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Genomic Approaches to Understand Sweetpotato Root Development In Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2012 Genomic approaches to understand sweetpotato root development in relation to abiotic factors Julio Solis Sarmiento Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Solis Sarmiento, Julio, "Genomic approaches to understand sweetpotato root development in relation to abiotic factors" (2012). LSU Doctoral Dissertations. 1217. https://digitalcommons.lsu.edu/gradschool_dissertations/1217 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. GENOMIC APPROACHES TO UNDERSTAND SWEETPOTATO ROOT DEVELOPMENT IN RELATION TO ABIOTIC FACTORS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Plant, Environmental & Soil Sciences by Julio Solis-Sarmiento B.S., Universidad Nacional Mayor de San Marcos, Peru, 1996 M.S., Universidad Peruana Cayetano Heredia, Peru, 2009 December 2012 Dedication I especially dedicate this study to my beloved wife, Jeny Perez To my mother, Benedicta Sarmiento; my father, Bernardino Solis; my grandmother, Alejandrina Chavez, and all my family, who supported me in every step. Finally, I dedicate this to my boss and friend, Don Labonte. ii Acknowledgments First and foremost, I extend my deepest gratitude to Dr Don Labonte, a boss that I will always remember. He is really the “sweetpotato guy” since he has the sweetness in his life and in his talking; Don has a unique patience that every worker would like to have in their boss. Don, as an advisor, was nice, and through his mentorship and patience, he taught me the unique world of sweetpotatoes (Louisiana, and in particular Chase and Baton Rouge). He gave me the freedom to do this job and learn more of bioinformatics. Don always encouraged me not to dream to solve a puzzle; instead to be a piece in the path of getting “Beauregard and Evangeline”. I just realized that sweetpotato breeders have the easiness to get the sweetness of the yams; you should know Dr. W. Gruneberg and Dr. R. Schafleitner to confirm this claim. I also extend special gratitude to the “Perl guy”, Dr. Steven Brandt for instant help in perl programming, Dr. Charles Cavanaugh and Dr. Jaime Prilusky, who provided invaluable support to the scripting and parsing required for decoding the sweetpotato transcriptome. I cannot forget to give my unique appreciation to Dr. Roland Schafleitner for providing me ~ 50% of data that motivated my life and stay at LSU, specifically for sharing the raw sweetpotato sequences from leaves and stem for our analysis. I also want to thank to Dr Niranjan Baisakh and Dr. Arthur Villordon for helping me to develop scientific skills, and all people involved in this work. Finally, I thank Nurit Firon, our collaborative partner in the BARD Project Number US-4015-07, “Molecular and anatomical characterization of sweetpotato storage root formation” that funded this work. I am very thankful to Dr. Simon Chang and Dr. Bin Li that served as committee members and for their support provided during my work. iii Table of Contents Dedication ....................................................................................................................................... ii Acknowledgments.......................................................................................................................... iii List of Tables ................................................................................................................................. vi List of Figures .............................................................................................................................. viii Abbreviations ................................................................................................................................. ix Abstract .......................................................................................................................................... xi Chapter 1. Literature Review .......................................................................................................... 1 1.1 Introduction ......................................................................................................................... 1 1.2 Genes involved in sweetpotato root development .............................................................. 2 1.3 Candidate genes associated with lignin metabolism ........................................................... 6 1.4 Candidate genes in hormone signaling ............................................................................... 8 1.5 Literature cited .................................................................................................................. 11 Chapter 2. Drought and Salt Stress on Sweetpotato Storage Root Development ......................... 16 2.1 Introduction ....................................................................................................................... 16 2.2 Materials and methods ...................................................................................................... 19 2.2.1 Plant material ........................................................................................................... 19 2.2.2 Delayed watering studies under greenhouse conditions .......................................... 19 2.2.3 Delayed watering studies under field conditions ..................................................... 20 2.2.4 Salt stress studies ..................................................................................................... 22 2.2.5 Gene expression profiling by quantitative reverse transcription PCR ..................... 22 2.2.6 Experimental design and statistical analyses ........................................................... 23 2.3 Results ............................................................................................................................... 24 2.3.1 Delayed watering studies under greenhouse conditions .......................................... 24 2.3.2 Delayed watering studies under field conditions ..................................................... 25 2.3.3 Gene expression under drought stress and salt stress .............................................. 25 2.4 Discussion ......................................................................................................................... 31 2.5 Literature cited .................................................................................................................. 42 Chapter 3. Comparative De Novo Assembly and Analysis of the Sweetpotato Transcriptome Identifies Candidate Genes for Regulatory Roles and Calcium Signaling in the Onset of Storage Root Formation................................................................................................ 48 3.1 Introduction ....................................................................................................................... 48 3.2 Materials and methods ...................................................................................................... 51 3.2.1 Biological material and RNA extraction .................................................................. 51 3.2.2 Transcriptome data, sequence analysis and assembly ............................................. 52 3.2.3 Functional annotation of the sequences ................................................................... 53 3.2.4 Quantitative RT-PCR analysis and reverse transcription PCR of selected unigenes ................................................................................................................ 54 iv 3.3 Results ............................................................................................................................... 56 3.3.1 Novel unique sequence for root libraries ................................................................. 58 3.3.2 Expression analysis of genes possibly involved in the onset of storage roots ......... 60 3.4 Discussion ......................................................................................................................... 79 3.5. Literature cited ................................................................................................................. 90 Chapter 4. Construction and application of a sweetpotato microarray for gene expression profiling during sweetpotato root development ................................................................ 96 4.1 Introduction ....................................................................................................................... 96 4.2 Materials and methods ...................................................................................................... 97 4.2.1 Biological sample and extraction of RNA ............................................................... 97 4.2.2 Custom microarray development and probe design................................................. 97 4.2.3 Sample preparation for microarray hybridizations, measurement of fluorescence signals and data analysis .................................................................
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