Resolving thePythium ultimum species complex by Quinn Eggertson A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs partial fulfillment of the requirements for the degree of Master of Science in Biology Carleton University Ottawa, Ontario ©2012 Quinn Eggertson Library and Archives Bibliotheque et Canada Archives Canada Published Heritage Direction du 1+1 Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-93569-9 Our file Notre reference ISBN: 978-0-494-93569-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Eggertson 2012 A b s t r a c t The Pythium ultimum species complex is comprised of two morphological varieties: var. ultimum and var. sporangiiferum. Previous efforts to resolve whether the two varieties are genetically distinct have produced conflicting results. Using 29 isolates of Pythium ultimum - twelve isolates of var. sporangiiferum and seventeen isolates of var. ultimum, multi-gene genealogies were created from both newly identified highly variable genes and previously identified genes. Observed conflict between species estimates given by nuclear and mitochondrial gene regions resulted in two separate studies. The first examined multiple nuclear genes to resolve the species complex, while the second tested the suitability of mitochondrial genes for phylogenetic analysis. Nuclear multi-gene genealogies found that the Pythium ultimum species complex is comprised of four genetically distinct species, while mitochondrial multi-gene genealogies indicate that the mitochondrial genome of Pythium ultimum is single copy, non-recombinant and uniparentally inherited. Q. Eggertson 2012 A cknowledgement I would like to thank C. Robin Buell and John Hamilton from Michigan State University for providing me with SNP data and allowing me access to the P. ultimum genome website before it was released. I would also like to express sincere gratitude towards Andre Levesque for being a supportive and engaged supervisor, Tara Rintoul for her support, patience and always appreciated advice, Gregg Robideau for his general helpfulness and for kindly lending me his primers for OCM1, and Rafik Assabgui, Julie Chapados, and Kasia Dadej for their quick sequencing. I’d also like to acknowledge Myron Smith, who taught me the basics of both genetics and mycology and Nicolas Rodrigue whose willingness and patience in explaining the nuances of bioinformatics software was appreciated. Last but not least, I would like to thank my family and friends for their support, encouragement and for only looking slightly pained when I tried to explain my research. Q. Eggertson 2012 T able of Contents Resolving the Pythium ultimum species complex .................................................................. i Abstract ............................................................................................................................ ii Acknowledgement .........................................................................................................iii Table of Contents ..........................................................................................................iv List of Tables .................................................................................................................. vi List of Figures ................................................................................................................. vi List of Appendices ..........................................................................................................vii Thesis Introduction .................................................................................................................... 1 Chapter 1: Resolving the Pythium ultimum species complex using nuclear multi-gene genealogies under the GCPSR ............................................................................................... 5 Introduction .....................................................................................................................5 Materials and Methods ................................................................................................ 7 In house BLAST, Identification and Selection o f Isolates ...........................................7 Acquisition o f Strains and Culture Conditions............................................................9 DNA Extraction............................................................................................................... 9 SNP Analysis and Gene Selection ............................................................................... 12 Primer Design :......................................... 13 Gene Amplification with PCR ......................................................................................15 Gel-Electrophoresis......................................................................................................15 Sequencing.....................................................................................................................18 Outgroups......................................................................................................................18 Sequence Editing, Alignment and Phylogenetic Analysis .........................................19 Gene and Species Tree Visualization ..........................................................................20 Results ............................................................................................................................. 23 Preliminary Screening of Isolates ............................................................................... 23 PCR and Sequencing Primer Performance............................................................... 23 Sequencing and Sequence Editing.............................................................................. 24 Sequencing Ambiguities............................................................................................... 25 Phylogenetic Trees........................................................................................................25 Discussion ........................................................................................................................33 - iv - Q. Eggertson 2012 Chapter 2: Examining mitochondrial and nuclear gene tree conflict in species level analysis of Pythium ultimum............................................................................................. 48 Introduction .................................................................................................................. 48 Materials and Methods .............................................................................................. 53 Selection of Isolates .................................................................................................. 53 SNP Analysis and Gene Selection ............................................................................ 53 Primer Design ........................................................................................................... 54 Gene Amplification with PCR ...................................................................................54 Sequencing.................................................................................................................54 Outgroups..................................................................................................................54 Sequence Editing, Alignment and Phylogenetic Analysis .......................................56 Pairwise Distances.................................................................................................... 57 Results .............................................................................................................................59 Gene Selection .................................................................................................... 59 Primer Design