfestansPhytophthora ramorumPhytophthora sojae
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Why it is important?Why it is important?
1) Diagnostic and species identification
2) Evolutionary history and hypothesis
3) Utilizing available genome sequences
Phytophthora InfestansPhytophthora ramorumPhytophthora sojae
4) Species complex studies initiated.
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ITS RegionsITS Regions
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ITS RegionsITS Regions
Highly conserved region, coding region
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ITS RegionsITS Regions
Highly conserved region, coding region
Variation, Key for diagnostics
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COXII RegionsCOXII Regions
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COXII RegionsCOXII Regions
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COXII RegionsCOXII Regions
Example)Phytophthora infestansPhytophthora mirabilisPhytophthora phaseoli
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Evolution of PhytophthoraEvolution of Phytophthora
B
A
C D E
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More complex, evolutionary More complex, evolutionary questionsquestions
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More complex, evolutionary More complex, evolutionary questionsquestions
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More complex, evolutionary More complex, evolutionary questionsquestions
Using full genome sequences, Alignment, EST sequence
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More complex, evolutionary More complex, evolutionary questionsquestions
Using full genome sequences, Alignment, EST sequence
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Criteria for choosing potential Criteria for choosing potential markersmarkers
P. ramorum (66.7Mbp)
P. sojae (86.0Mbp)
High similarity coding regions
Regions of lower similarity or
Predicted Introns
Similarity to ORF in other
eukaryotes
Presence of corresponding EST
sequences
Full genome sequence in 2006
at JGI
Full genome sequence in 2006
at JGI
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229 72 27 7
Potential Informative
Homologous from other
eukaryotes
Regions of high
conservation identified
Primer design
Criteria for choosing potential Criteria for choosing potential markersmarkers
Tested by PCR
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More loci, More accurate?More loci, More accurate?
(82+2) * (1+7) = 672672 * 2 = 1,344 (sequences)
What is hypothesis on this?
8,700 nucleotide sites
60S B-tublin EnolaseHeat shock
proteinLarge subunit TigA Ef-1a
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DNA amplification and DNA amplification and sequencingsequencing
http://www.phytophthoradb.org
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Phylogenetic analyses and Phylogenetic analyses and programsprograms
Maximum Likelihood
Maximum Parsimony
Bayesian Analyses
PAUP, PHYLIP MrBayes
7 loci sequences
Multiple sequence alignment with clustalX
Preliminary phylogenetic tree using NJ with a Kimura two parameter nucleotide substitution model
Modeltest, Akaike Information Criterion, GARLI
Flat Dirichlet, Tracer version, BEASTAlternative topological hypotheses in PAML
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Phytophthora DatabasePhytophthora Database
Global Phytophthora NetworkGlobal Phytophthora Network
Plant Pathogen Database Plant Pathogen Database
and Genomic Initiativeand Genomic Initiative
at Penn State University since at Penn State University since 20052005
Thank you.Thank you.