GENOMIC MAPPING OF LEAF RUST AND STEM RUST RESISTANCE LOCI IN DURUM WHEAT AND USE OF RAD-GENOTYPE BY SEQUENCING FOR THE STUDY OF POPULATION GENETICS IN PUCCINIA TRITICINA A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Meriem Aoun In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department Plant Pathology November 2016 Fargo, North Dakota North Dakota State University Graduate School Title GENOMIC MAPPING OF LEAF RUST AND STEM RUST RESISTANCE LOCI IN DURUM WHEAT AND USE OF RAD-GENOTYPE BY SEQUENCING FOR THE STUDY OF POPULATION GENETICS IN PUCCINIA TRITICINA By Meriem Aoun The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Maricelis Acevedo Chair Dr. James A. Kolmer Dr. Elias Elias Dr. Robert Brueggeman Dr. Zhaohui Liu Approved: 11/09/16 Dr. Jack Rasmussen Date Department Chair ABSTRACT Leaf rust, caused by Puccinia triticina Erikss. (Pt), and stem rust, caused by Puccinia graminis f. sp. tritici Erikss. and E. Henn (Pgt), are among the most devastating diseases of durum wheat (Triticum turgidum L. var. durum). This study focused on the identification of Lr and Sr loci using association mapping (AM) and bi-parental population mapping. From the AM conducted on the USDA-National Small Grain Collection (NSGC), 37 loci associated with leaf rust response were identified, of which 14 were previously uncharacterized. Inheritance study and bulked segregant analysis on bi-parental populations developed from eight leaf rust resistance accessions from the USDA-NSGC showed that five of these accessions carry single dominant Lr genes on chromosomes 2B, 4A, 6BS, and 6BL. The other three accessions have Lr genes with more complex inheritance. All eight accessions carry different genes than those already mapped in durum cultivars except one accession with Lr61. Linkage mapping in two of the bi-parental populations showed that the gene in PI 209274 (LrCA) was mapped to 6BS between SNPs IWA3298 and IWB39456, while the gene in PI 192051 (LrPort) was mapped to 4AL, flanked by IWA4254 and IWA8341. Resistance to Pgt-race TTKSK was also observed in PI 534304 and PI 192051. PI 534304 was found to carry Sr13 on chromosome 6AL, while PI 192051 carries a novel Sr gene (SrPort) mapped to 7AS flanked by IW8390 and IWA1805. The genotype PI 192051 has an additional QTL (QSr.ndsu-5B) to Pgt races in a field trial in Ethiopia in 2016. The QSr.ndsu-5B was mapped to 5BL and delimited by IWA6992 and IWA2181. The study of virulence diversity in Pt isolates collected from several countries identified seven races among 51 isolates collected from durum wheat and 21 races among 40 isolates collected from common wheat. The phylogeny study on 30 Pt isolates based on the Restriction-Associated DNA (RAD)-Genotype by Sequencing (GBS), clustered the isolates into eight clades, with higher iii diversity in the SNP genotypes in common wheat isolates compared to that in durum wheat isolates. RAD-GBS is identified as a suitable and informative genotyping technique to study the population genetics of Pt. iv ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisor, Dr. Maricelis Acevedo for her guidance, encouragement, understanding, support, and mentorship during my doctoral studies at the Department of Plant Pathology. With deep sense of gratitude, I would like to express my sincere thanks to Dr. James Kolmer at the United States Department of Agriculture, Cereal Disease Laboratory (USDA-CDL, Saint Paul, Minnesota) for providing constructive guidance throughout the period of my research study. His wealth of knowledge in wheat leaf rust was an asset to my research work. I am indebted to the other members of my committee, Dr. Elias Elias, Dr. Robert Brueggeman, and Dr. Zhaohui Liu for their valuable suggestions, encouragements, and constructive feedback. I would also like to thank the department chair, Dr. Jack Rasmussen who supported my stay at North Dakota State University (NDSU). Special thanks to the faculty of the Department of Plant Pathology at NDSU for mentoring me into a deeper thinking plant pathologist. I extend my thanks to all the members of the wheat rust laboratory, Matthew Breiland, Amanda Swank, Zennah Kosgey, Albert Kertho, Jason Zurn, and Sheshanka Dugyala for always being positive and supportive. I would like also to thank Jon Richards and Tom Gross at the Barley Pathology Lab at NDSU for their help in the genotyping of P. triticina isolates. My appreciation goes to the students and staff of the Department of Plant Pathology for providing me a friendly environment to perform my research. I am very grateful for the support from our collaborators at the USDA-CDL at Saint Paul, Minnesota, Dr. Matthew Rouse for his help in screening two mapping populations with stem rust and Dr. Jerry Johnson and Dr. Les Szabo for their help in the phylogeny analysis. Many thanks goes to our collaborators at CIMMYT- Mexico, Dr. Julio Huerta-Espino for providing me with some P. triticina isolates for this project, v Dr. Amor Yahyaoui for his continuous encouragement and advice, Dr. Karim Ammar and Dr. Alexander Loladze for their help in conducting leaf rust nursery in Mexico. I am also indebted to Dr. Steven Xu from the USDA–ARS, Cereal Crops Research Unit in Fargo for providing me with germplasm from NSGC and Dr. Shiaoman Chao from USDA-ARS Small Grain Genotyping Lab in Fargo for her constructive feedback and genotyping of the durum wheat populations. I am deeply grateful to the collaborators from the Ethiopia Institute for Agricultural Research Center in Debre Zeit and ICARDA-Morocco for their help in carrying out rust nurseries. My thanks go to the North Dakota Wheat Commission and State Board of Agricultural Research and Education for funding my research project. I would like to thank my friends Sahar Arabiat, Zennah Kosgey, Nivi Abraham, Hazila Khalid, and Serap Vatansever who encouraged me and made my stay in Fargo enjoyable and memorable. Many thanks go to Dr. Mohamed Somo who helped me in so many ways during my stay in Fargo. I also appreciate the long distance support of my friends Ada Metaliu, Asma Benali, Shaimaa Helmy, Amal Kahla, Janet Kanani, Samira Kharchoufi, Soukaina Ben Othmen, and Suad Khaled who motivated me during my entire stay in Fargo. I am indebted to my parents and my siblings who provided me endless love, encouragement, care, patience, and support during the entire period of my academic life. vi DEDICATION To my beloved parents Ibrahim Aoun and Mahbouba Aoun and to my brothers and sisters Mouna, Mohamed, Marwa, Maha, Makrem, and Mehdi who always inspire me. vii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGMENTS .............................................................................................................. v DEDICATION .............................................................................................................................. vii LIST OF TABLES ....................................................................................................................... xiii LIST OF FIGURES ...................................................................................................................... xv LIST OF APPENDIX TABLES .................................................................................................. xvi LIST OF APPENDIX FIGURES................................................................................................ xvii GENERAL INTRODUCTION ....................................................................................................... 1 CHAPTER I. LITERATURE REVIEW ......................................................................................... 5 Evolution of durum wheat ........................................................................................................... 5 Origin and domestication of durum wheat .................................................................................. 7 Dissemination of domesticated durum wheat ............................................................................. 8 Wheat rusts ................................................................................................................................ 10 Taxonomy and disease cycle ................................................................................................. 10 Leaf rust ................................................................................................................................. 12 Leaf rust importance and symptoms .................................................................................. 12 Puccinia triticina hosts, biology, and epidemiology ......................................................... 13 Population genetics of Puccinia triticina ........................................................................... 14 Characteristics of P. triticina populations in durum wheat ............................................... 17 Leaf rust resistance in durum wheat .................................................................................. 19 Wheat stem rust ..................................................................................................................... 22 Importance, symptoms,