Genomic and Breeding Resources to Produce Seeded and High Biomass Interspecific Hybrids of Napiergrass and Pearl Millet
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GENOMIC AND BREEDING RESOURCES TO PRODUCE SEEDED AND HIGH BIOMASS INTERSPECIFIC HYBRIDS OF NAPIERGRASS AND PEARL MILLET By DEV RAJ PAUDEL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Dev Raj Paudel To my late Mom ACKNOWLEDGMENTS I wish to express my appreciation to the members of my advisory committee: Dr. Fredy Altpeter, Dr. Jianping Wang, Dr. Patricio Munoz, Dr. Calvin Odero, and Dr. Salvador Gezan, who have guided, supported, and encouraged me throughout the course of my research project. Sincere thanks to my research advisor Dr. Fredy Altpeter for allowing me to join his group and pursue the work described here. Thank you for all the support, guidance, and mentorship that you have provided during my graduate studies. I am truly inspired by your professionalism and leadership role. I am particularly indebted to my co-advisor Dr. Jianping Wang who provided me with space in her lab to do my experiments and provided me with opportunities to develop skills in molecular and computational biology. Your mentorship has been an invaluable gift over the past couple of years. One day, I hope to inspire others as you've inspired me. I would like to gratefully acknowledge the University of Florida Graduate School Fellowship for funding the first four years of my PhD. I am thankful to the Graduate School Doctoral Dissertation Award for funding my final semester. I am grateful to the Florida Plant Breeders Working Group for providing funds for this research. I was grateful to be surrounded by many wonderful people during my journey. Many thanks to talented undergraduates, Fan Wen, Erik Hanson, and Stephanie Maya, who helped not only with the experiments, but also helped me learn and hone my mentorship skills. In addition, I would like to express my gratitude to the following people: • Dr. Baskaran Kannan without whose help, support, and wisdom, this work wouldn’t have been completed. 4 • Staff at Plant Science Research & Education Unit, Citra, FL for coordinating and helping in planting, managing, and harvesting of research plots. • Dr. Calvin Odero, Venancio Fernandez, Raphael Mereb Negrisoli, and Nikol Havranek at the Everglades Research and Education Center for providing support to do fieldwork in Belle Glade, FL. • Dr. Tina Strauss, Dr. Eshan Gurung, Er. Prasan Gurung, and Dr. Laxman Adhikari for your continued support, valuable insights in my research, and friendship. • Members of the Altpeter Lab: Derek Hurley, Dr. Saroj Parajuli, Dr. Ratna Karan, Dr. Simon Gere, and Bryant Brown, for helping during field work and research activities. • Members of the Wang Lab: Dr. Liping Wang, Dr. Xiping Yang, Dr. Ze Peng, Dr. Zhou Hai, Aleksey Kurashev, Dr. Yu-Chien Tseng, and Dr. Song Jian for helping in fieldwork, lab experiments, as well as discussions related to research that were instrumental in providing insights into some of the work described in this document. • Marco Sinche for kindly providing yield and flowering data of mapping experiments. • Dr. Rajeev Varshney for providing the pearl millet reference genome sequence. • Dr. Karen-Harris Schultz for providing napiergrass sequences. • Cynthia Hight for playing a phenomenal role in supporting academic endeavors and ensuring that I was on track every semester. 5 • Members of the Plant Science Council for guidance, support, friendship, and humor that has helped me survive graduate school. • Nepalese community in Gainesville, FL for always being there for me. Obtaining this advanced degree was only possible due to the sheer sacrifice, patience, unconditional love and understanding, long-term support, and encouragement in all aspects of my career and life from the love of my life, my dear wife, Ashmita Guragain, my father Bala Bhadra Paudel, mother late Dhan Kumari Paudel, brother Dipendra Paudel, sister-in-law Kalpana Sapkota, and niece Deleena Paudel. I am thankful to my parents-in-law and family, and other relatives for persistent encouragement and support. Finally, this dissertation is dedicated to my true champion and idol, my mother, late Dhan Kumari Paudel, whom I lost during the final semester. She was my confidant and mentor. She was the epitome of love, sacrifice, simplicity, wisdom, and strength, to whom I owe everything. Life isn't the same without you. I hope I have made you proud. 6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 12 LIST OF ABBREVIATIONS ........................................................................................... 17 ABSTRACT ................................................................................................................... 18 CHAPTER 1 BACKGROUND ...................................................................................................... 20 Introduction ............................................................................................................. 20 Botany .............................................................................................................. 21 Napiergrass Ancestry ....................................................................................... 22 Napiergrass in the United States ...................................................................... 23 Pearl Millet ........................................................................................................ 24 Interspecific Hybrids of Napiergrass and Pearl Millet (PMN Hybrids) ............... 25 Cytoplasmic Male Sterility (cms) ...................................................................... 25 Molecular Tools Applied in Plant Breeding ....................................................... 27 Genotyping by Sequencing (GBS) ................................................................... 29 Target Enrichment Sequencing ........................................................................ 30 Quantitative Trait Loci Analysis ........................................................................ 30 Biomass yield ................................................................................................... 31 Flowering Time ................................................................................................. 31 Genes Related to Flowering ............................................................................. 33 Objectives ............................................................................................................... 34 2 SURVEYING THE GENOME AND CONSTRUCTING A HIGH-DENSITY GENETIC MAP OF NAPIERGRASS (CENCHRUS PURPUREUS SCHUMACH.) ......................................................................................................... 37 Introduction ............................................................................................................. 38 Methods .................................................................................................................. 41 Napiergrass Genome Survey ........................................................................... 41 SSR Identification and Marker Development .................................................... 42 Plant Materials and DNA Extraction ................................................................. 43 Genotyping-by-sequencing ............................................................................... 43 Comparative Genomics .................................................................................... 43 Sequence Analysis and SNP Calling ................................................................ 44 Linkage Map Construction ................................................................................ 45 Comparison Between Napiergrass and Pearl Millet Genome........................... 46 7 Results .................................................................................................................... 46 Napiergrass Genome Survey ........................................................................... 46 Genotyping-by-sequencing ............................................................................... 47 SNP Calling by Various SNP Callers ................................................................ 49 Genetic Linkage Map Construction .................................................................. 49 Comparison Between Genomes of Napiergrass and Pearl Millet ..................... 51 Discussion .............................................................................................................. 52 3 MAPPING QTLS CONTROLLING FLOWER NUMBER AND FLOWERING TIME IN NAPIERGRASS ........................................................................................ 84 Introduction ............................................................................................................. 84 Materials and Methods............................................................................................ 87 Development of a Mapping Population ............................................................. 87 Phenotyping the Mapping Population ............................................................... 88 Genetic Map ....................................................................................................