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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M Repository SCREENING POTATO GENOTYPES FOR ANTIOXIDANT ACTIVITY, IDENTIFICATION OF THE RESPONSIBLE COMPOUNDS, AND DIFFERENTIATING RUSSET NORKOTAH STRAINS USING AFLP AND MICROSATELLITE MARKER ANALYSIS A Dissertation by ANNA LOUISE HALE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2003 Major Subject: Genetics SCREENING POTATO GENOTYPES FOR ANTIOXIDANT ACTIVITY, IDENTIFICATION OF THE RESPONSIBLE COMPOUNDS, AND DIFFERENTIATING RUSSET NORKOTAH STRAINS USING AFLP AND MICROSATELLITE MARKER ANALYSIS A Dissertation by ANNA LOUISE HALE Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: J. C. Miller, Jr Jim J. Giovannoni (Chair of Committee) (Member) Jeffrey D. Hart R. Daniel Lineberger (Member) (Member) Stephen King Tim Davis (Member) (Head of Department) Geoffrey M. Kapler (Chair of Genetics Faculty) December 2003 Major Subject: Genetics iii ABSTRACT Screening Potato Genotypes for Antioxidant Activity, Identification of the Responsible Compounds, and Differentiating Russet Norkotah Strains Using AFLP and Microsatellite Marker Analysis. (December 2003) Anna Louise Hale, B.S., Texas A&M University Chair of Advisory Committee: Dr. J. Creighton Miller, Jr. Total antioxidant activity and total carotenoid levels were evaluated for more than 100 common potato (Solanum tuberosum, L.) cultivars grown in the United States, advanced breeding lines from several Western U.S. breeding programs, and 47 related, tuber-bearing species. An initial assessment of variability for antioxidant activity provided baseline information to be used for potential potato promotion and for the development of new varieties with greater human health benefits. Wide variability in antioxidant levels provided evidence of genetic control of this trait, indicating that it could be possible to breed for enhanced levels of antioxidant compounds in potato. Accessions, varieties, and advanced breeding lines identified in the broad screen as having high antioxidant activity and high total carotenoid levels, were fine screened via HPLC to determine specific phenolic and carotenoid compounds present in potato. The objective of the study was to identify parents for use in the Texas breeding program to develop potato varieties containing increased levels antioxidant compounds. In the broad screen for total antioxidant activity, the 47 related, tuber-bearing species showed a wider range of variability than the cultivated varieties and breeding iv lines. Based on the DPPH assay, antioxidant activity ranged from 103-648 uM trolox equivalents in the cultivated varieties and advanced breeding lines, while that of the wild species was 42-892. HPLC analysis revealed that the phenolic content of the species, and their cultivated counterparts, was primarily composed of caffeic and chlorogenic acids. Other phenolics identified were p-coumaric acid, rutin hydrate, vanillic acid, epicatechin, t-cinnamic acid, gallic acid, and salicylic acid. The highest phenolic content discovered in the accessions was five-fold higher than the highest of the cultivated genotypes. Carotenoid analysis revealed lutein in the accessions, but the yellow-flesh breeding lines were much higher in carotenoids. In addition to the work conducted on antioxidants, an attempt was made to separate intraclonal variants of the potato cultivar Russet Norkotah. Eleven microsatellite primers and 112 AFLP primer combinations failed to produce any reproducible polymorphisms. The inability to detect differences between the clones could be due to the tetraploid nature of the clones or epigenetic differences not detected by the procedures utilized in this study. v ACKNOWLEDGMENTS I would like to express my sincere thanks and appreciation to my committee chairman, Dr. J. Creighton Miller, Jr. for his patience and guidance during my years as a graduate student. Dr. Miller’s enthusiasm for this research encouraged me to complete this project. He will always be remembered as a wonderful advisor, a great role model, and a good friend. Dr. Jeannie Miller, Creighton’s wife, is a truly remarkable woman. Her assistance in editing everything I’ve written, including, presentations, abstracts, web pages, and this dissertation is greatly appreciated. I would also like to thank my committee members for their unique contributions to this project. I have greatly admired Dr. Jim Giovannoni since our initial encounter. Jim has taught me a great deal about molecular genetics, but more importantly, he represents the type of professional that I strive to become some day. Despite being well known in the world of science, Jim never seems to forget about the little people. His patience, knowledge, and his willingness to serve on my committee, even after moving to New York, are greatly appreciated. I would like to thank Dr. Jeffrey Hart for his statistical help on a number of projects as well as his wonderful sense of humor. Dr. Hart’s enthusiasm for this project when it was becoming routine for me, made it exciting once again. Dr. Daniel Lineberger, in addition to his scientific advice, taught me how to design web pages, and encouraged my artistic side. His novel approaches to problems and his ability to predict the “next big thing” are greatly admired. Steve King’s vi enthusiasm for this project, his advice, and his and willingness to join the committee on short notice are greatly appreciated. In addition to my committee members, a number of other faculty members have had a significant impact on me during graduate school. Dr. Jerry Taylor, in addition to his help with quantitative genetics, was very supportive of me both in and out of the classroom. He is full of great advice and will always be remembered as a good friend. Dr. Jim Wild has encouraged me since my freshman year at A&M, and his trust in me is greatly appreciated. I would like to express my appreciation to the people who I’ve worked with in Dr. Miller’s lab including Doug Scheuring, and my fellow graduate students, Lavanya Reddivari, Tyann Blessington, and Ndambe Nzaramba. None of this would have been possible without our student workers Kristin Sikorski, Dean Whittaker, Pamela Cifra, Christine Johnson, Tori Meyer, Jenny DeMarko, and Mark Tietz. I would like to extend my special thanks and love to my husband, Eric, my parents, and my grandfather, for their patience, love, understanding, and support through the years. Finally, I would like to thank the friends that I’ve met at Texas A&M (especially Ashlee McCaskill and Shanna Moore) for their support. vii TABLE OF CONTENTS Page ABSTRACT…………… ................................................................................................. iii ACKNOWLEDGMENTS..................................................................................................v TABLE OF CONTENTS................................................................................................ vii LIST OF FIGURES…….................................................................................................. xi LIST OF TABLES…….. ............................................................................................... xiii CHAPTER I INTRODUCTION......................................................................................1 Statement of the Problem ...............................................................3 II LITERATURE REVIEW...........................................................................4 Introduction ....................................................................................4 History of the Potato ..........................................................4 Potato Production in Texas ................................................8 Nutritional Value of the Potato ........................................11 Part #1. Screening Potato Genotypes for Antioxidant Activity and Identification of the Responsible Compounds ......................14 Micronutrients and Their Importance to Human Health..14 Antioxidants .....................................................................15 Phenolics and Polyphenols...............................................19 Carotenoids.......................................................................23 Antioxidants in Potatoes...................................................31 Phenolics in Potatoes........................................................32 Carotenoids in Potatoes....................................................38 viii CHAPTER Page Part #2 - Differentiating 7 Russet Norkotah Strains Using AFLP Marker Analysis ................................................................49 Karyological and Isozyme Analyses ................................50 Restriction Fragment Length Polymorphisms (RFLP) ....51 Randomly Amplified Polymorphic DNA (RAPD) ..........52 Amplified Fragment Length Polymorphisms (AFLPs)....53 Microsatellites ..................................................................56 III INTRASPECIFIC VARIABILITY FOR ANTIOXIDANT ACTIVITY IN POTATO (S. TUBEROSUM L.) ........................................................60 Introduction ..................................................................................60 Materials and Methods.................................................................62 Plant Materials..................................................................62

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