Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations January 2016 COMBINATIONS OF MUTATIONS WITHIN PATHWAY PROVIDE FOR COMBINED TRAIT PHENOTYPES IN LINES WITH SACPD MUTATIONS IN SOYBEAN Erik Lewis Gaskin Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Recommended Citation Gaskin, Erik Lewis, "COMBINATIONS OF MUTATIONS WITHIN PATHWAY PROVIDE FOR COMBINED TRAIT PHENOTYPES IN LINES WITH SACPD MUTATIONS IN SOYBEAN" (2016). Open Access Theses. 1115. https://docs.lib.purdue.edu/open_access_theses/1115 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 12/26/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Erik Lewis Gaskin Entitled COMBINATIONS OF MUTATIONS WITHIN PATHWAY PROVIDE FOR COMBINED TRAIT PHENOTYPES IN LINES WITH SACPD MUTATIONS IN SOYBEAN For the degree of Master of Science Is approved by the final examining committee: Karen Hudson Chair Katy Rainey David Rhodes To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Karen Hudson, Katy Rainey Joseph Anderson 11/18/2016 Approved by: Head of the Departmental Graduate Program Date i COMBINATIONS OF MUTATIONS WITHIN PATHWAY PROVIDE FOR COMBINED TRAIT PHENOTYPES IN LINES WITH SACPD MUTATIONS IN SOYBEAN A Thesis Submitted to the Faculty of Purdue University by Erik L. Gaskin In Partial Fulfillment of the Requirements for the Degree of Master of Science December 2016 Purdue University West Lafayette, Indiana ii For cleaner veins. iii ACKNOWLEDGEMENTS I would like to thank everyone that assisted me in accomplishing this goal of achieving my degree. Without Karen Hudson, I would not have even had this opportunity. Her knowledge in both the theoretical and technical applications of my studies was the foundation for my own development in understanding. It was her guidance that kept me on track to finish this project. My praise goes out to Militza Carrero-Colón, our laboratory technician. She not only taught me most of my laboratory skills, but also pushed me to develop my independence. An excellent role model, I could fill this page and more with positive remarks just about her. David Schlueter, our 'go-to-field guy,' was always a huge help in any task that needed done. Never one to turn way from a job of any difficulty, he has surely helped my studies progress further than I could have without him. A special thank you to everyone else that assisted to me in my studies. From our various undergraduate students that collected and process samples, to everyone that helped me through my personal struggles at home, everyone has been beautifully amazing. I am grateful to have had them as not only part of my scholarly career, but my life in general. With every ounce of sincerity, I thank everyone who has helped me in accomplishing this end. iv TABLE OF CONTENTS Page LIST OF TABLES .............................................................................................................. v LIST OF FIGURES ........................................................................................................... vi LIST OF ABBREVIATIONS ........................................................................................... vii ABSTRACT ..................................................................................................................... viii CHAPTER 1. INTRODUCTION .................................................................................... 1 CHAPTER 2. RESULTS ............................................................................................... 20 CHAPTER 3. DISCUSSION ......................................................................................... 47 CHAPTER 4. MATERIALS AND METHODS ........................................................... 52 REFERENCES ................................................................................................................. 59 v LIST OF TABLES Table .............................................................................................................................. Page 1.1 SACPD-C Mutant Lines ............................................................................................. 15 2.1Mutant SNP Markers and Specifications ..................................................................... 25 * 2.2 FAD2-1AW194STOP x SACPD-CA218E Fatty Acid Composition ................................... 26 * 2.3 FAD2-1AW194STOP x SACPD-CA218E Protein and Oil Composition ........................... 27 * 2.4 FAD2-1AP284S x SACPD-CA218E Fatty Acid Composition ......................................... 28 * 2.5 FAD2-1AP284S x SACPD-CA218E Protein and Oil Composition ................................. 29 * 2.6 FAD2-1AL41F x SACPD-CA218E Fatty Acid Composition .......................................... 30 * 2.7 FAD2-1AL41F x SACPD-CA218E Protein and Oil Composition ................................... 31 2.8 FAD3AW81STOP x SACPD-CY211C Fatty Acid Composition ......................................... 32 2.9 FAD3AW81STOP x SACPD-CY211C Protein and Oil Composition .................................. 33 * 2.10 FAD3CP267S x SACPD-CA218E Fatty Acid Composition .......................................... 34 * 2.11 FAD3CP267S x SACPD-CA218E Protein and Oil Composition ................................... 35 2.12 Composition of FAD2-1AL41F x SACPD-CA218E, Mayaguez 201 ............................. 36 2.13 Composition of FAD3AW81STOP x SACPD-CY211C, Mayaguez 2015........................ 37 2.14 Composition of FAD3CP267S x SACPD-CA218E, Greenhouse 2015 ........................... 38 vi LIST OF FIGURES Figure ............................................................................................................................. Page 1.1 Stearic Acid Molecule with Carbon Numbering ....................................................... 16 1.2 Fatty Acid Biosynthesis Pathway .............................................................................. 17 1.3. Sequence Alignment of SACPD-A/B/C, Arabidopsis, and Castor Bean ................... 18 1.4 SACPD-CA218E x Prize F2 Stearic Acid Percentage ..................................................... 19 2.1 FAD2-1AW194STOP x (SACPD-CA218E x Prize) FA Composition Per Genotype ........... 39 2.2 FAD2-1AP284S x (SACPD-CA218E x Prize) FA Composition Per Genotype ................ 40 2.3 FAD2-1AL41F x (SACPD-CA218E x Prize) FA Composition Per Genotype.................. 41 2.4 FAD3AW81STOP x SACPD-CY211C FA Composition Per Genotype .............................. 42 2.5 FAD3CP267S x (SACPD-CA218E x Prize) FA Composition Per Genotype ................... 43 2.6 Genotypic and Phenotypic Distribution of FAD2-1AL41F x SACPD-CA218E ....................... 44 2.7 Genotypic and Phenotypic Distribution of FAD3AW81STOP x SACPD-CY211C ................... 45 2.8 Genotypic and Phenotypic Distribution of FAD3CP267S x SACPD-CA218E .......................... 46 63 vii LIST OF ABBREVIATIONS ACP - Acyl-carrier protein CTAB - Cetyltrimethyl Ammonium Bromide EDTA - Ethylenediaminetetraacetic acid ER - Endoplasmic Reticulum FA(S) - Fatty acid (synthesis) FAD2 - Fatty acid desaturase 2 FAD3- Fatty acid desaturase 3 GMO - Genetically Modified Organism KAS - Beta-ketoacyl-ACP synthase NIR - Near Infrared Spectroscopy SACPD - Delta-9-stearoyl-acyl-carrier protein desaturase TAE - Tris base, acetic acid, EDTA buffer TBE - Tris, borate, EDTA buffer TE - Tris EDTA buffer USB - United Soybean Board W82 - Williams 82 viii ABSTRACT Gaskin, Erik L. M.S., Purdue University, December 2016. Combinations of Mutations Within Pathway Provide for Combined Trait Phenotypes in Lines with SACPD Mutations in Soybean. Major Professor: Karen Hudson. Stearic acid is one of the five major fatty acids produced in soybean. It is an 18 carbon fully saturated lipid and is known for neutral or positive effects on LDL cholesterol when consumed by humans. Unfortunately, stearic acid only accounts for about 4% of the total seed oil produced in commodity soybean. Previous work has shown that stearic acid can reach levels as high as 28% of the total oil fraction when SACPD-C, the gene responsible for most of the stearic acid variation in soybean seed, is knocked out. In order to increase stearic acid content and create soybeans with improved utility based on fatty acid composition, we combined SACPD-C mutations with other mutations in the fatty acid biosynthetic pathway. When combined, double mutant progeny carrying mutant alleles of SACPD-C and FAD2-1A do not have elevated levels of oleic acid. However, sacpdc fad3 double mutants have statistically significantly elevated levels of stearic acid and statistically significantly lower linolenic acid. 1 CHAPTER 1. INTRODUCTION At just under 4 billion bushels produced by the U.S. in 2014 (USDA-NASS, 2016) soybean (Glycine max) is a crop that most families rely on for many reasons, whether they are aware of it or not. Soybean is extremely useful, in that the seeds can be consumed as food for both animals and humans but the oils produced by the seed can also be extracted and used in the food industry