TRANSGENE STABILITY, TRANSPOSON ACTIVATION AND POTENTIAL FOR GENE TAGGING IN PEANUT (Arachis hypogaea L). by MANJU CHANDRAN (Under the Direction of Peggy Ozias-Akins) ABSTRACT Crop improvement in peanut (Arachis hypogaea L.) is aimed at improving yield, product quality, reduction in seed allergen content and resistance to biotic and abiotic stresses. Application of RNA interference (RNAi) was successful in developing a transgenic line B11.1.1/11 with complete reduction of Ara h 2 and reduced levels of Ara h 6, two major seed allergens. The transgene was stable across three transgenic generations tested as Ara h 2 signal was absent and Ara h 6 signal was reduced in quantitative western blot assay. Stability of transgene silencing to altered soil sulfur, specifically on partially silenced Ara h 6, was tested by growing the transgenic line under three soil sulfur levels. Transgenic lines showed no Ara h 2 expression and significantly reduced Ara h 6 expression under different sulfur levels, providing further evidence for stability of the transgene and the effectiveness of RNAi even under nutritional conditions optimal for synthesis of sulfur-rich proteins. High soil sulfur concentrations significantly increased the levels of Ara h 2 and Ara h 6 in non-transgenic lines compared to low sulfur. Ara h 3 levels were significantly reduced under low sulfur in both transgenic and non-transgenic lines and Ara h 1 levels were unaffected by sulfur. Activation of the peanut transposon, AhMITE1, was studied under two stress situations, namely ethylmethane sulfonate (EMS) mutagenesis and prolonged tissue culture. Transposon display provided evidence that the peanut Miniature Inverted-repeat Transposable Element (MITE) was activated under both stresses and variation was observed with regard to the different stress situations and genotypes studied (‘Tifrunner’ and ‘Florunner’). Peanut MITE sequence capture, Sanger sequencing, and primer design to MITE and flanking DNA sequences enabled the development of 17 markers polymorphic across 29 peanut genotypes. Sequences flanking MITEs showed similarity to protein coding regions via BLAST searches. High throughput Illumina sequencing with five genotypes was carried out to recover additional flanking sequence for marker development and gene identification. Stability of protein silencing across generations and under different nutritional conditions showed that the polyploid peanut genome is amenable to stable transgene expression. Genome instability associated with MITE transposons may occur under conditions of stress and may provide a source of positive and negative variation for gene functional analysis and breeding. INDEX WORDS: Peanut transgene stability, sulfur and transgene stability, transposon activation and stress, transposon-based sequence capture. TRANSGENE STABILITY, TRANSPOSON ACTIVATION AND POTENTIAL FOR GENE TAGGING IN PEANUT (Arachis hypogaea L). by MANJU CHANDRAN B.Sc. (Agri.), Kerala Agricultural University, 1993 M.Sc. (Hort.), Kerala Agricultural University, 1996 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2013 © 2013 Manju Chandran All Rights Reserved TRANSGENE STABILITY, TRANSPOSON ACTIVATION AND POTENTIAL FOR GENE TAGGING IN PEANUT (Arachis hypogaea L). by MANJU CHANDRAN Major Professor: Peggy Ozias-Akins Committee: Joann Conner Cecilia McGregor John Beasley Arthur Grider Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2013 DEDICATION I dedicate my dissertation to my family, friends and all those individuals who have believed and encouraged me throughout my life. iv ACKNOWLEDGEMENTS At the very outset I thank Dr. Peggy Ozias-Akins, my major Professor & chair of committee for her guidance, support and encouragement. Through her approach and way of dealing she taught me how to be a true professional without losing personal touch. I sincerely appreciate her patience, especially with my ‘writing skills’, and all her contribution of time for review, ideas and funding which make my Ph.D. life insightful and an amazing learning experience . I would like to thank my committee members Dr. Joann Conner, for her guidance in molecular techniques, which helped me many times when I was challenged, Dr. John Beasley for his expertise in field studies, Dr. Arthur Grider for his guidance in protein work and Dr. Cecilia McGregor for her thought-provoking suggestions. I would like to thank Brian Abernathy for helping with the Bioinformatic works related to my research. I have immensely enjoyed working with all the professionals (present and past) in Dr. Ozias- Akins’ lab for the last four years of my research in Tifton. I thank Dr. Ye Chu who had helped me with many aspects of this research. I would like to express my gratitude to Ms. Evelyn Morgan and Mr. Greg Thomas for the technical support. v Many thanks to Joe, Anjan, Jackie, Jane, Congling, Bindu, Murug, Rattan, Walid, Guna, Brant, Yufang, Madhu, Lekshmi, Mrs. & Mr. Rajeev and members of The Tift Indian Group for their positive energy and constant encouragement. I also extend my appreciation to staff members of the Department of Horticulture and NESPAL for the friendship and help, especially Mary Jane and Evelyn Folds. Running short of words to express my gratitude for my loving, supportive, encouraging, and patient daughters, Nayana and Nirupama, as well as my husband who have stood with me, at all times. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ...................................................................................................................... xii CHAPTER 1 INTRODUCTION .........................................................................................................1 References ................................................................................................................4 2 LITERATURE REVIEW ..............................................................................................9 Introduction ..............................................................................................................9 Distribution and Classification of Transposable Elements ......................................9 Classification of MITEs .....................................................................................12 Activation of Transposons ...................................................................................15 Association of MITEs with Gene Rich Areas of Genomes ...................................18 MITEs as Molecular Markers ..............................................................................19 Target Enrichment and Next Generation Sequencing to Develop Molecular Markers and Identify Genes ...........................21 Next Generation Sequencing Platforms .................................................................21 Transposons as Insertional Mutagens and Gene Tagging ......................................24 Silencing of Transgenes in Plants ..........................................................................26 Effect of Environmental Factors on Genome Stability and Expression of Transgenes .....................................................................................28 vii Sulfur Metabolism in Plants...................................................................................31 Plant Sulfur Nutrition and its Effect on Seed Protein Content ..............................32 Conclusion ........................................................................................................36 References ........................................................................................................36 3 STABILITY OF SEED ALLERGEN CONTENT DUE TO ALLERGEN GENE SILENCING IN MULTIPLE GENERATIONS OF PEANUT...................................54 Abstract ..................................................................................................................54 Introduction ............................................................................................................55 Materials and Methods ...........................................................................................58 Results ........................................................................................................62 Discussion ........................................................................................................63 Conclusion ........................................................................................................68 References ........................................................................................................69 4 EFFECT OF SOIL SULFUR ON EXPRESSION OF ALLERGEN GENES IN TRANSGENIC PEANUT SILENCED FOR ARA H 2 AND ARA H 6 ..............83 Abstract ..................................................................................................................83 Introduction ............................................................................................................84 Materials and Methods ...........................................................................................90 Results ........................................................................................................95