CHARACTERIZATION OF OVERWINTERING PERENNIALITY IN NAPIERGRASS: TRADITIONAL AND MOLECULAR APPROACHES A Dissertation by CHARLIE D. DOWLING, III Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Russell W. Jessup Committee Members, Byron L. Burson Jamie L. Foster Raul I. Cabrera Head of Department, David D. Baltensperger December 2015 Major Subject: Plant Breeding Copyright 2015 Charlie D. Dowling, III ABSTRACT Napiergrass (Pennisetum purpureum Schum.) is a tropical grass limited in adaptation to the southern United States, and efforts to expand its area of production as a quality forage crop as well as a high-biomass biofuel crop are needed. Phenotypic selection and screening of napiergrass S1 inbred lines and F1 hybrids for increased winter hardiness (USDA Winter Hardiness Zones 8b to 7b) demonstrated that napiergrass is more winter hardy than expected. Variation in hardiness zone 7b winter weather patterns at Vernon, TX, and Alma, AR, resulted in 40% survival across genotypes and no winter survival, respectively, even though the minimum temperatures at both locations were similar. Winter survival in this species is associated with rhizome development. Therefore, rhizome candidate gene expression assays using quantitative polymerase chain reaction (qPCR), on fall and spring sampled rhizomes for genes associated with overwintering, rhizome development, and rhizome proliferation resulted in two cloned genes, APETELA2 (AP2) and Rare Cold Inducible 1 (RCI1) conferring detectable changes in gene expression patterns. Spring sampled rhizomes showed the greatest fold change (FC), specifically in the zone of cell division across all genotypes. Attempts to transfer genes associated with cold-tolerance from oriental fountaingrass (Pennisetum orientale L.C. Rich.) and buffelgrass [Cenchrus ciliaris (L.) syn Pennisetum ciliare Link] into napiergrass via wide hybridization were not successful because no interspecific hybrids were recovered. Analysis of pollen tube growth and pollen-pistil interactions did not reveal barriers to these hybridizations, but post- ii fertilization events may be the reason for the failure to recover hybrids and this needs to be investigated. iii DEDICATION I would like to dedicate this work to my parents, Charlie and Debbie Dowling, who have been a great reinforcement to me through the years. My dad told me to go to college if I did anything while he was lying on what we thought was his deathbed. Well, Dad, I did, and here I am 15 years later. Thank you for your prayers, emotional and financial support, as well as giving me the basic knowledge and skills to be successful in this world. I would also like to thank Grams Berit and Slim for all of their loving kindness and support as well. I could not have made it this far without your continuous cheer and encouragement. iv ACKNOWLEDGEMENTS I would like to thank my committee: Dr. Jessup, Dr. Burson, Dr. Foster, and Dr. Cabrera for their guidance through my research. There were many people that helped make this achievement possible throughout my time at Texas A&M. I owe my deepest appreciation to Dr. Jessup and Dr. Burson who mentored me and encouraged me as a student of the plant sciences. I also would like to thank Dr. Smith, Dr. Hatch, Dr. Murray, Dr. Hallmark, Dr. Cothren, Dr. Ibrahim, Dr. Rooney and all the other professors inside and outside of the Dept. of Soil & Crop Sciences who challenged and developed my knowledge and level or understanding. Thank you to Dr. Esten Mason and Dennis Motes at the University of Arkansas, Dr. Darius Malinowski at Vernon, Dr. Jim Heitholt at Texas A&M University-Commerce, Greg Wilson, Tony Watson, Mason Kearns, Daniel Mowry, Aurelie Collee, Morgan Carlson, Al Nelson, all at the Burleson County TAMU Field Lab, Ms. Judy, Ms. Glenda, Amanda Ray, Ms. Francis, Juan Cadeza, and Shea for all of your assistance, guidance, and support while studying and conducting research at Texas A&M University. Thanks to all of the friends and confidants that kept me sane, happy, and motivated throughout my studies. Thank you to the Department of Soil & Crop Sciences and BP for financial support. v TABLE OF CONTENTS Page ABSTRACT............................................................................................................... ii DEDICATION........................................................................................................... iv ACKNOWLEDGEMENTS...................................................................................... v TABLE OF CONTENTS.......................................................................................... vi LIST OF FIGURES................................................................................................... viii LIST OF TABLES.................................................................................................... ix CHAPTER I LITERATURE REVIEW........................................................................ 1 The Genus Pennisetum............................................................................. 1 Pearl Millet.............................................................................................. 2 Napiergrass.............................................................................................. 3 Oriental Fountaingrass and Buffelgrass.................................................. 5 Cytogenetics of Pearl Millet, Napiergrass, Buffelgrass, and Oriental Fountaingrass.......................................................................................... 7 Objectives............................................................................................... 10 II INTERSPECIFIC HYBRIDIZATION BETWEEN NAPIERGRASS AND OTHER PENNISETUM SPECIES.................................................. 11 Introduction............................................................................................. 11 Materials and Methods............................................................................ 15 Results and Discussion............................................................................ 19 Conclusions............................................................................................. 33 III IMPROVED OVERWINTERING IN NAPIERGRASS......................... 34 Introduction.............................................................................................. 34 Materials and Methods…......................................................................... 40 Results and Discussion…......................................................................... 45 Conclusions….......................................................................................... 63 vi IV CONCLUSIONS...................................................................................... 64 REFERENCES.......................................................................................................... 66 APPENDIX A............................................................................................................ 90 APPENDIX B............................................................................................................ 103 vii LIST OF FIGURES FIGURE Page 1 Pollen stainability of oriental fountaingrass ‘Cowboy’............................... .. 22 2 Pollen tube of oriental fountaingrass accession 433 that stopped growing within 12 hours after pollination ................................................................... 29 3 Images of irregular pollen tube growth in pistils from intra- and interspecific pollinations ............................................................................... 31 4 Relative expression of the AP2 gene in rhizomes of the 12TX09 and 12TX10 genotypes at Commerce, TX ........................................................... 56 5 Relative expression of the RCI2 gene in rhizomes of the 12TX09 and 12TX10 genotypes at Commerce, TX ........................................................... 57 6 Relative expression of the AP2 and RCI2 genes in rhizomes of the 12TX09 and 12TX10 genotypes at Commerce, TX. in the fall of 2013 ....... 103 7 Relative expression of the AP2 and RCI2 genes in rhizomes of the 12TX09 and 12TX10 genotypes at Commerce, TX. in the spring of 2014 ............................................................................................................... 104 8 Relative expression of the AP2 and RCI2 genes in the region of cell division for 12TX09, 12TX10, 12TX18, and Merkeron at College Station, TX. in the fall of 2013 ................................................................................... 105 9 Relative expression of the AP2 and RCI2 genes in the region of cell elongation for 12TX09, 12TX10, 12TX18, and Merkeron at College Station, TX. in the fall of 2013 ...................................................................... 106 10 Relative expression of the AP2 and RCI2 genes in the region of cell division for 12TX09, 12TX10, 12TX18, and Merkeron at College Station, TX in the spring of 2014 ............................................................................... 107 11 Relative expression of the AP2 and RCI2 genes in the region of cell elongation for 12TX09, 12TX10, 12TX18, and Merkeron at College Station, TX in the spring of 2014 .................................................................. 108 viii LIST OF TABLES TABLE Page 1 Plant materials used as pollen parents in wide crosses and for pollen