coi barcoding of oomycetes and the design of arrays for environmental samples andré lévesque,...
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![Page 1: COI barcoding of oomycetes and the design of arrays for environmental samples André Lévesque, Gregg Robideau and Wen Chen Biodiversity, AAFC and Biology](https://reader035.vdocuments.us/reader035/viewer/2022062519/56649e415503460f94b32aa3/html5/thumbnails/1.jpg)
COI barcoding of oomycetes and the design of arrays for environmental samples
André Lévesque, Gregg Robideau and Wen ChenBiodiversity, AAFC and
Biology Department, Carleton University
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Lévesque, C.A., and de Cock, A.W.A.M. 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycol. Res. 108:1363-1383.
Evolution of all the species of the genus Pythium(ITS and LSU)
Arthur de Cock, CBS, Netherlands,collection of
« ex-type » cultures
pythiosis
mycoparasite
mycoparasites
Other species are in the saprophyte to facultative plant parasite spectrum
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Gregg RobideauM.Sc. Candidate(since Sept 2007)Dept. of BiologyCarleton University
• ~700 COI sequences of oomycetes obtained to date
• Over 80 species of Pythium, 20 species of Phytophthora, and several other genera such as Achlya, Peronophythora, and Saprolegnia
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COI Pythium Phylogeny: Tree Top Half
K
D
AC
C
A
B
B
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Pythium attrantheridium vs. P. intermedium
Nucleotide Substitutions (x100)0
2.9
2
Py_attrantheridium_DAOM230388_COI.seqPy_attrantheridium_DAOM230386_COI.seqPy_attrantheridium_DAOM230384_COI.seqPy_attrantheridium_DAOM230385_COI.seqPy_attrantheridium_DAOM230387_COI.seqPy_attrantheridium_DAOM230383_COI.seqPy_intermedium_CBS13687_COI.seqPy_intermedium_CBS26638_COI.seqPy_intermedium_DAOM229146_COI.seqPy_ultimum_CBS39851_COI.seq
• P. attrantheridium is a recently described species, closely related to P. intermedium
• ITS sequence divergence between these two species is only 5%• COI sequence divergence is 2.5-4.4%
ITS COI
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There is an analogy between barcoding marker gene(s) for all species and the sequencing all the genes of one species (genomics)
• Identify Up and Down regulated genes.
Some techniques:EST’s and arrays
• Quantify most important genes affected
RT-qPCR
• Identify « Up and Down » regulated species.
Some techniques:PCR/cloning/sequencingand arrays
• Quantify most important species affected
(RT)-qPCR
There are also analogies with functional genomics:
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James T. Tambong, A.W.A.M. de Cock, N.A. Tinker, and C.A. Lévesque. 2006. Oligonucleotide array for identification and detection of Pythium species. Applied & Environmental Microbiology 72:2691-2706
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Soil processing with Pythium ITS array
13 Pythium species found by array hybridization
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Direct processing of environment samples -Comparing treatments
Compacted
Non-Compacted
Non-CompactedCompacted
500 gallons tanks at the front and rear of tractor
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Non-compacted Soil Compacted Soil (4 years)
Chemiluminogrammes showing hybridization patterns in soil compaction experiments
Phy
toph
thor
a P
ythi
um
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
A Un1B Un3 Oom0 Oom1 Py/Ph Phy71 mult1 mastG InPar IrSyl irre13 AcOli fila6 fila8 ariGG GraIGB CaToG suDiP DIG
B vex5 boreG ulti-5 iwa8 heViG und7 marsG jasm MacIrr irre8 paro1 aca6 moG apDiG diss arist4 GraInG caten sulca0
C vex1 nMaz5 ulti-3 iwa9 heNaG un7B acrog uncin inter2 sylva1 paroe2 oli8a adhae flevo dissB aristC infla torul sulca1 InGrn1
D cucur borea ulti1-1 viIwa het3 helica echin polyma inter4 sylva2 cylin oli8b monos aquat diss6 arist grami2 torulB dissi0 grand
E vexa3 ostra ulti1-6 paddi het13 anand nRosLk masto4 interTy sylvat5 polymo olig2 tracG apler disso7 aristB grami0 torul5 dissi6 insid5
F nReh2 oedoc ulti1-7 iwa156 het18 undul9 minus masto7 newInter mamil irreg2 olig4 hypo pachy myrio3 grami8 angus pyrillo insid9
G indig nMaz1 ulti2-7 vio159 vio78-1 orthG parvu marsi macro8 debar irreg10 acan4 hypo delie2 aphan2 myrio5 volut8 pyrilup
H helioG chama ulti2-4 splen1 vio78-3 nunn middl macro6 spino7 irreg9 acan8 trach delie3 aphan3 myrio6 perii volut9 vante4
I nReh1 helio ulti2-6 splen5 nagae perpl multi9 nMaz6 macro7 spino8 nIrreg acan9 perip delie4 aphan4 scler pluri volut4 vante7 rostr
J Un1B Un3 Oo0 Oo15 Py/Phy Phy71 Phy68 Phy3 Phy84 G17 G17-8 G9 cii/cp G15 Haloph Un1B
K G5-6 G5 camb7B G7-8 G7 G19 G16 infe1 n_para later frag/syr mega2 meg19 meg/G15 ma/tar8 insol DIG
L G4-81 cin61 frag6 camb9B syrin6 cryp0 cryp7 iranic palmi7 infest3 nico hiber citri mega14 mega69 kats5 ma/tartr7 G3
M mega.gs cinn13 frag8 camb9 syrin0 cryp12 cryp/24 cland palmi4 infes0 cact3 cact2 bot/mea humic gon/aq kats7 baham polym G21 G vex
N mel/si/spi cinn17 frag9 camb7 porri0 cryp17 eryth tenta megak8 mirab cact9 pseu6 cap/mex capsi peron0 quer/qui epist avice macr/ric kandel
O vignae ci pa17 frag7 cambi6 porri8 cryp2 medic megak1 phaseo idaei pseu7 cap/citro cii/pd90 peron5 ilicis boehm vesic sojae opercPhy
toph
thor
a P
ythi
um
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
A Un1B Un3 Oom0 Oom1 Py/Ph Phy71 mult1 mastG InPar IrSyl irre13 AcOli fila6 fila8 ariGG GraIGB CaToG suDiP DIG
B vex5 boreG ulti-5 iwa8 heViG und7 marsG jasm MacIrr irre8 paro1 aca6 moG apDiG diss arist4 GraInG caten sulca0
C vex1 nMaz5 ulti-3 iwa9 heNaG un7B acrog uncin inter2 sylva1 paroe2 oli8a adhae flevo dissB aristC infla torul sulca1 InGrn1
D cucur borea ulti1-1 viIwa het3 helica echin polyma inter4 sylva2 cylin oli8b monos aquat diss6 arist grami2 torulB dissi0 grand
E vexa3 ostra ulti1-6 paddi het13 anand nRosLk masto4 interTy sylvat5 polymo olig2 tracG apler disso7 aristB grami0 torul5 dissi6 insid5
F nReh2 oedoc ulti1-7 iwa156 het18 undul9 minus masto7 newInter mamil irreg2 olig4 hypo pachy myrio3 grami8 angus pyrillo insid9
G indig nMaz1 ulti2-7 vio159 vio78-1 orthG parvu marsi macro8 debar irreg10 acan4 hypo delie2 aphan2 myrio5 volut8 pyrilup
H helioG chama ulti2-4 splen1 vio78-3 nunn middl macro6 spino7 irreg9 acan8 trach delie3 aphan3 myrio6 perii volut9 vante4
I nReh1 helio ulti2-6 splen5 nagae perpl multi9 nMaz6 macro7 spino8 nIrreg acan9 perip delie4 aphan4 scler pluri volut4 vante7 rostr
J Un1B Un3 Oo0 Oo15 Py/Phy Phy71 Phy68 Phy3 Phy84 G17 G17-8 G9 cii/cp G15 Haloph Un1B
K G5-6 G5 camb7B G7-8 G7 G19 G16 infe1 n_para later frag/syr mega2 meg19 meg/G15 ma/tar8 insol DIG
L G4-81 cin61 frag6 camb9B syrin6 cryp0 cryp7 iranic palmi7 infest3 nico hiber citri mega14 mega69 kats5 ma/tartr7 G3
M mega.gs cinn13 frag8 camb9 syrin0 cryp12 cryp/24 cland palmi4 infes0 cact3 cact2 bot/mea humic gon/aq kats7 baham polym G21 G vex
N mel/si/spi cinn17 frag9 camb7 porri0 cryp17 eryth tenta megak8 mirab cact9 pseu6 cap/mex capsi peron0 quer/qui epist avice macr/ric kandel
O vignae ci pa17 frag7 cambi6 porri8 cryp2 medic megak1 phaseo idaei pseu7 cap/citro cii/pd90 peron5 ilicis boehm vesic sojae operc
Spot intensities analyzed using Genepix Pro 3.0 software
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rostratum
heterothallicum
torulosum
sylvaticum
Non-compactedCompacted
a
b
a
a
b
a
b
a
0
10000
20000
30000
40000
50000
60000
Mean signals (in quanta)
Effect of soil compaction on populations of different Pythium species (hybridization signals of species-specific oligonucleotides spotted on DNA array)
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DNA arrays developed so far by our group
• Pythium and Phytophthora – ITS• Fusarium – EF1• Penicillium – Beta Tubulin• Apple, greenhouse and cranberry pathogens - ITS
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Design of COI array for Penicillium
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Signature Oligo (“Sigoli”) software developed for identifying unique sequence regions in large databases before using Array Designer
Collaboration with Dr. Verónica Dahl, Department of computing sciences,
Simon Fraser University
Student: Manuel Zahariev
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Dr. Wen Chen
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Acknowledgements
• NSERC and Genome Canada for financial support• Nicole Desaulniers and Rafik Assabgui
for technical support