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Modification of Fatty Acid Composition in Soybean Seeds to Improve

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Modification of Fatty Acid Composition in Soybean Seeds to Improve MODIFICATION OF FATTY ACID COMPOSITION IN SOYBEAN SEEDS TO IMPROVE SOYBEAN OIL QUALITY AND FUNCTIONALITY A Dissertation Presented to The Faculty of the Graduate School At the University of Missouri In Partial Fulfillment Of the Requirements for the Degree DOCTOR OF PHILOSOPHY By Anh TUNG PHAM Dr. Kristin Bilyeu, Dissertation Supervisor July 2011 The undersigned, appointed by the dean of the Graduate School, have examined the Dissertation entitled MODIFICATION OF FATTY ACID COMPOSITION IN SOYBEAN SEEDS TO IMPROVE SOYBEAN OIL QUALITY AND FUNCTIONALITY Presented by Anh Tung Pham A candidate for the degree of DOCTOR OF PHILOSOPHY OF PLANT BIOLOGY AND GENETICS And hereby certify that, in their opinion, it is worthy of acceptance. Dr. Kristin D. Bilyeu Dr. David A. Sleper Dr. J. Grover Shannon Dr. Emmanual Liscum ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Kristin Bilyeu not only for giving me the opportunity to join her research group and conduct exciting research in plant biology and genetics, but also for her continuous advising, support, and encouragement during my doctoral studies. The quality of this study was greatly influenced by her help in avoiding adversities and in solving problems which arose during the course of the research project, and also by her research ethics and her perseverance for a deeper understanding of this project. I would also like to thank Dr. Grover Shannon for his collaboration and contribution to my dissertation projects. I would like to thank Dr. David Sleper for your guidance and support. I would like to thank Dr. David Sleper, Dr. Grover Shannon, Dr. Mannie Liscum for serving on my doctoral committee. Also, I would like to thank all the professors from different educational levels who have encouraged me or inspired me, one way or another, to appreciate academia and to pursue an advanced degree. I greatly acknowledge the help of the current and past members of Dr.Shannon‟s group and Dr. Bilyeu‟s group: Dr. Jeong-Dong Lee for his helpful insights and for supplying the germplasm and segregating populations used in this study, Dr. Jason Gillman who share with me your deep knowledge and your willingness to help with all the difficulties that I encountered during the course of the study. My thanks also are for Kerry Clark, Christi Shannon, Stewart Selves and Paul Little for helping me with field works, protein, oil and fatty acid analysis. Thanks and good luck to my colleagues Ashley ii Tetlow, Katherine Hagely, and Julian Lenis. You have created a family-like asmosphere that makes me enjoy and appreciate our friendship. At last, many thanks to all friends that I was fortunate to meet in Columbia MO and had so much fun with. I am so happy that we shared a part of our “journeys” and I really hope to stay in touch. I am especially thankful to all you that our friendship has already lasted so long and your support has not changed, although so far away. I am also grateful to my family, especially my parents, Binh Nguyen and Minh Pham, and my brother, Linh Pham, for their love and understanding. I deeply wish that in the near future I will be able to share more time with you. Finally, I would like to thank my husband, Giang Nguyen for being there to share my excitement and disappointment, encourage me and keep my motivation at all times, and make me smile even those days that “everything went wrong” iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii CHAPTER 1 Literature review .......................................................................................... 1 Figure ....................................................................................................................... 49 Literature cited ........................................................................................................ 51 CHAPTER 2 Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait ................................................................................ 69 Abtract ...................................................................................................................... 71 Introduction ............................................................................................................. 72 Materials and methods ............................................................................................ 77 Population development......................................................................... 77 DNA isolation and PCR for sequencing of FAD2-1A and FAD2-1B .... 79 Sequence analysis .................................................................................. 79 FAD2-1B allele specific molecular marker assay ................................. 80 FAD2-1A allele specific molecular marker assay for 17D .................... 81 FAD2-1A allele specific molecular marker assay for M23 ................... 81 Fatty acid, protein and oil determination ............................................... 82 Population genotyping ........................................................................... 83 Results ..................................................................................................................... 84 iv Identification of mutant alleles of FAD2-1B in soybean lines PI 283327 and PI 567189A ..................................................................................... 84 The PI 283327 FAD2-1B allele is associated with an increase in seed oleic acid content ................................................................................... 86 Combinations of mutations in FAD2-1A and FAD2-1B produce high oleic acid levels in the seed oil .............................................................. 87 Excess desaturase activity: A single wild-type FAD2-1 allele prevents high oleic acid accumulation ................................................................. 90 The high oleic acid phenotype is stable in plants grown in alternate environments .......................................................................................... 91 Full fatty acid profiles and total oil and protein content ........................ 92 Discussion ............................................................................................................... 94 Figures ................................................................................................................... 100 Tables .................................................................................................................... 108 Literature cited ...................................................................................................... 111 CHAPTER 3 A novel FAD2-1A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content ................................... 115 Summary ............................................................................................................... 116 Introduction ........................................................................................................... 117 Materials and method ............................................................................................ 121 DNA isolation and sequencing of FAD2-1A and FAD2-1B ................ 121 Population development....................................................................... 121 v FAD2-1A allele specific molecular marker assay ................................ 122 Fatty acid, protein and oil determination ............................................. 123 Population genotyping using SimpleProbe assay ................................ 123 Design of field experiments ................................................................. 124 Statistical analysis ................................................................................ 125 Results ................................................................................................................... 126 Identification of novel alleles of FAD2-1A and FAD2-1B in soybean plant introductions ............................................................................... 126 Combinations of novel mutant alleles of FAD2-1A with mutant FAD2- 1B alleles produce high oleic acid levels in the seed oil ...................... 128 Full fatty acid profiles and total oil and protein content ...................... 130 Discussion ............................................................................................................. 133 Figures ................................................................................................................... 137 Tables .................................................................................................................... 139 Liturature cited ...................................................................................................... 145 CHAPTER 4 Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil .................................................................................. 148 Summary ............................................................................................................... 149 Introduction ........................................................................................................... 150 Materials and methods .......................................................................................... 155 Population development......................................................................
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