Panicum Virgatum L.) For
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MOLECULAR PHYLOGENETIC ANALYSIS, GENETIC MAPPING, AND IMPROVEMENT OF SWITCHGRASS (PANICUM VIRGATUM L.) FOR BIOENERGY AND BIOREMEDIATION TO EXCESS PHOSPHORUS IN THE SOIL by ALI M. MISSAOUI (Under the Direction of Joseph H. Bouton) ABSTRACT Research was conducted to explore the genomic organization of switchgrass (Panicum virgatum L.) and its potential for bioenergy and bioremediation to excess P in the soil. The utility of nrDNA ITS1-5.8S-ITS2 region and chloroplast trnL(UAA) intron in determining relatives of switchgrass in the genus Panicum were evaluated using 42 Panicum taxa. The ITS sequences exhibited higher divergence than trnL(UAA) and provide potential in resolving the classification of this genus. Alignment of trnL(UAA) sequences from 34 switchgrass accessions revealed a 49 nucleotide-deletion (∆350-399) specific to lowland accessions, which can be used for the classification of upland and lowland germplasm. The extent of genetic diversity in 21 upland and lowland switchgrass genotypes was investigated using 85 RFLP probes. Jaccard and Dice distances showed a high genetic diversity between and within ecotypes. The segregation and linkage of 224 single dose restriction fragments (SDRF) generated from 99 RFLP probes in 85 progenies of two tetraploid (2n = 4x = 36) parents (Alamo x Summer) indicated that switchgrass is an autotetraploid with high degree of preferential pairing. The recombinational length of switchgrass genome is 4617 cM. Greenhouse and field investigation of the genetic variation and heritability of P uptake in 30 genotypes under fertilizer rates of 450 mg P and 200 mg N Kg-1 soil showed that switchgrass accumulates high levels of P (0.76 % in the greenhouse and 0.36% in the field). P uptake was correlated more with biomass production (r= 0.65 to 0.90) and less with P concentration (r= 0.10 to 0.42). Expected gain from selection for P concentration is low (1 to 2%). A substantial progress can be achieved through selection for higher biomass. Effectiveness of the honeycomb selection design in identifying superior genotypes for biomass production in switchgrass was evaluated at 1.2 m inter-plant spacing. In four field experiments, yield of half-sib lines derived from polycrossing 15 genotypes selected for high yield was on average higher than the yield of half-sib lines derived from 15 genotypes selected for low yield from Alamo and Kanlow nurseries. This suggests that identifying superior genotypes at 1.2 m spacing using the honeycomb method is possible. INDEX WORDS: Switchgrass, Panicum virgatum, bioenergy, nrDNA, ITS, chloroplast, trnL(UAA), phylogeny, SDRF, genetic diversity, genetic mapping, RFLP, polyploid mapping, phosphorus, P uptake, bioremediation, honeycomb design. MOLECULAR PHYLOGENETIC ANALYSIS, GENETIC MAPPING AND IMPROVEMENT OF SWITCHGRASS (PANICUM VIRGATUM L.) FOR BIOENERGY AND BIOREMEDIATION TO EXCESS PHOSPHORUS IN THE SOIL by ALI M. MISSAOUI B.S., Oregon State University, 1986 M.S., Texas Tech University, 1998 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2003 © 2003 Ali M. Missaoui All Rights Reserved MOLECULAR PHYLOGENETIC ANALYSIS, GENETIC MAPPING AND IMPROVEMENT OF SWITCHGRASS (PANICUM VIRGATUM L.) FOR BIOENERGY AND BIOREMEDIATION TO EXCESS PHOSPHORUS IN THE SOIL by ALI M. MISSAOUI Major Professor: Joseph H. Bouton Committee: H. Roger Boerma Andrew H. Paterson Peggy Ozias-Akins David E. Kissel Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2003 iv ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. Joe Bouton who has provided me with support throughout this work along with the freedom to choose my research topics. I am grateful to the members who served on my committee and took the time to review this lengthy document including Dr. Andrew H. Paterson, Dr. Peggy Ozias-Akins, Dr. Miguel Cabrera, and Dr. David E. Kissel. Special thanks are given to Dr. Roger Boerma for his assistance and critical insights into my work. I also express my deep appreciation to the people who have helped during all phases of this project. I remain very grateful to Dr. Glenn Burton and his family for their support through the “Glenn and Helen Burton Feeding the Hungry Scholarship”, the Department of Crop and Soil Sciences, and the United States Department of Energy, Environmental Sciences Division, Oak Ridge National Laboratory for the financial support of this work Finally, I am deeply indebted to my family and especially my wife Wided for their constant support, encouragement and love. Their inspiration remains my main support throughout all my endeavors. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS............................................................................................... iv LIST OF TABLES............................................................................................................. ix LIST OF FIGURES .......................................................................................................... xii CHAPTER 1 INTRODUCTION .............................................................................................1 2 GENOME ANALYSIS OF POLYPLOIDS USING MOLECULAR MARKERS: A LITERATURE REVIEW ....................................................3 Genetic and evolutionary aspects of polyploidy ...........................................3 Molecular markers and their importance in genome analysis.....................12 Linkage mapping.........................................................................................37 Genetic mapping in polyploids....................................................................45 References ...................................................................................................63 3 MOLECULAR PHYLOGENETIC ANALYSIS OF THE COMPLEX PANICUM (PANICOIDEAE, PANICEAE): UTILITY OF CHLOROPLAST DNA SEQUENCES AND RIBOSOMAL INTERNAL TRANSCRIBED SPACERS.....................................................................104 Abstract .....................................................................................................105 Introduction ...............................................................................................105 Materials and methods...............................................................................113 vi Results .......................................................................................................115 Discussion .................................................................................................119 References .................................................................................................123 4 MOLECULAR INVESTIGATION OF THE GENETIC VARIATION AND POLYMORPHISM IN SWITCHGRASS (PANICUM VIRGATUM L.) CULTIVARS AND DEVELOPMENT OF A DNA MARKER FOR THE CLASSIFICATION OF SWITCHGRASS GERMPLASM .....................137 Abstract .....................................................................................................138 Introduction ...............................................................................................140 Materials and methods...............................................................................144 Results .......................................................................................................149 Discussion .................................................................................................153 References .................................................................................................158 5 GENETIC LINKAGE MAPPING IN SWITCHGRASS (PANICUM VIRGATUM L.) USING DNA MARKERS..............................................173 Abstract .....................................................................................................174 Introduction ...............................................................................................175 Materials and methods...............................................................................179 Results .......................................................................................................183 Discussion .................................................................................................191 References .................................................................................................198 6 PHOSPHORUS NUTRITION AND ACCUMULATION IN PLANTS: A LITERATURE REVIEW ..........................................................................219 vii Introduction ...............................................................................................219 Phosphorus in the soil................................................................................219 P uptake across the plasma membrane ......................................................222 Destiny of P transported into the cell ........................................................225 Control of P uptake activity.......................................................................227 Phenotypic and genetic differences in P uptake by plants ........................230 Environmental aspects of phosphorus.......................................................233 Potential use of crop species for phytoremediation to excess phosphorus in the soil................................................................................235 Genetic manipulation to increase P uptake in plants.................................237 References .................................................................................................241