University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses, Dissertations, and Student Research in Agronomy and Horticulture Department Agronomy and Horticulture 12-2018 Generation and Evaluation of Modified opaque-2 (o2) Popcorn Suggests a Route to Quality Protein Popcorn (QPP) Ying Ren University of Nebraska-Lincoln Follow this and additional works at: http://digitalcommons.unl.edu/agronhortdiss Part of the Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Genetics Commons, Other Plant Sciences Commons, and the Plant Breeding and Genetics Commons Ren, Ying, "Generation and Evaluation of Modified opaque-2 (o2) Popcorn Suggests a Route to Quality Protein Popcorn (QPP)" (2018). Theses, Dissertations, and Student Research in Agronomy and Horticulture. 155. http://digitalcommons.unl.edu/agronhortdiss/155 This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses, Dissertations, and Student Research in Agronomy and Horticulture by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. GENERATION AND EVALUATION OF MODIFIED OPAQUE-2 (o2) POPCORN SUGGESTS A ROUTE TO QUALITY PROTEIN POPCORN (QPP) by Ying Ren A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Agronomy and Horticulture (Plant Breeding and Genetics) Under the Supervision of Professor David R. Holding Lincoln, Nebraska December, 2018 GENERATION AND EVALUATION OF MODIFIED OPAQUE-2 (o2) POPCORN SUGGESTS A ROUTE TO QUALITY PROTEIN POPCORN (QPP) Ying Ren, Ph.D. University of Nebraska, 2018 Advisor: David R. Holding I have been working on a Quality Protein Popcorn breeding project where QPM conversion is carried out simultaneously for several elite popcorn germplasms. During my study in the graduate program, I led the following aspects of the Quality Protein Popcorn Breeding Project: 1. Identified suitable QPMs as opaque-2 allele donors. 2. Examined the feasibility of quick introgression of the opaque-2 allele into popcorn lines via marker-assisted selection. 3. Monitored modification by SDS-PAGE zein profiling and light box phenotypic selection to make sure multiple modifier loci for opaque-2 were incorporated each time generation advancement was carried out. 4. Carried out high-throughput DNA extraction using the BioSprint Workstation and performed genotyping to guide field selection. 5. Principle Component Analysis (PCA) of amino acid profiles from UPLC (Ultra Performance Liquid Chromatography) of candidate QPP lines along with corresponding parental lines (QPM and popcorn). (in collaboration with Dr. Ruthie Angelovici from University of Missouri Columbia, MO) DEDICATION iii I would like to dedicate this work to all my family members who have always believed in me and have been supportive unconditionally in every aspects of my life, my advisors Dr. Xiyun Song Dr. David Holding for their generous impartion of knowledge, patient guidance during my pursuit of degrees and rigid attitude toward scientific research, and to all of my lovely friends for their companionship. Ying Ren iv Acknowledgements I'm indebted to Dr. David Holding for his support and encouragement. He sets good examples for me in both academia and life. I was fortunate to get onboard with this project led by him and gained great experience in performing scientific research. Great thanks to all of my committee members, Dr. Rodriguez, Dr. Clarke, Dr. Schnable and Dr. Rose who provided all the help I need to be on the right track toward graduation and a Ph.D. degree in Agronomy and Horticulture. Many thanks to ConAgra Foods for its support in funding and providing popcorn germplasms. Also, I would like to thank the North Central Regional Plant Introduction Station for providing QPM germplasms for research. Thanks for the collaborators who have made this research possible, Dr. Oscar Rodriguez from ConAgra, Dr. Ruthie Angelovici and Dr. Abou Yobi from University of Missouri Columbia, Dr. Devin Rose and Leandra Marshall from University of Nebraska-Lincoln. I feel grateful to Christine Smith, Dr. Kyla Morton, Dr. Aixia Li and Dr. Shangang Jia for their help with field work and Mike Livingston for his help with advice on high-throughput DNA extraction and genotyping. I also would like to express my thanks to instructors whom I learned a lot from in class including Dr. Eskridge from Department of Statistics, Dr. Hyten and Dr. Amundsen from Department of Agronomy and Horticulture, Dr. Cerrutti from School of Biological Sciences. Thanks for the knowledge and experience they’ve imparted to me. Thanks are due to my family, my friends and my two dog companions for their v unconditional care and support in all aspects as always. They have been my tutors for love, faith and how to lead a life. My life would have been a lot less happy without any of them in it. I dedicate this dissertation to all these people in my life. vi Funding Acknowledgement This research was financially supported by ConAgra Foods. vii Preface The results presented in Chapter 2, Chapter 3 and Chapter 4 were accepted for publication in Frontiers in Plant Science-Plant Breeding. (Y. Ren, A. Yobi, L. Marshall, R. Angelovici, O. Rodriguez and D. R. Holding, “Generation and evaluation of modified opaque-2 popcorn suggests a route to Quality Protein Popcorn,” (accepted for publication in Frontiers in Plant Science-Plant Breeding, Nov 20th, 2018).) viii Table of Contents LISTS OF MULTIMEDIA OBJECTS CHAPTER 1…………………………………………………………………...xi CHAPTER 2…………………………………………………………………...xi CHAPTER 3………………………………………………………………….xii CHAPTER 1 Literature Review Literature Review……………………………………………………………….1 1. Maize…………………………………………………………………………2 1.1 Maize kernel…………………………………………………………………3 1.1.1 Structure of a maize kernel…………………………………………………3 1.1.2 Kernel composition………………………………………………………3 1.2 Maize protein………………………………………………………………4 1.2.1 Zeins………………………………………………………………………4 1.2.2 Non-zeins…………………………………………………………………9 2. opaque-2 mutants and Quality Protein Maize………………………………9 2.1 opaque-2 mutants…………………………………………………………9 2.2 Quality Protein Maize (QPM) ……………………………………………20 2.3 Comparison of o2, QPM with wild type……………………………………29 3 Mechanism of amino acids change and protein quality improvement………31 3.1 Mechanism of amino acid changes in o2…………………………………31 3.1.1 Pathway for lysine synthesis and degradation……………………………31 3.1.2 Biosynthesis of other amino acids………………………………………37 3.2 Protein quality improvement………………………………………………38 ix 3.2.1 Conventional approach for protein quality improvement………………38 3.2.1.1 Marker-assisted breeding for gene introgression………………………38 3.2.1.2 QPM conversion studies…………………………………………………41 3.2.2 Application of transgenic approach for protein quality improvement………42 4. Popcorn………………………………………………………………………43 4.1 Types of popcorn……………………………………………………………44 4.2 Popcorn quality………………………………………………………………44 4.3 Popping process and mechanism for popping………………………………45 4.4 Popcorn breeding for incorporation of desirable traits……………………..51 4.5 Quality Protein Popcorn (QPP)……………………………………………55 Figures and Tables……………………………………………………………57 References……………………………………………………………………......67 CHAPTER 2 Design of breeding scHeme for QPP development Introduction…………………………………………………………………….108 1. Materials and ethods…………………………………………………......110 1.1 Plant materials……………………………………………………………110 1.2 Methods……………………………………………………………………111 2. Breeding scheme and selection procedure…………………………..…..113 2.1 Breeding cheme……………………………………………………………114 2.2 Genotypic and phenotypic selection methods applied in the breeding process…………………………………………………………………………114 Figures and Tables……………………………………………………………117 References……………………………………………………………………..132 x CHAPTER 3 Analysis of QPP developed between tHree QPM and four popcorn parental lines 1. Selection of BC2F4 population with full endosperm modification………….137 2. Confirmation of o2 genotype in BC2F4 population with full modification….137 3. Amino acids profiling revealed high lysine in all six different BC2F5 o2 introgressions…………………………………………………………………..137 4. Selection of fully modified o2 popcorn can result in equivalent popping to popcorn parents…………………………………………………………...…..140 Figures and Tables……………………………………………………….......142 References…………………………………………………………………….156 CHAPTER 4 Discussion of tHe impediments of QPP breeding and solutions, its prospective and future plans 1. Discussion on the impediments of QPP breeding and solutions……………....158 2. QPP prospective and future plans………………………………….…….....160 References………………………………………………………………….165 CHAPTER 5 Appendix 1. R codes for data analysis and presentation………………………………...171 1.1 PCA analysis of protein-bound amino acids (PBAA) ………………………171 1.2 PCA analysis of free amino acids (FAA) …………………………………...172 1.3. Comparison of log2 value of ratio (o2 /wild type ratio) for 15 amino acids using protein-bound amino acid (PBAA) measurements ……………………..173 1.4 Comparison of the relative concentration of total protein………………...175 1.5 Popping volume comparison……………………………………...………176 xi Lists of Multimedia Objects CHAPTER 1 Introduction Figure 1. Structure of a maize kernel (Hoseney et al., 1986).…………………………57 Figure 2. Regulatory effects of the Opaque-2 gene (Prioul et al., 2008).……………..58 Figure 3. A proposed model for PBF and O2 controlling kernel nutritional quality and yield in maize (Zhang