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EMS Induced Mutations in Dhurrin Metabolism and Their Impacts on Sorghum Growth and Development Jenae Lavon Skelton Purdue University

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EMS Induced Mutations in Dhurrin Metabolism and Their Impacts on Sorghum Growth and Development Jenae Lavon Skelton Purdue University Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations Summer 2014 EMS induced mutations in dhurrin metabolism and their impacts on sorghum growth and development Jenae LaVon Skelton Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Part of the Agriculture Commons, and the Agronomy and Crop Sciences Commons Recommended Citation Skelton, Jenae LaVon, "EMS induced mutations in dhurrin metabolism and their impacts on sorghum growth and development" (2014). Open Access Theses. 685. https://docs.lib.purdue.edu/open_access_theses/685 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. i EMS INDUCED MUTATIONS IN DHURRIN METABOLISM AND THEIR IMPACTS ON SORGHUM GROWTH AND DEVELOPMENT A Thesis Submitted to the Faculty of Purdue University by Jenae L. Skelton In Partial Fulfillment of the Requirements for the Degree of Master of Science August 2014 Purdue University West Lafayette, Indiana ii ACKNOWLEDGEMENTS First, I would like to thank Dr. Herbert Ohm for giving me the opportunity to start my graduate program at Purdue in his wheat breeding program. I would also like to thank my current advisor, Dr. Mitchell Tuinstra, for giving me the opportunities to continue my graduate studies in his corn and sorghum lab, as well as my other committee members, Dr. Jeff Volenec and Dr. Michael Mickelbart. I would like to thank Ag Alumni Seed for allowing me to plant a replicate of my field study at the nursery in Romney, IN, and for partly funding my research along with the USDA National Institute of Food and Agriculture, USDA-ARS Award No. 58-3620- 8-688 from which my project was funded. I would like to give a hearty thanks to Dr. John Burke and his laboratory group from USDA in Lubbock, TX for running the dhurrin HPLC for my tissue samples. A huge thank you goes out to my fellow lab mates, technicians, and other Purdue employees who helped me with both projects, especially: Andy Linvill, Elizabeth Buescher, Maria Mateos-Hernandez, Nicola Carraro, Nick Babcock, Ray Lindsey, Shaylyn Wiarda, Joshua Fitzgerald, Melissa McDonald, Kirsten Thomas, Rima Thapa, Amanda Easterly, Alex Renaud, Molly McKneight, Charles Quaye, Ron Steiner, and many others. iii I would especially like to thank my parents, my fiancé, my family and friends who supported me during my graduate studies. Without their support, I might not have had the strength and motivation to keep going. iv TABLE OF CONTENTS Page LIST OF TABLES .............................................................................................................. v LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................. x ABSTRACT ............................................................................................................... x CHAPTER 1. LITERATURE REVIEW ...................................................................... 1 1.1 Introduction ............................................................................................... 1 1.2 Dhurrin Metabolism in Sorghum .............................................................. 2 1.2.1 Dhurrin Biosynthesis and Catabolism in Sorghum .............................3 1.2.2 Dhurrin as an Herbivory Defense Compound .....................................6 1.2.3 Effects of Reduced Dhurrin Concentration .........................................7 1.3 Stay-Green in Sorghum ............................................................................. 8 1.3.1 Relationship between Stay-Green and the Nitrogen Status of the Plant………….. ................................................................................ 12 1.3.2 Relationship Between Dhurrin and Stay-Green ................................13 CHAPTER 2. GENETIC ANALYSIS OF EMS INDUCED DHURRIN MUTANTS ………………………………………………………………………..14 2.1 Introduction ............................................................................................. 15 2.2 Materials and Methods ............................................................................ 18 2.2.1 Identification of Dhurrin Mutants .....................................................18 2.2.1.1 EMS Sorghum Population Development ......................................... 18 2.2.1.2 Genetic Screen for HCN Production ................................................ 18 2.2.2 Identification of Causal Mutations ....................................................20 2.2.2.1 DNA Extraction and Sequencing ..................................................... 20 2.2.2.2 Bioinformatics .................................................................................. 21 v Page 2.2.2.3 Co-segregation Analysis .................................................................. 22 2.2.3 Dhurrin Content of EMS Mutants .....................................................25 2.3 Results ..................................................................................................... 26 2.3.1 Identification of Acyanogenic Mutants .............................................26 2.3.2 Candidate SNPs Identified ................................................................27 2.3.3 Co-Segregation of Mutation with KASP Markers ............................28 2.3.4 Dhurrin Concentration ......................................................................29 2.4 Discussion ............................................................................................... 30 CHAPTER 3. PHENOTYPIC ANALYSIS OF THE IMPACT OF DHURRIN MUTANTS ON SORGHUM GROWTH AND DEVELOPMENT ................................. 33 3.1 Introduction ............................................................................................. 33 3.2 Materials and Methods ............................................................................ 37 3.2.1 Objective 1: Characterizing Growth and Development of Dhurrin Mutants .......................................................................................................... 37 3.2.2 Objective 2: Dhurrin Content and Stay-green ...................................40 3.2.3 Statistical Analyses ................................................................................. 44 3.3 Results ..................................................................................................... 45 3.3.1 Growth and Development Characteristics of Parent Lines ...............45 3.3.2 Growth and Development Characteristics of F2 Families .................53 3.3.3 Stay-Green under Drought Conditions .............................................63 3.3.3.1 Dhurrin Concentration ...................................................................... 63 3.3.3.2 Characterization of Stay-Green ........................................................ 64 3.4 Discussion ............................................................................................... 66 REFERENCES ............................................................................................................. 70 APPENDICES Appendix A SAS Code ................................................................................................ 79 Appendix B Data Files................................................................................................. 85 VITA ........................................................................................................... 158 vi LIST OF TABLES Table .............................................................................................................................. Page Table 1.1 Summary of stay-green traits according to their classification as functional or cosmetic and a description of distinguishing characteristics. ............................................. 9 Table 2.1 KASP marker analyses of parent lines and segregating F2 plants indicating the pedigree, HCN production characteristics based on the Feigl-Anger Assay, and the CYP79A1 genotype for each sample. ............................................................................... 29 Table 2.2 Mean dhurrin concentration values for parents BTx623 and EMS 2447, wild- type and acyanogenic F2 plants, and acyanogenic maize. The statistic groupings are based on Tukey’s studentized range test.. ................................................................................... 30 Table 3.1 Chlorophyll content index (CCI) averages of the four parent lines at Purdue University Agronomy Center for Research and Education (ACRE) and Ag Alumni in Romney, IN. The means were compared using a t-test and analyzed by separate location at each sampling time. Means with a different statistical grouping letter (Group) are significantly different from each other based on a least significant difference (LSD) value... ............................................................................................................................... 48 Table 3.2 Height means of the four parent lines at Purdue University Agronomy Center for Research and Education (ACRE) and Ag Alumni in Romney, IN. The means were compared using a t-test and analyzed by separate location at each sampling time. Means with a different statistical grouping letter (Group) are significantly different from each other based on a least significant difference (LSD) value.................................................49 Table 3.3 Leaf number means
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