Identification of novel sources of variation for the improvement of cold germination ability and early seedling vigor in upland cotton (Gossypium hirsutum L.) by Ritchel Bueno Gannaban, B.S. A Thesis In Plant and Soil Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCES Approved Dr. Rosalyn B. Angeles-Shim Chair of Committee Dr. Benildo G. de los Reyes Dr. Brendan R. Kelly Dr. Endang Septiningsih Mark Sheridan Dean of the Graduate School August, 2019 Copyright 2019, Ritchel Bueno Gannaban Texas Tech University, Ritchel Bueno Gannaban, August 2019 ACKNOWLEDGMENTS It is my pleasure to acknowledge everyone whose efforts and contributions led me to the completion of this research study. Without the support and encouragement, I would not have been able to complete this very important chapter of my life’s journey. I would like to thank my thesis advisor, Dr. Rosalyn B. Angeles-Shim of the Department of Plant and Soil Science at Texas Tech University. Dr. Shim provided the opportunity for me to pursue my graduate studies. With her guidance, I managed to survive the grueling life of a graduate student. Dr. Shim’s office was always open whenever I had a question about my research or writing. She consistently allowed this paper to be my own work, but steered me in the right direction whenever she thought I needed it. Not only she is a very considerate adviser but also a confidant who never fails to see the potential in every person. It is truly an honor to work under a great mentor. I would also like to acknowledge my committee members, Dr. Benildo G. de los Reyes and Dr. Brendan Kelly of the Department of Plant and Soil Science at Texas Tech University and to Dr. Endang Septiningsih of the Department of Soil and Crop Sciences at Texas A&M University for their guidance. I am gratefully indebted to the valuable comments towards the completion of my academic courses and on this thesis. I would also like to thank my dear friends and lab mates, Puneet Kaur Mangat and Joshua James Singleton for their passionate participation and input, my research experiments could not have been successfully conducted. I am also deeply appreciative of Dr. Junghyun Shim, Dr. Cade Colden, and Jake Sanchez for imparting me their expertise to process my data. Above all, I am grateful for the friendship and camaraderie. It means a lot to me. Finally, I must express my very profound gratitude to my family. To my parents, Mr. and Mrs. Fernando Gannaban and to my loving partner, Garret Greene for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without them. Thank you. ii Texas Tech University, Ritchel Bueno Gannaban, August 2019 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................... ii ABSTRACT .......................................................................................................... vi LIST OF TABLES .............................................................................................. vii LIST OF FIGURES ........................................................................................... viii CHAPTER I: LITERATURE REVIEW .............................................................1 Cotton biology and economic importance ...................................................1 Challenges in cotton production ..................................................................2 Measures to address cold sensitivity in upland cotton .................................5 Genetic variation to improve cold tolerance in upland cotton .....................6 Pre-breeding activities towards the use of germplasm for cold improvement of cotton .................................................................................8 Research Goals...........................................................................................10 CHAPTER II: IDENTIFICATION OF NOVEL SOURCES OF GENETIC VARIATION FOR THE IMPROVEMENT OF COLD GERMINATION ABILITY IN UPLAND COTTON (Gossypium hirsutum L.)...........................11 Introduction ......................................................................................................11 Materials and Methods .....................................................................................13 Plant materials ............................................................................................13 Plant propagation and genomic DNA extraction .......................................13 SSR genotyping .........................................................................................14 Genetic diversity analysis ..........................................................................14 Viability seed testing..................................................................................15 Cold germination screening .......................................................................15 Cold germination parameters .....................................................................16 Statistical analysis ......................................................................................16 Results and Discussion ....................................................................................17 Genetic diversity analysis using SSR markers...........................................17 Germination ability of the test germplasm under low temperature stress.......................................................................................19 Patterns of variation in the cold germination ability of the test germplasm ..................................................................................................21 Conclusion .......................................................................................................22 CHAPTER III: IDENTIFICATION OF NOVEL SOURCES OF GENETIC VARIATION TO ENHANCE SEEDLING VIGOR OF UPLAND COTTON (Gossypium hirsutum L.) UNDER LOW TEMPERATURE STRESS ............32 Introduction ......................................................................................................32 iii Texas Tech University, Ritchel Bueno Gannaban, August 2019 Materials and Methods .....................................................................................33 Plant materials ............................................................................................33 Morphological measurements ....................................................................34 Physiological measurements ......................................................................34 Biochemical assays ....................................................................................35 Statistical analysis ......................................................................................36 Results and Discussion ....................................................................................36 Morphological response of cotton seedlings to low temperatures .............36 Physiological response of cotton seedlings to low temperatures ...............39 Cellular response of cotton seedlings to low temperatures ........................40 Conclusion .......................................................................................................43 CHAPTER IV: COMPARATIVE TRANSCRIPTOME PROFILING OF UPLAND COTTON (Gossypium hirsutum L.) UNDER LOW TEMPERATURE STRESS.................................................................................54 Introduction ......................................................................................................54 Materials and Methods .....................................................................................56 Plant materials ............................................................................................56 RNA extraction ..........................................................................................56 Data analysis .............................................................................................57 Gene enrichment analysis ..........................................................................57 Identification of differentially expressed genes associated with cold stress ...........................................................................................58 Results and Discussion ....................................................................................58 RNA sequencing of different samples and data analysis ...........................58 Identification of DEGs in response to cold stress in SA-0718 and SA-3781 ...........................................................................58 Patterns of gene expression in response to cold stress in SA-0718 and SA-3781 ...........................................................................59 Gene ontology analysis of DEGs in cotton subjected to cold stress ..........60 Differential gene expression patterns of Gossypium hirsutum L. in response to cold stress............................................................................61 Conclusion .......................................................................................................63 REFERENCES .....................................................................................................72 APPENDICES ......................................................................................................87 Appendix A Description and