AN ABSTRACT OF THE THESIS OF Dabao Sun Lu for the degree of Master of Science in Botany and Plant Pathology presented on September 6, 2016. Title: Genome Organization in the Ectomycorrhizal Truffle Rhizopogon vesiculosus. Abstract approved: ______________________________________________________ Joseph W. Spatafora Rhizopogon vesiculosus is a common ectomycorrhizal (EM) symbiont of Pseudotusga menziesii (Douglas-fir) in the coast range of the Pacific Northwest. The species has been studied for its systematics, genet size, population structure, and competitive ability in several field and experimental studies. This thesis seeks to provide a more thorough characterization of the genome of R. vesiculosus using bioinformatic analyses with the objective of improving our understanding of its genomic architecture and to obtain a more robust reference for future studies on Rhizopogon. The analysis was facilitated by Chromatin conformation capture assay (Hi-C) sequencing which enabled the assembly of our previous draft genome into 10 chromosome level linkage groups. Kingdom wide comparative genomic studies in fungi suggest that there are particular signatures in ectomycorrhizal genomes including an increase of transposable elements (TE) and lineage specific small secreted proteins (SSPs). This observation was true for R. vesiculosus as we mined the genome for TEs and SSPs. We sought to classify the TEs, but found that a large proportion of them had no hits to curated TE databases. The abundance of SSPs fit the profile of other EM fungi with approximately 200 SSPs and half of them being species specific. We identified species specific genes (SSG) and orthologous clusters shared with the sister genus Suillus, along with clusters unique to genus Rhizopogon and R. subgenus Villosuli, as well as the mating loci and centromeric regions. The long-range contiguity allowed us to map all these elements together with single nucleotide polymorphisms (SNPs). We hypothesized that the SSPs, TE, and lineage specific genes would cluster more around the ends of our linkage groups where recombination rate tends to be elevated, and that the centromeres would be highly enriched for TEs, but the pattern turned out to be more complex. Centromeres were regional in nature, comprising an average length of 40,000 bp and a gene density similar to the remainder of the genome. Most chromosomes contained regions that were enriched for species specific genes, but these regions were not clustered towards the ends of linkage groups. However genome mapping revealed higher SNP densities in TEs and species specific genes, in contrast to reduced levels in the orthologs shared with Suillus, suggestive of a “two-speed-genome” with heterogeneous rates of evolution. Moreover, the transition/transversion ratio was found to be significantly higher in TEs, which we interpreted as a possible signature of genome defense mechanisms, highlighting the many opposing evolutionary forces that a genome must balance. This work represents a significant step forward in the genomics of Rhizopogon, and we have demonstrated the utility of Hi-C sequencing for improving assemblies of dikaryotic fungi. A number of bioinformatics pipelines were developed or refined, and these may serve to streamline future Hi-C analysis of other genomes. ©Copyright by Dabao Sun Lu September 6, 2016 All Rights Reserved Genome Organization in the Ectomycorrhizal Truffle Rhizopogon vesiculosus by Dabao Sun Lu A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 6, 2016 Commencement June 2017 Master of Science thesis of Dabao Sun Lu presented on September 6, 2016 APPROVED: Major Professor, representing Botany and Plant Pathology Head of the Department of Botany and Plant Pathology Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Dabao Sun Lu, Author ACKNOWLEDGEMENTS I feel privileged for having been given the opportunity to embark on what will be remembered as three enjoyable and formative years as a student at Oregon State University where I have been able to grow significantly as a person and was molded as a scientist. The many people who in some way have contributed to this thesis and experience outnumber the literature references, and I will therefore limit myself to mention those who have been most directly involved with my project. First and foremost I would like to thank my advisor Dr. Joseph Spatafora. I am truly grateful for his willingness to take me on in his lab and believing that I had what it took to undertake and complete graduate studies in mycology, where many others, including myself, at times doubted so. His way of thinking about mycology has been mind-opening, and his passion for it is contagious. His personal support has been crucial for navigating me through what at times can be rough waters in the graduate school research experience, and I would never have felt as much at home here without him and his wife Elizabeth’s hospitality and care. I am grateful to the whole Spatafora lab for being the friendly and creative working environment which it is. Among lab members I credit Alija Mujic for introducing me to the wonders of the hypogeous world, and I am particularly appreciative for all the hours he spent patiently mentoring me in every aspect of mycology, and for being a dear friend. I am thankful to Kathryn Bushley and Alisha Quandt for showing me the ropes of molecular biology and making someone far away from home feel welcome through their cheerful presence. I thank Derek Johnson for significantly reducing the shock factor in my introduction to the world of bioinformatics, and for keeping me company in the small hours before deadlines. I will never forget our many discussions and shared frustration over poorly documented code. I thank Rheannon Arvidson and Ying Chang for being good lab citizens, for helpful suggestions that improved this project along the way, and for sharing my preference for tea. Thank you to Kazuya Tsukagoshi for brightening up the days with his good humor and friendship. A special thanks to Brittny Gardner and her zealous spirit which helped me get through what otherwise would have been insurmountable amounts of labwork, for her efforts to improve my English in spite of any tangible results, and for showing me Oregon outside Corvallis. I am indebted to our collaborators Ivan Liachko and Shawn Sullivan who provided the data, without their expertise and initiative this project would never have happened. I know my stay here would not have been as streamlined without Dianne Simpson, Carleen Nutt, and Blaine Baker in the BPP department handling the administrative affairs. It would not have been as good of a learning experience without the numerous instructors I was able to interact with through my coursework, and most importantly it would not have been as enjoyable without all my fellow students in BPP. I am thankful for the international office at OSU, and in particular I want to recognize Emiko Christopherson and Marigold Holmes for helping me through copious amounts of paperwork, and I also thank Kelly Bonin and her colleagues at the IIE for working with me the entire time. I thank my committee members, Jane Smith, Nikolaus Grunwald and David Myrold, for all their support, and for understanding the unpredictable nature of biological research. I would like to acknowledge Christopher Sullivan for all the hard work he puts in to keep the CGRB computational cluster running, which my thesis has heavily relied on. There are many foundations from which I have received support. Foremost, I am indebted to the Fulbright program and Fulbright Norway for making the prospect of pursuing a graduate program in the US a financially possible option for me in the first place. In addition to Fulbright, the support I received from Dr. Haavard Kauserud among several professors at the University of Oslo increased my confidence enough to apply for graduate programs, and provided me with the momentum to venture all the way across the Atlantic and the North American continent in search for new pastures. I received the Haakon Styri Award from the American Scandinavian Foundation during my second year. The scholarship is administered by the Norway America Association, and was key for continuing my funding, and also contributed significant amounts to my research funds. Among other things, it facilitated a visit to the Bruns lab at UC Berkeley where Sara Branco generously and patiently took her time to train me in several bioinformatic pipelines containing aspects which have been important in my thesis work. I thank NATS and all their members for constituting both a monetary, intellectual, and recreational resource. I have received financial support from NATS through the Henry Pavelek Memorial Scholarship Fund and a student scholarship which sent me truffling to the Sierra Nevada. I am also grateful to OMS and SOMA for generously supporting me through their student scholarships. I received the GSA and Anita Summers travel grant from the BPP to present this project at the annual meeting of the Mycological Society of America at Berkeley in 2016, and I would like to extend my gratitude for the administers, founders, and fundraisers for these funds. During my last year, I have received funding through working as a research assistant for the Oregon State University Herbarium, and I would like to thank Dr. Richard Halse and all the other herbarium workers for making it a peaceful and friendly working environment. The herbarium position involves teaching the mycology class, which proved to be a great learning experience much thanks to Dr. Dan Luoma and Chris Bennemann. I thank Joey Deshields, Elva Manquera, and Eric Renteria for hunting Rhizopoogon in the field with me, although the samples we collected did not get to play a role in this project, the cultures we obtained from them represent valuable additions that will likely play a role in future Rhizopogon projects.