COI barcoding of oomycetes and the design of arrays for environmental samples André Lévesque, Gregg Robideau and Wen Chen Biodiversity, AAFC and Biology Department, Carleton University

Evolution of all the species of the genus Pythium (ITS and LSU) 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) pythiosis mycoparasites Arthur de Cock, CBS, Netherlands, collection of « ex-type » cultures mycoparasite Other species are in the saprophyte to facultative plant parasite spectrum

M.Sc. Candidate (since Sept 2007) Dept. of Biology Carleton University Gregg Robideau M.Sc. Candidate (since Sept 2007) Dept. of Biology Carleton 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 A C B A B C

Pythium attrantheridium vs. P. intermedium 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 are also analogies with functional genomics: There is an analogy between barcoding marker gene(s) for all species and the sequencing all the genes of one species (genomics) There are also analogies with functional 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/sequencing and arrays Quantify most important species affected (RT)-qPCR

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 Non-Compacted Compacted Non-Compacted Compacted 500 gallons tanks at the front and rear of tractor

Compacted Soil (4 years) Chemiluminogrammes showing hybridization patterns in soil compaction experiments Non-compacted Soil Compacted Soil (4 years) Spot intensities analyzed using Genepix Pro 3.0 software

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

Simon Fraser University Student: Manuel Zahariev 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