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INHERITANCE of CHLOROPLAST DNA (Cpdna) in LOBELIA SIPHILITICA

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INHERITANCE of CHLOROPLAST DNA (Cpdna) in LOBELIA SIPHILITICA INHERITANCE OF CHLOROPLAST DNA (cpDNA) IN LOBELIA SIPHILITICA A thesis submitted to the Kent State University Honors College in partial fulfillment of the requirements for General Honors by Alicia Durewicz May, 2012 Thesis written by Alicia Durewicz Approved by ________________________________________________________________, Advisor _____________________________________, Chair, Department of Biological Sciences Accepted by _____________________________________________________, Dean, Honors College ii TABLE OF CONTENTS LIST OF FIGURES............................................................................................................iv LIST OF TABLES...............................................................................................................v ACKNOWLEDGMENTS..................................................................................................vi CHAPTER I. INTRODUCTION....................................................................................1-9 II. METHODS...........................................................................................10-27 III. RESULTS.............................................................................................28-32 IV. CONCLUSION AND DISCUSSION..................................................33-41 REFERENCES.............................................................................................................42-45 APPENDICES..............................................................................................................46-52 A. List of parental sequence motifs found.................................................46-48 B. List of crosses, with parent and offspring sequence motifs..................49-52 iii LIST OF FIGURES Figure 1. Lobelia siphilitica........................................................................................9 Figure 2. Map of Lobelia siphilitica populations surveyed.......................................11 Figure 3. Schematic of the chloroplast psbK-rps16 region.......................................12 Figure 4. Direction and location of amplification and sequencing primers..............18 Figure 5. Repeat unit transition matrix......................................................................22 Figure 6 Types of organellar inheritance.................................................................25 Figure 7. Evidence for heteroplasmic offspring........................................................26 Figure 8. Frequency of inheritance types observed...................................................30 Figure 9. Diversity of inheritance types among crosses............................................32 iv LIST OF TABLES Table 1. List of primers used in sequencing and polymerase chain reactions.........17 Table 2. Unique minisatellite repeat units found in this study.................................21 Table 3. Unique minisatellite repeat motifs.............................................................23 v ACKNOWLEDGMENTS It is a pleasure to thank all those who have made my senior honors thesis possible. First and foremost, I owe my deepest gratitude to my Advisor, Dr. Andrea Case. Her continuous motivation, patience, and guidance has made this project possible and a success. I would also like to thank the members of my defense committee, Dr. Soumitra Basu, Dr. Gail Fraizer, and Dr. Helen Piontkivska, for their assistance with this endeavor. Many thanks towards Dr. Christina Caruso and the University of Guelph for use of their greenhouse and facilities, and Dr. Eric Knox for sharing his knowledge about Lobelia siphilitica's chloroplast genome. I am also grateful to graduate students Hannah Madson and Eric Floro for their immeasurable assistance and advice. Thank you to the Honor's College for providing undergraduates with this opportunity to challenge themselves and show their fullest potential. Lastly, I would like to thank my family and friends, but especially my parents and brother. They have been by my side throughout this entire process and have provided me with everlasting love, understanding, and support. Thank you all, without your encouragement and collaboration, this thesis would not have been possible. Thank you again. vi CHAPTER I INTRODUCTION In eukaryotes, such as plants and animals, modes of inheritance differ among the distinct genomes within the cell. In general, nuclear genomes are inherited bi-parentally. Mendelian laws state that half of the offspring's nuclear DNA is inherited from the mother, and the other half is inherited from the father. However, organellar (mitochondria and chloroplast) genomes are usually uniparentally inherited. This is considered to be a rule with few exceptions across all eukaryotes (Birky, 1995), and forms the basis for many assumptions about the evolution of cytoplasmic and nuclear genomes. In 1909, two researchers (Baur, 1909 and Correns, 1909 as cited in Birky, 1995 and Xu, 2005) were the first to document patterns of organellar inheritance in two plant species: Pelargonium zonale (Geraniaceae) and Mirabilis jalapa (Nyctaginaceae). Correns (1909, as cited in Birky, 1995) observed chloroplast inheritance by mating wild type (green) mothers with mutant (variegated green and white) males. His findings showed uniparental inheritance (receiving only one parent's genome) of the chloroplast phenotype. All of the offspring displayed the mother's green phenotype and did not show evidence of the father's variegated phenotype. It was Baur (1909, as cited in Birky, 1995) who noticed offspring of P. zonale inherited chloroplasts from either the mother only, the father only, or from both parents. Much later, Kuroiwa et al. (1992) discovered that the variegation in these, and many other plants, was due to a mutation in the chloroplast genome. Since mitochondria and chloroplasts play a vital role in the life of a cell, 1 2 mutations can possibly increase or decrease the fitness and survival of individuals. The idea of alternative inheritance patterns led researchers to focus on the distinct genomes located in these important organelles, and discover the possibilities of why different patterns exist. The main hypothesis for uniparental cytoplasmic inheritance is based on the consequences of interactions between the nucleus and the organelles to the organism (Law and Hutson, 1992; Burt and Trivers, 2006). Burt and Trivers (2006) posit that during early evolution of the cell, if two precursors to organelles (mitochondrial precursors in their review) were present in the cytoplasm, the more "selfish" precursor would have proliferated. By "selfish" genome, they mean, the smaller of the two, which replicates the quickest, and becomes the more dominant because of its replication advantage. However, this "selfish" genome could have been detrimental to the cell. If it replicated faster because it was missing important genes, such as DNA coding for metabolic processes, the host cell would be at a disadvantage. Thus, natural selection favored mechanisms enforcing uniparental inheritance. Cells with only one organellar genome would have no within-individual conflict, and have a higher chance of passing on this pattern of inheritance to future generations. Limiting the presence of these "selfish" genomes is not the only benefit to uniparental inheritance. Heteroplasmy, the occurrence of two or more unique organellar genomes, may also have detrimental effects on an individual even if they replicate at the same rate. The first consequence of having two distinct organellar genomes could result in miscommunication between the nuclear genome and the organellar genomes in 3 question. Schwartz and Vissing (2002) published data on a 28-year old man who had paternal mitochondria in his muscle cells, but maternal mitochondria in all other cells. This would not normally be a problem, but the paternal line had a two-nucleotide base pair (bp) deletion that caused him to have troubles with exercising. Additional studies have associated many mitochondrial diseases with heteroplasmy (reviewed in Chinnery and Turnbull, 2000 and Chinnery et al., 2002). In these cases, a mutant and a wild-type mitochondrial DNA (mtDNA) coexist within the cells. It is these mtDNA mutations, which have been linked to a variety of diseases such as Kearns-Sayre syndrome (Zeviani et al., 1988; Moraes et al., 1989), diabetes, and deafness (Reardon et al. 1992). The second consequence of heteroplasmy does not harm the individual, but rather, changes how biologists track the evolution of a species through individual lines of descent (Galtier et al., 2009). Geneticists assume uniparental inheritance when using organellar genetic markers to study maternal lineages of a single species or find a single most recent common ancestor of multiple species. In human studies, the mitochondrial DNA is used to ascertain a person's nationality of origin (Ayala, 1995; Behar et al., 2008). In most cases, uniparental inheritance of organellar genomes is maternal. However, if heteroplasmy is present, the mode of inheritance is not necessarily uniparental. Although heteroplasmy can be produced de novo through mutation within a host, it seems more likely that both haplotypes will be passed on to subsequent generations if the heteroplasmy results from a previous occurrence of bi-parental inheritance or paternal leakage (when an individual inherits the organellar genome from both the father and the mother). There have been few studies looking at how 4 heteroplasmy arises (Pearl et al., 2009). But, for a de novo mutation
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