1 ABSTRACT 2 OLSON, ERIC LEONARD. Characterization of Stem Rust Resistance in US Wheat 3 Germplasm. (Under the direction of Gina Brown-Guedira.) 4 5 In 1999 in Uganda a race of stem rust, Puccinia gramins f. sp. tritici was 6 identified with virulence to Sr31. This race, designated as TTKS based on the North 7 American nomenclature system, combined Sr31 virulence with virulence to the majority 8 of Triticum aestivum L. derived stem rust resistance genes. The development of resistant 9 cultivars is needed as TTKS may reach global dispersal due to its unique virulence to 10 multiple known and unknown resistance genes and widespread cultivar susceptibility. 11 The ability to detect the presence of specific stem rust resistance genes using molecular 12 markers presents a viable method for identifying resistance to race TTKS in the absence 13 of the pathogen itself. The frequency of DNA markers associated with resistance genes 14 Sr24, Sr26, Sr36, and Sr1RSAmigo which confer resistance to TTKS was assessed in 15 diverse wheat cultivars and breeding lines from breeding programs throughout the United 16 States. The reliability of these markers in predicting the presence of the resistance genes 17 in diverse germplasm was evaluated through comparison with phenotypic data. 18 Introgression of undeployed seedling resistance genes is necessary to improve the 19 availability of resistance to TTKS. The stem rust resistance gene Sr22 confers resistance 20 to TTKS. Sr22 is present on a chromosomal translocation derived from Triticum 21 boeoticum Boiss. which is homoeologous to the A genome of T. aesitivum Linkage 22 analysis of SSR loci on 7AL was done to identify the loci most closely linked to Sr22. 23 Individuals with reduced T. boeoticum segments due to recombination between wheat 24 chromosome 7AL and the Sr22 introgression were identified with SSR markers in F2:3 25 populations of crosses between the germplasm stock Sr22Tb and the hard winter wheat 1 lines 2174 and Lakin. From analysis of F3:4 populations derived from F2 recombinants, 2 F3:4 individuals with further reduced translocation segments have been identified. 3 Recombinant lines with reduced translocations will provide a more agronomically 4 desirable source of Sr22 stem rust resistance in hard winter wheat germplasm that can be 5 readily deployed utilizing molecular markers. The identification of molecular markers 6 efficacious for the selection of genes for resistance to TTKS will hasten the development 7 of resistant cultivars. 8 Characterization of Stem Rust Resistance in US Wheat Germplasm 1 2 by 3 Eric Leonard Olson 4 5 A thesis submitted to the Graduate Faculty of 6 North Carolina State University 7 in partial fulfillment of the 8 requirements for the Degree of 9 Master of Science 10 11 12 Crop Science 13 14 15 Raleigh, North Carolina 16 17 18 2009 19 20 21 APPROVED BY: 22 23 24 25 26 _______________________ _________________________ 27 Dr. David Marshall Dr. James B. Holland 28 29 30 31 32 33 34 __________________________ 35 Dr. Gina Brown-Guedira 36 Chair of Advisory Committee 37 38 39 40 41 42 1 2 BIOGRAPHY 3 4 Eric Leonard Olson was born in Dodgeville, WI in 1980 to Leonard and Catherine 5 Olson. Eric, the oldest of three siblings, lived and worked on the family dairy farm for 6 many years. The best years of his life were spent working beside his brother, father and 7 grandfather on the farm. Attending the University of Wisconsin in Platteville, Eric 8 developed a love of science and a desire to make meaningful contributions to agriculture 9 through science. Opportunity for graduate studies at North Carolina State University was 10 available and in 2007 Eric began work on an MS degree with Dr. Gina Brown-Guedira. 11 In January of 2009 Eric will begin a Phd. program at Kansas State University working 12 with Dr. Michael Pumphrey and Dr. Bikram Gill. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ii 1 2 ACKNOWLEDGEMENTS 3 4 I would like to thank most of all those few who let me believe that graduate 5 school was a possibility. Thank you to my family for teaching me how to work hard and 6 for being there for me, always. Thank you to Dr. Gina Brown-Guedira for the 7 opportunity to do challenging and meaningful work. I am grateful to Jared Smith and 8 Kim Howell for sharing their valuable technical expertise. I sincerely thank Dr. Michael 9 Pumphrey for contributing populations and providing phenotypic evaluations. Thank 10 you to my committee members Dr. Jim Holland and Dr. David Marshall. A special 11 thanks to Dr. Gina Brown-Guedira and Dr. David S. Marshall for the opportunity to 12 travel to Kenya. Many thanks to all who took time to listen and helped me learn through 13 meaningful discussion. 14 15 iii 1 TABLE OF CONTENTS 2 3 Page 4 LIST OF TABLES…………………………………………………………………..........vi 5 6 LIST OF FIGURES……………………………………………………………..….........vii 7 8 CHAPTER I. Literature Review.………………………………………………………….1 9 Importance of Wheat Cultivation to Humans….………………………………… 2 10 Significance of Wheat………………………………………………….....2 11 Wheat Evolution and Cytogenetics…………………………………………..........4 12 Origins of modern wheat…………………………………………….........4 13 Allopolyploidy……….……………………………………………………7 14 The Use of Wheat Relatives in Breeding for Disease Resistance………………...8 15 Wheat germlplasm resources.....…………………...…………………...…8 16 Introgression Methods…………………………………………………….9 17 Ph1 Mutants……………………………………………………….9 18 Gametocidal Genes……………………………………..………..10 19 Radiation…………………………………………………………11 20 The StemRust Pathogen………………………………………………………….12 21 Historical Impact…………………………………………………………12 22 Life Cycle…………………………………………………………….......13 23 Infection Process…………………………………………………………16 24 Physiologic Races………………………………………………………..17 25 Population Genetics and Evolution………………………………………18 26 New Highly Virulent Pgt Race…………………………………………..19 27 Stem Rust Resistance…………………………………………………………….22 28 Sr and Avr gene interaction………………………………………………22 29 Stem Rust Resistance Genes………..……………………………………22 30 Resistance to Ug99…………………………………………………..…. 24 31 Development of Resistant Germplasm………...………………………...23 32 References……………………………………..…………………………………28 33 34 CHAPTER II. Genotyping of U.S. Wheat Germplasm for Presence of Stem Rust 35 Resistance Genes Sr24, Sr36 and Sr1RSAmigo 36 Abstract…………………………………………………………………………..47 37 Introduction………………………………………………………………………48 38 Materials and Methods…………………………………………………………...51 39 Plant Materials…………………………………………………………...51 40 iv Marker Analyses…………………………………………………………52 1 Phenotypic Analysis……………………………………………………...54 2 Results………………………………………….………………………………...55 3 Discussion………………………………………………………………………..61 4 References………………………………………………………..…..…….…….65 5 6 7 CHAPTER III. Genetic Characterization of Stem Rust Resistance Gene Sr22 8 Abstract………………………………………………..…..………………..……76 9 Introduction……………………………………………………..…..……....…....77 10 Materials and Methods………………………………………..…..…….…...…...81 11 Plant Materials………………………………………..…..…………...…81 12 Stem Rust Evaluations…………………………………..…..…………...83 13 Molecular Marker Analyses…………………………..…..……………...84 14 Results…………………………..…..……………………………………………86 15 Phenotypic Evaluation…………...……………..…..……………………86 16 Genetic and Ph y s i c a l Mapping of Sr22Tb Introgression.………...…...…87 17 Linkage Analysis of F3:4 Recombinant populations…………...…………90 18 Identification of Recombinants………………………………………...93 19 Discussion………………………………………………………………………..94 20 References………………….…………………………………………………….98 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 v LIST OF TABLES 1 2 CHAPTER II. Genotyping of U.S. Wheat Germplasm for Presence of Stem Rust 3 Resistance Genes Sr24, Sr36 and Sr1RSAmigo 4 5 Table 1. Number of U.S. wheat lines from different regions and market classes 6 having stem rust resistance genes Sr24, Sr36 and Sr1RAm , and Sr31 identified 7 with molecular markers.……………….…………………………………………71 8 9 Table 2. Species of origin, chromosomal location, diagnostic markers and 10 expected size in base pairs of amplified fragments for selection of Sr24, Sr36, 11 Sr1RAmigo, and Sr26.…….………………………….….......……..………………71 12 13 Table 3. Effective Sr1RAmigo resistance in the presence 14 of Sr24 virulence……………………………………………………...……….…71 15 16 Table 4. Stem rust resistance gene pyramids present……………………………72 17 18 Table 5. Lines resistant to TTKSK and TTKST without marker alleles for 19 Sr24, Sr36 or Sr1RAmigo..........................................................................................72 20 21 CHAPTER III. Genetic Characterization of Stem Rust Resistance Gene Sr22 22 23 Table 1. Markers used for linkage analysis of Sr22, primer sequences, parent 24 allele sizes in base pairs and primer annealing temperatures (Tm) in 25 degrees Celsius ……………………………………………………………..…102 26 27 Table 2. Segregation of Sr22 in F2:3 and F3:4 populations…….………………103 28 29 Table 3. Alleles of the Sr22Tb donor parent, the cultivar Steinwedel and 30 the hard winter wheat cultivars 2174 and Lakin for six markers.........................104 31 32 33 34 vi LIST OF FIGURES 1 2 CHAPTER II. Genotyping of U.S. Wheat Germplasm for Presence of Stem Rust 3 Resistance Genes Sr24, Sr36 and Sr1RSAmigo 4 5 Figure 1. PCR amplification of markers for selection of 6 Sr36, Sr1RAmigo, Sr24 and Sr26…………....……………………………………..73 7 8 Figure 2. PCR amplification of BARC71 for the identification of Sr24 and 9 differentiation between the 3DL/3Ae translocation derived from ‘Agent’ and the 10 1BL∙1BS-3Ae translocation derived from ‘Amigo’ of Sr24……...……………...74 11 12 CHAPTER III. Genetic Characterization of Stem Rust Resistance Gene Sr22