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University of Florida Thesis Or Dissertation Formatting EVOLUTION OF THE GENETIC CONTROL OF LEAF DEVELOPMENT WITH AN EMPHASIS ON CARNIVOROUS PITCHER PLANTS By NICHOLAS WILLIAM MILES 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 2013 1 © 2013 Nicholas William Miles 2 To those who believed in me, especially my Mom and Dad, Heather-Rose, and Susan Spaulding 3 ACKNOWLEDGMENTS I would like to thank my advisors, Pam and Doug Soltis, for their tireless work and support. This dissertation is testament to the saint-like patience they have had with me over the years, and especially this year. I would also like to thank my committee members, Drs. David Oppenheimer and Mark Settles, for the integral part they have played in the forming of my ideas. Both of them and their labs have shared resources that I cannot thank them for enough. Drs. Tsukaya and Yamaguchi, who showed genuine rectitude as hosts to me in a foreign country and enabled me to collect data that I had strived to do for 6 years, I am forever indebted to. The members of the Soltis Lab, who I now view in likeness of family members, I will remember for the rest of my life with great sentiment. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 12 Angiosperm Leaves as a System for the Study of Evolutionary Development (Evo-Devo) .......................................................................................................... 12 Nucleotide Sequencing Technology Leafs Out ....................................................... 15 Evo-Devo Studies of Carnivorous Pitcher Plants .................................................... 16 2 NEO- AND SUB-FUNCTIONALIZATION OF THE PLANT BODY PATTERNING GENE CLASS III HD-ZIP CORRELATES WITH LAND PLANT EVOLUTION ........ 19 Introductory Remarks.............................................................................................. 19 Methods .................................................................................................................. 24 Sequence Retrieval .......................................................................................... 24 Alignment ......................................................................................................... 25 Phylogenetic Analyses ..................................................................................... 25 Results .................................................................................................................... 26 BLAST results .................................................................................................. 26 Phylogenetic analyses ...................................................................................... 26 Discussion .............................................................................................................. 29 3 GENOMIC COEVOLUTION OF HD-ZIP III RELATED PATHWAY GENES ........... 46 Introductory Remarks.............................................................................................. 46 Methods .................................................................................................................. 51 Sequence retrieval ........................................................................................... 51 Alignment ......................................................................................................... 53 Phylogenetic analyses ...................................................................................... 53 Arabidopsis microarray expression .................................................................. 53 Results .................................................................................................................... 54 Paralog numbers .............................................................................................. 54 Pathway proportionality .................................................................................... 54 Lineage evolution ............................................................................................. 55 Discussion .............................................................................................................. 56 5 4 LEAF EVO-DEVO OF THE OF CARNIVOROUS PITCHER PLANT, CEPHALOTUS FOLLICULARIS (CEPHALOTACEAE) .......................................... 77 Introductory Remarks.............................................................................................. 77 Methods .................................................................................................................. 80 RNA extraction and gene amplification ............................................................ 80 Sequence alignment and phylogenetic analysis ............................................... 81 In situ hybridization ........................................................................................... 81 Probe construction ..................................................................................... 81 Tissue fixation ............................................................................................ 82 Hybridization .............................................................................................. 82 Results .................................................................................................................... 83 Discussion .............................................................................................................. 83 5 CONCLUSION ........................................................................................................ 96 APPENDIX A FURTHER CARNIVOROUS PLANT EXPERIMENTS ............................................ 99 AS2 ......................................................................................................................... 99 VIGS Development ............................................................................................... 100 LIST OF REFERENCES ............................................................................................. 107 BIOGRAPHICAL SKETCH .......................................................................................... 116 6 LIST OF TABLES Table page 2-1 GenBank accession numbers of sequences from previous studies of Floyd et al. (2006) and Prigge and Clark (2006) .......................................................... 33 2-2 Sampled Gymnosperm Taxa from 1KP Data.................................................... 34 2-3 Sampled Monilophyte taxa from 1KP data ....................................................... 35 3-1 Number of paralogs of the 4 gene families in the 6 sampled angiosperm genomes. ............................................................................................................ 60 3-2 Proportions of the number of paralogs for each of the 4 gene families in the genomes of the 6 sampled angiosperm genomes. ............................................. 61 3- 3 Places in the genomes of the sampled angiosperm genomes that contain the tandem precursor miR166 sequences. ......................................................... 61 4-1 Nucleotide sequence of primers used to amplify HistoneH4 and YABBY genes in Cephalotus follicularis. ......................................................................... 86 7 LIST OF FIGURES Figure page 1-1 Genetic model controlling leaf development and polarity in angiosperms.. ........ 18 2- 1 Evolution of HD-ZIP III and ZPR in land plants.. ................................................. 36 2-2 Protein domain structure of HD-ZIP III and ZPR................................................. 38 2-3 Phylogenetic tree of the topology found to have the highest likelihood by RAxML for sampled vascular plant HD-ZIP III sequences. ................................. 39 2-4 Subtree of HD-ZIP III vascular plant phylogeny containing lycophtye and PaleoHDZ3 clades. ............................................................................................. 40 2-5 Subtree HD-ZIP III vascular plant phylogeny containing gymnosperm ZPR clade. .................................................................................................................. 42 2-6 Subtree HD-ZIP III vascular plant phylogeny containing monilophyte RBVC8 as well as gymnosperm and angiosperm C8 clades. .......................................... 43 2-7 Gymnosperm RBVC8 as well as gymnosperm and angiosperm RBV subtree of of HD-ZIP III vascular plant phylogeny. .......................................................... 44 2-8 Alignment of gymnosperm ZPR sequences and the Homeodomain-Leucine- Zipper Region of HD-ZIP III. ............................................................................... 45 3-1 Phylogeny of major Angiosperm lineages and eudicot orders with the placement of sampled taxa. ................................................................................ 62 3-2 Phylogeny and ancestral genome evolution
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