HYBRIDIZATION OF TETRAPLOID HIGHBUSH CULTIVARS WITH DIPLOID VACCINIUM ELLIOTTII By ELLIOT NORDEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017 1 © 2017 Elliot Norden 2 To my family and to my friends 3 ACKNOWLEDGMENTS I thank my parents for their continued support throughout my life. I thank my mentors, Dr. Paul Lyrene and Dr. Jose Chaparro, for their support on my project and expert advice and insights. In addition, I thank both of them for showing me the beauty of plant breeding, and pushing me to complete this degree and be a better person. I express gratitude to my committee members, Dr. Paul Lyrene, Dr. Jose Chaparro and Dr. Pam Soltis. I thank, Dr. James Olmstead, Werner Collante, and Dr. Kenneth Quesenberry. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 INTRODUCTION .................................................................................................... 11 2 INTER-SPECIFIC HYBRIDIZATION AND CROSSING BEHAVIOR OF VACCINIUM ELLIOTTII WITH SOUTHERN HIGHBUSH CULTVARS ................... 14 Materials and Methods............................................................................................ 17 Studies with Vaccinium elliottii .......................................................................... 17 Inter-specific hybridization with Vaccinium corymbosum ........................... 17 Flower studies ............................................................................................ 17 Leaf studies ................................................................................................ 18 Flow cytometry analyses ............................................................................ 19 Cytological studies ..................................................................................... 20 Crossing Studies .............................................................................................. 21 Intercrosses for F2 generation ................................................................... 21 Backcrosses ............................................................................................... 22 Berry studies .............................................................................................. 22 Statistical analysis ...................................................................................... 23 Results and Discussion........................................................................................... 23 Studies with Vaccinium elliottii .......................................................................... 23 Interspecific hybridization data ................................................................... 23 Putative F1 hybrid selection ........................................................................ 24 Morphological Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii) .................................................................................................... 25 Flower characteristics ................................................................................ 25 Leaf characteristics .................................................................................... 26 Berry characteristics .................................................................................. 27 Fertility Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii) ........................................................................................................... 28 Cytogenetic Studies ......................................................................................... 29 Chromosome Counting .................................................................................... 31 N-37 ........................................................................................................... 31 N-32 ........................................................................................................... 31 Crossing Data 2016 .......................................................................................... 31 5 Intercrosses ............................................................................................... 31 Backcrosses ............................................................................................... 32 Crossing Data 2017 .......................................................................................... 33 Intercrosses ............................................................................................... 33 Backcrosses ............................................................................................... 33 Conclusions ............................................................................................................ 34 3 PLOIDY LEVEL DETERMINATION IN COLCHICINE-TREATED VACCINIUM ELLIOTII SEEDLINGS AND SUBSEQUENT INTERSPECIFIC HYBRIDIZATION BETWEEN HIGHBUSH BLUEBERRY CULTIVARS AND AUTOTETRAPLOID V. ELLIOTTII ......................................................................... 47 Materials and Methods............................................................................................ 48 Materials ........................................................................................................... 48 Flow Cytometry Analyses ................................................................................. 49 Stomata Analyses ............................................................................................ 51 Pollen Analyses ................................................................................................ 51 Interspecific Hybridization of Colchicine-Derived Tetraploid Vaccinium elliottii with Tetraploid Highbush Blueberry Cultivars ..................................... 52 Results and Discussion........................................................................................... 52 Colchicine Treatment ....................................................................................... 52 Flow Cytometry Data ........................................................................................ 53 Stomata and Pollen Size Data .......................................................................... 54 Interspecific Hybridization of Colchicine-Derived Autotetraploid Vaccinium elliottii with Tetraploid Highbush Cultivars ..................................................... 55 Conclusions ............................................................................................................ 57 LIST OF REFERENCES ............................................................................................... 63 BIOGRAPHICAL SKETCH ............................................................................................ 66 6 LIST OF TABLES Table page 2-1 Results from 14 tetraploid highbush x Vaccinium elliottii crosses done in the spring of 2014. All highbush flowers were pollinated with pollen bulked from diploid Vaccinium elliottii clonesz. ....................................................................... 37 2-2 Results from the 2016 crosses including: intercrossing of F1 hybrids and b c backcrossing of F1 hybrids to diploid VE (Vaccinium elliottii) and tetraploid VC (Vaccinium corymbosum) varieties. .................................................................... 38 2-3 Leaf, flower, and berry characteristics of three taxa: V. elliottii (VE), southern highbush (HB) and highbush cultivars x V. elliottii F1 hybrids (HB x VE). ........... 39 2-4 Comparative analysis of DNA content of etiolated tissue versus Non-etiolated tissue from V. elliottii (VE), highbush (HB) and highbush x V. elliottii F1 hybrids using flow cytometry. .......................................................................................... 40 2-5 Genome size estimation by flow cytometry of VC (Vaccinium corymbosum) x VE (Vaccinium elliottii) F1 hybrids utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Pollen size of VE selections was also used as a predictor of ploidy level. Pollen shed and % fully formed tetrads were used as indicators of fertility. ............................................................................................ 41 2-6 Results from the 2017 crosses, including: Intercrosses of F1 hybrids to obtain F2 populations and backcrosses to VCb (Vaccinium corymbosum) varieties........... 43 2-7 Comparative success of Highbush x Highbush, Vaccinium elliottii x Vaccinium elliottii, and Highbush x Vaccinium elliottii crosses 2014z. ................................. 43 3-1 Population one, genome size estimation by flow cytometry of colchicine-treated V. elliottii clones utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Stomata size and pollen size were also used as predictors of ploidy level. ................................................................................................................... 58 3-2 Population two, genome size estimation by flow cytometry of colchicine-treated V.