IDENTIFICATION OF PATHOGENIC RACES AND MICROSATELLITE MARKERS OF TILLETIA CARIES (D.C.) TUL. & C. TUL. AND MAPPING OF A COMMON BUNT RESISTANCE GENE IN WINTER WHEAT By GLAFERA JANET B. MATANGUIHAN ii A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Crop and Soil Sciences MAY 2011 © Copyright by GLAFERA JANET MATANGUIHAN, 2011 All Rights Reserved iii © Copyright by GLAFERA JANET MATANGUIHAN, 2011 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of GLAFERA JANET B. MATANGUIHAN find it satisfactory and recommend that it be accepted. __________________________________________ Stephen S. Jones, Ph.D., Chair __________________________________________ 98 Timothy D. Murray, Ph.D. __________________________________________ Scot H. Hulbert, Ph.D. __________________________________________ Lori M. Carris, Ph.D. __________________________________________ Xianming M. Chen, Ph.D. ii ACKNOWLEDGEMENTS Pursuing a Ph.D. degree has been one of the most rewarding and enriching experiences of my life, and there are many people who have helped make it so. First of all, I thank Dr. Stephen S. Jones, for giving me the opportunity to be a graduate student in his lab. He is an excellent, generous mentor who genuinely cares and values his staff and students. The training I have received from him is invaluable. I could not have wished for a better adviser. I am deeply grateful to the members of my committee for investing their time in my research and guiding me with their expertise. I also thank Blair Goates, who has been a most generous source of seeds, races, and technical advice, and Dr. Deven See, for his time and guidance in the analysis of my SSR data. He has made it possible for me to finish the third chapter of my dissertation. I also 10 thank Dr. Bob Allan for answering my questions and offering insights into my work, and 4 Dan Dreesmann for always finding space for me in the Plant Growth Facilities. I also thank the members of the Jones‟ Lab (past and present) – Meg Gollnick and Kerry Balow, for their friendship and support in various ways. Meg made the lab feel like a family to me. Steve Lyon was always willing to help me in my field experiments. I thank Dr. Kevin Murphy for writing the organic grant that got me here in the first place, giving me feedback on my research proposal in my first year, and agreeing to be a co- author. I thank Dr. Matt Arterburn for his enthusiasm and generosity when he taught me how to do PCR and AFLP analysis. I also thank all the time slip students who have assisted me in DNA extraction and scoring of bunt incidence in the field – Shane Eucker, Jake Bopray, Trevor Fox, Rachel Daniel, and Sam Turner. iii I thank my fellow Mount Vernon Plant Breeding students – Jeff Endelman, Karen Hills and Lucas Patzeck - for their friendship and input in my research, and I thank the Mount Vernon NWREC staff for logistical support which they gave so willingly: Jeanne Burrit, Ashley Bentley, Jamie Anderson and Kate Gleissner. I thank the Cereal Disease lab for the camaraderie, support and research help in so many ways: HongYan Sheng, Kathy Klos, Julie Evans, Henry Wetzel, and Megan Robinson. They have regarded me as one of their lab members and I deeply appreciate that. I also thank the Winter Wheat lab for the use of their PCR machines and genotyping software. I also thank my fellow graduate students – Renuka Attanayake, Evans Njambere, Xiaodong Bao, Ebrahim Bebiker, Grant Poole, and Jane Stewart - for encouragement 10 4 when times got tough, and for sharing their knowledge; Avi Alcala and Anne Tarun for their prayers, encouragement and friendship- they have become like dear sisters to me. Most of all, I thank my family for their love, prayers, support and encouragement. My parents have instilled in me a love for learning and a drive for excellence. My siblings have always taken joy in my achievements, and I in theirs. I could not have finished this without the love and prayers of my husband, his wisdom and counsel. My sons are my inspiration. Through it all, like a golden thread interwoven into everything – God‟s faithfulness! iv IDENTIFICATION OF PATHOGENIC RACES AND MICROSATELLITE MARKERS OF TILLETIA CARIES (D.C.) TUL. & C. TUL. AND MAPPING OF A COMMON BUNT RESISTANCE GENE IN WINTER WHEAT Abstract by Glafera Janet B. Matanguihan, Ph.D. Washington State University May 2011 Chair: Stephen S. Jones Common bunt, caused by Tilletia caries and T. laevis, is one of the most devastating seedborne diseases of wheat. In conventional agriculture, the disease is managed almost 10 4 exclusively with chemical seed treatments. However, synthetic chemicals are prohibited in organic agriculture, and growers must rely heavily on resistant cultivars to manage the disease. To facilitate breeding for resistance under organic systems, the resistance genes in 14 winter wheat cultivars were identified by inoculating these with 40 pathogenic races of common bunt. A resistance gene model was constructed based on their disease reaction to the races, and the reaction of 15 bunt differential cultivars. Eight cultivars were postulated to possess new bunt resistance genes or new gene combinations. To test this model, F2 populations of these cultivars were analyzed for goodness of fit to the expected segregation ratio. The F2 population of Lewjain x Elgin showed a 3:1 ratio (resistant: susceptible), indicating that Lewjain has a single major gene for bunt resistance. Wheat microsatellite or simple sequence repeats (SSR) markers were used to v map this gene to its chromosomal location. A total of 763 SSR markers were screened on the parents, and 349 polymorphic SSR loci were screened on the mapping population. The marker wmc112, located on chromosome 2D, was linked with the resistance trait with a distance of 37 cM. This is an initial indication of the location of the bunt resistance gene in Lewjain. Madsen, Finch and Masami have resistance genes worth investigating further since these could be different from that of Lewjain. For effective deployment of resistance genes, it is important that races of the pathogen be identified and monitored. Towards this goal, races of the pathogen present in Washington, Oregon, California and Idaho were identified by inoculating 12 T. caries collections on 15 common bunt differential cultivars. Results indicate the presence in Washington of a new pathogenic race which has the broadest virulence spectrum compared to known 10 races. Microsatellite loci were also isolated from T. caries, and 12 polymorphic 4 microsatellite markers were developed for future genetic diversity and population genetics studies. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT .........................................................................................................................v LIST OF TABLES ...............................................................................................................x LIST OF FIGURES .......................................................................................................... xii CHAPTER 1. CONTROL OF COMMON BUNT IN ORGANIC WHEAT Abstract ......................................................................................................................1 Introduction ................................................................................................................2 Host resistance ............................................................................................................9 Seed treatments ........................................................................................................26 The challenge remains ..............................................................................................33 References ................................................................................................................41 10 4 2. A NEW PATHOGENIC RACE OF TILLETIA CARIES WITH THE BROADEST VIRULENCE SPECTRUM OF KNOWN RACES Abstract ....................................................................................................................58 Introduction ..............................................................................................................59 Collection of common bunt samples ........................................................................62 Determination of virulence patterns .........................................................................63 New pathogenic race ................................................................................................67 Possibility of other new races ...................................................................................68 Discussion ................................................................................................................68 Literature cited .........................................................................................................76 3. RESISTANCE TO COMMON BUNT IN WINTER WHEAT Abstract ....................................................................................................................82 vii Introduction ..............................................................................................................84 Materials and methods .............................................................................................88