Genetics, Sources, and Mapping of Stem Rust Resistance in Barley A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Austin Joel Case IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Adviser: Dr. Brian J. Steffenson April 2017 © Austin Joel Case, April 2017 Acknowledgements Many thanks to my major advisor Dr. Brian Steffenson for providing an enjoyable and challenging project. Dr. Steffenson always believed in me and was a stalwart ally even when projects half a world away were not going as planned. Thanks are owed to my committee members, Dr. Matthew Rouse, Dr. Ruth Dill-Macky, and Dr. Kevin Smith. A great debt is owed to fellow graduate students in the Steffenson lab: Dr. Bullo Mamo, Dr. Matthew Haas, Shuyi Huang, Fazal Manan, and Grete Slaugh. A special thanks is owed to Matthew Haas who proved that Minnesota nice is actually nice. Thanks to Steffenson lab personnel past and present: Matthew Martin, Tamas Szinyei, Dr. Jeness Scott, Jamie Simmons, Cole Welchlin, Dachi Jguniashvili, Yuan Chai, Dr. Oadi Maty, Dr. Ahmad Sallam, and Ryan Johnson. Special thanks to Matthew Martin for his ability to organize projects in the face of overwhelming chaos; to Tamas Szinyei for having an ability to find any seed packet in any box no matter how old; and to Dr. Ahmad Sallam for assistance with data analysis. I would also like to thank Dr. Jeness Scott for her early morning coffee talks, even if they were only occasionally productive. I would also like to thank the countless undergraduates for their time and efforts. If there was a hall of fame for undergraduate assistants, then Katie Ring is a first ballot no contest hall of famer, all other undergraduate students will fail to measure up. I would also like to thank Ryan Gavin, although not the best worker, he was always a pleasure to work with. Additionally, I would like to thank Felipe de Silva, Andra Bates, and Margaret Krause for being my mentees and allowing me to develop my management skills. I wish to thank the entire Plant Pathology Department, which has been a great home for these past five years. Thanks to Stephanie Dahl for her professional management of the BSL-3 facility. I would like to thank Dr. Sridhar Bhavani and Dr. Godwin Macharia for assistance with field nurseries in Kenya and for making me feel welcome while I was there. Thanks to Dr. Harold Bockelman for always being generous with seed. Thanks to Dr. Gina Brown-Guedira and Dr. Priyanka Tyagi for assistance with Genotype by i Sequencing Analysis. I would also like to thank my family. Thanks to my parents and grandparents, who have always supported me in chasing my dreams, even when it meant living away from home. Thanks to my wife Lisa and my son Wyatt, who have supported me throughout this journey and have always, supported me without question. This could not have been done without your love and support. ii Dedication I dedicate this to my family who has supported me without question. iii Abstract Stem rust is a devastating disease of cereal crops worldwide. In barley (Hordeum vulgare), the disease is caused by two pathogens: Puccinia graminis f. sp. secalis (Pgs) and Puccinia graminis f. sp. tritici (Pgt). In North America, the stem rust resistance gene Rpg1 has protected barley from losses for more 60 years; however, widely virulent Pgt races from Africa in the Ug99 group threaten the crop. To identify novel quantitative trait loci (QTL) for stem rust resistance, bi-parental and association mapping studies were undertaken in the Barley iCore Collection (BCC) held by the USDA National Small Grains Collection. Association mapping studies of the BCC were conducted for seedling resistance to Pgt race TTKSK (Ug99 group) in the greenhouse and adult plant resistance (APR) to Pgt TTKSK composite in Njoro, Kenya and Pgt race QCCJB in St. Paul, MN. A major effect QTL (Rpg-qtl-5H-11_11355) for APR in all locations was identified on chromosome 5H. This QTL represents a unique locus for APR and has been confirmed in other studies. Subsequently, 290 of the most resistant BCC accessions, the BCC Selects (BCCS), were screened for resistance Pgt races MCCFC, QCCJB, HKHJC, TTKSK, TTKST, TKTTF, and TRTTF, and also Pgs isolate 92-MN-90. From this investigation, four BCCS accessions were postulated to carry Rpg1, 14 to carry Rpg2, 91 to carry Rpg3, four to carry rpg4/Rpg5, and 59 to carry potentially novel resistance genes. To map the APR genes of Rpg2 and Rpg3 in Hietpas-5 (CIho 7124) and GAW-79 (PI 382313), respectively, two biparental populations were developed with Hiproly (PI 60693), a stem rust susceptible accession. Both populations were phenotyped to the domestic Pgt races of MCCFC, QCCJB, and HKHJC in St. Paul and to Pgt TTKSK composite in Njoro. In the Heitpas-5/Hiproly population, a major effect QTL was identified on chromosome 2H, which is proposed as the location for Rpg2. In the GAW-79/Hiproly population, a major effect QTL was identified on chromosome 5H and is the proposed location for Rpg3. The resistance sources identified and characterized in this study enhance barley breeding programs focused on stem rust resistance. iv Table of Contents Acknowledgements ............................................................................................................ i Dedication ......................................................................................................................... iii Abstract ............................................................................................................................. iv Table of Contents .............................................................................................................. v List of Tables ..................................................................................................................... x List of Figures ................................................................................................................. xiv List of Online Resources ............................................................................................... xxii Chapter 1 General Introduction and Literature Review .............................................. 1 Barley Cultivation and Uses ........................................................................................................ 2 Barley Biology ............................................................................................................................. 2 Barley Genetics ........................................................................................................................... 3 Barley Genetic Resources ............................................................................................................ 4 Genetics of Disease Resistance ................................................................................................... 5 Stem Rust Biology ....................................................................................................................... 6 Races of Stem Rust ...................................................................................................................... 7 History of Stem Rust on Barley ................................................................................................... 8 Breeding for Stem Rust Resistance in Barley............................................................................ 10 Resistance Gene Rpg1 ............................................................................................................... 12 v Resistance Gene Complex rpg4/Rpg5 ....................................................................................... 13 Other Resistance Genes ............................................................................................................. 14 Stem Rust QTLs ........................................................................................................................ 15 Introduction to Genetic Mapping .............................................................................................. 17 Linkage Estimation .................................................................................................................... 17 Genetic Markers ........................................................................................................................ 19 Bi-parental Mapping Populations .............................................................................................. 20 Genome-Wide Association Study ............................................................................................. 22 Chapter 2 Genome-Wide Association Study of Stem Rust Resistance in a World Collection of Cultivated Barley...................................................................................... 25 Introduction ............................................................................................................................... 26 Materials and Methods .............................................................................................................. 32 Plant Materials ...................................................................................................................... 32 Genotyping ............................................................................................................................ 33 Adult Plant Rust Evaluations in Kenya ................................................................................. 33 Adult Plant Rust Evaluations in the United States ...............................................................