Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 166-188.

Thesis (Ph. D.)--Washington State University, May 2009. / Title from PDF title page (viewed on June 8, 2009). "School of Electrical Engineering and Computer Science." Includes bibliographical references.

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 94-108).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 199-226). Also available in print.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 41-43). Also available in electronic version.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome. / AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen rue. In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom. BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24 BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal. Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir 'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in 'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is. Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is. Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat. Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese DNA vry is, ontwerp. Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en volgordebepaling van die druif chloroplasgenoom.

For my dissertation research, I used a model system of bacteria and bacteriophage to study patterns of genome evolution. I performed whole-genome sequencing of replicate populations to determine the genetic changes responsible for a repeatable pattern of coevolution between bacteria and phage. I found that genetic changes conferring resistance in bacteria negatively impacted other traits such as growth rates and sensitivity to antibiotic. Different resistance mutations varied in the magnitude of their pleiotropic costs, and this resulted in a fixation bias favoring mutations that minimized pleiotropic effects. I manipulated the environment and found that differential pleiotropy between environments drove repeatable evolution at different genetic scales. Finally, I explored theoretically how bacteria, phage, and resource interact through a dynamic system of feedbacks. I used a mathematical model to describe priority effects in evolution, where the expected fate of a beneficial mutation varies depending upon whether it appears before or after a competing mutation. / 10000-01-01

Many insects and other arthropods have symbiotic microorganisms that may influence key facets of their biology. Cardinium hertigii is an intracellular bacterial symbiont, (phylum Bacteroidetes) of arthropods and nematodes. This versatile symbiont has been shown to cause three of four reproductive manipulations of their arthropod hosts known to be caused by symbionts: parthenogenesis induction (PI), where genetic males are converted into genetic females; feminization, where genetic males become functional females; and cytoplasmic incompatibility (CI), the symbiont-induced death of offspring from matings of infected males and uninfected females. Here, I explored the evolution of this symbiont and its reproductive manipulations, and found that closely related Cardinium strains have a tendency to associate with closely related hosts and the reproductive manipulations do not display a clear phylogenetic signal. To further understand the possible genes underlying these reproductive manipulations, I sequenced four Cardinium genomes and compared these with the two genomes analyzed in the literature. In these comparisons, I found that, although closely related Cardinium strains tend to reside in closely related hosts, there is no evidence for a suite of genes associated with host specificity, as few differences separate two strains residing in different host orders, suggesting that ecological opportunity for horizontal transmission may be more limiting to Cardinium than genomic capability. I additionally identify some genes that may be associated with the Cardinium’s ability to induce PI and CI in its wasp host. Overall, this dissertation has led to a better understanding of Cardinium and its effects on its hosts.

Doctor of Philosophy / Genetics Interdepartmental Program / Christopher Toomajian / Secondary metabolites (SMs), low molecular weight molecules that are not essential for normal organism growth and development, may confer a selective advantage in some environments. Fungal SMs are structurally and functionally diverse and include mycotoxins, plant regulators and pigments, and the genes that work together in SM biosynthetic pathways are physically clustered in the genome. Fusarium, a genus of filamentous fungi, is noted for SM production, especially mycotoxins, which may contribute to plant pathogenesis. Fusarium species exhibit differences in their SM profiles, and comparative genomics studies have found corresponding differences in the SM gene clusters in some Fusarium species. The investigation of differences in the genomes and SM gene clusters between closely related species, such as F. proliferatum and F. fujikuroi, may help explain their phenotypic divergence, including differences in SM profiles. In addition, the study of intra-species SM variation may indicate how SM loci affect a pathogen’s fitness traits. My research includes three main projects that address different aspects of Fusarium SM variability. To carry out my projects, I established a feasible Genotyping-by-Sequencing (GBS) protocol for Fusarium. One project explored the genetic bases underlying phenotypic divergence related to SM profiles and pathogenicity between F. proliferatum and F. fujikuroi using a quantitative genetics approach. Specifically, I 1) constructed the first high density genetic map based on progeny from an interspecific cross between these two species; and 2) detected a novel regulatory locus for gibberellic acid production and identified a region affecting onion virulence that includes the fumonisin gene cluster. The second project characterized the F. proliferatum parent genome from the previous cross and its SM gene clusters using a comparative genomics approach. Specifically, I 1) assembled the F. proliferatum genome into 12 chromosomes with a combined length of ~43 Mb; 2) annotated this assembly and characterized its 50 SM gene clusters; and 3) detected over 100 F. proliferatum specific genes that might play roles in this species’ host specificity and plant pathogenicity. The third project used a population genomics approach to explore how different F. graminearum chemotypes, or isolates classified based on the accumulation of alternate trichothecene toxin types, may differ for fitness traits and whether trichothecene genes are directly responsible for these differences. Specifically, I 1) genotyped over 300 F. graminearum strains from New York and the upper Midwest in the U.S. and from South America using our GBS protocol; 2) detected two major subpopulations that were correlated, though imperfectly, to the predicted 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON) chemotypes in the U.S.; 3) identified a rapid linkage disequilibrium decay over a few tens of kb followed by a slower decay to background levels over a distance of 200 kb to 400 kb in selected subpopulations in the U.S.; and 4) found that neither chemotype has a clear fitness advantage in a small set of isolates from New York, but that isolates belonging to one genetic subpopulation may on average have a fitness advantage over isolates from the other subpopulation.

Gene duplications generate critical components of genetic variation that can be selected upon to affect phenotypic evolution. The angiosperm GATA transcription factor family has undergone both ancient and recent gene duplications, with the HAN-like clade displaying divergent functions in organ boundary establishment and lateral organ growth. To better determine the ancestral function within core eudicots, and to investigate their potential role in floral diversification, I conducted HAN-like gene expression and partial silencing analyses in the asterid species petunia (Petunia x hybrida). My results indicate duplication of HAN-like genes at the base of Solanaceae followed by expression diversification within the flower. Although no aberrant phenotypes were apparent following single gene knockdowns, silencing of both paralogs lead to leaf senescence. Together with other functional studies, these data suggest a possible ancestral role for HAN-like genes in core eudicot shoot apical meristem development, followed by functional diversification following both speciation and duplication.