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

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

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

COI Pythium Phylogeny: Tree Top Half

K

D

AC

C

A

B

B

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

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:

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

Soil processing with Pythium ITS array

13 Pythium species found by array hybridization

Direct processing of environment samples -Comparing treatments

Compacted

Non-Compacted

Non-CompactedCompacted

500 gallons tanks at the front and rear of tractor

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

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)

DNA arrays developed so far by our group

• Pythium and Phytophthora – ITS• Fusarium – EF1• Penicillium – Beta Tubulin• Apple, greenhouse and cranberry pathogens - ITS

Design of COI array for Penicillium

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

Dr. Wen Chen

Acknowledgements

• NSERC and Genome Canada for financial support• Nicole Desaulniers and Rafik Assabgui

for technical support