Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...

The evolution of plant genomes is notable for manifesting a cycle of whole genome doubling, fractionation (gradual loss of redundant genes) and speciation. The thesis is based on a branching process model of the doubling and fractionation process, integrated with a standard model of sequence divergence. The immediate application of this work is to account for the distribution of sequence similarity for duplicate gene pairs, both within plant genomes and between two related plant genomes in terms of a cycle of polyploidization, fractionation and speciation. We derive a mixture distribution for duplicate gene pair similarities generated by speciation and/or repeated episodes of polyploidization. We account not only for the timing of these events in terms of local modes or peaks of the component distributions, but also their volume, or amplitude, and variance. We outline how to infer the parameters of the model. We illustrate with analyses of the distribution of homolog similarities in a number of plant families: Brassicaceae, Solanaceae and Malvaceae. To our knowledge, this is the first method to account for the volume of the component normals of a distribution of similarities, preliminary to an evolutionarily meaningful inference procedure. In addition, we solve the problem of identifying the ploidy level of a series of two or three polyploidizations by invoking the observed and predicted gene triple profiles for each model, i.e., by calculating the probability of the four types of triple with origins in one or the other event, or both.

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Ethanol tolerance in Saccharomyces cerevisiae is a complex polygenic trait. As a toxin, ethanol damages multiple cell constituents as well as being both a substrate and product of the metabolism of S.cerevisiae. This complexity has made ethanol tolerance difficult to study. Deletion screens have identified hundreds of genes that impair ethanol tolerance by their absence and hence might help survival in high ethanol environments. Similarly, expression studies have revealed genes that respond to ethanol shock, but it is unclear whether those genes are likely targets for improvement of ethanol tolerance in strains growing normally. In addition, those yeasts that are currently commercially exploited for their high ethanol tolerance in the brewing and bioethanol industries are commonly aneuploid or polyploid which makes it difficult to correlate particular features of their genotype with the ethanol tolerant phenotype. Experimental evolution can reveal genetic changes that change competitive fitness. It is practical to run numerous competitions in parallel between isogenic S.cerevisiae strains for hundreds of generations under ethanol stress, after which whole genome sequencing can identify the genetic changes. Fluorescent tagging of those strains can reveal small changes in population dynamics. We propagated 144 populations in batch culture for between 100 and 200 generations under 4 ethanol regimes (0%, 4.5%, 6.5% and ramped 0-10%). We monitored the progress of evolution by using mixtures of two fluorescently tagged, but otherwise isogenic, haploid, hoΔ (site-specific endonuclease deletant) founder strains (DeLuna et al 2008). Population dynamics measured using fluorescently labelled strains indicated that changes had occurred in competitive fitness due to adaptive evolution. Cell-size measurement and flow cytometry showed that evolved populations were diploid or triploid and the transition to higher ploidy occurred more rapidly with increasing ethanol stress. During the experimental evolution three strains evolved the capacity to grow on organic acids. We have sequenced the complete genomes of eight evolved strains. These strains are confirmed as being diploid, but not aneuploid. Sequencing of evolved strains revealed mutations that have not been previously characterised in deletion or expression studies of ethanol or organic acid tolerance in S.cerevisiae. Both increasing ploidy, to produce triploids as well as diploids, and the acquisition of organic acid tolerance under ethanol stress are unexpected outcomes that have implications for future work.

Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources. / Ph. D. / Despite their humble appearance and routine consumption, potatoes have a complex genetic structure and a life cycle capable of both sexual reproduction through flowers, fruit and seed, and asexual reproduction through the tubers which also comprise the edible product. From an agronomic perspective, one of the most important qualities of a potato tuber is size, a feature influenced by genetics and environment. Cell-to-cell variation for the amount of DNA per cell, one component that influences tuber size, is known to occur, yet our ability to measure DNA content in starchy tuber cells has been obscured by debris generated through routine preparation techniques. We present and evaluate a new method for measuring the DNA content of potato tuber cells, which provides reliable results across a range of different potato varieties and species. ‘Jumping genes’ also known as transposons, first reported in maize but now known to occur in most advanced plant and animal species, have been found to comprise ~5% of the recently sequenced potato genome. We show that a particular class of transposons is more likely to occur adjacent or actually in certain types of genes, such as those which confer resistance to disease, where they may have meaningful effects on how those genes operate. We then proceed to predict the current activity of the various families of these jumping genes to understand how they continue to alter the genetic landscape of potato. Finally we identify a particular gene which dictates flower color in potato (purple vs. white). We demonstrate that several transposons occur in some forms of the flower color gene. Originally we hypothesized that transposons were associated with the turning off of the purple flower color form; however, on closer examination, we could express the white flower form in transgenic plants that were originally white-flowered and convert them to have purple flowers, demonstrating that even the white flower form was functional. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the availability of the special strains of potatoes with reduced chromosome number and the publication of the potato genome.

Contact zones of cytotypes harbour a unique opportunity to study the dynamics and evolution of mixed-ploidy systems. Butomus umbellatus (flowering rush) is one of a few species in which diploid and triploid cytotype is present in nature. The first known mixed-ploidy populations of B. umbellatus were discovered in south-eastern Slovakia during the previous studies. Common occurrence of the species in an area with substantially natural dynamics of wetland ecosystems represents a unique model system for unbiased comparisons of traits and genetic diversity between diploid and triploid individuals along with their dispersal capabilities on a landscape level. The main aim of of this thesis is to uncover the truth behind evolutionary processes that are taking place in the contact zone of diploid and triplioid cytotype of B. umbellatus in south-eastern Slovakia. Using flow cytometry and molecular methods (microsatellites) we will test whether this is a primary or secondary contact zone and assess the level of genetic variability between individuals and populations of both cytotypes. Using the analysis od microsatellite loci we discovered that the contact zone of cytotype is of both primary and secondary character and gene flow between cytotypes was also detected. These results were supported by high production and...

[Abstract] This thesis is aimed at better understanding of cytotype co-existence in mixed- ploidy populations with an emphasis on a microevolutionary processes behind it. Our past knowledge was based on a few thoroughly investigated model taxa like Chamerion angustifolium and Heuchera grossulariifolia, but some generalizations seem to be premature in the light of new findings. A detailed research of other taxa included in the thesis showed that polyploid complexes can vary dramatically in their ability to cope with the co-existence of cytotypes in mixed-ploidy popu- lations. Whereas mixed-ploidy populations are virtually lacking in some species (an example being Vicia cracca, Paper III.), ploidy-heterogeneous populations are very common in others, maintained by free mating interactions and the absence of reproductive isolation among cytotypes (e.g. Pilosella echioides, Paper II.). The strenght and cumulative effect of various breeding barriers (both pre- or post- zygotic) govern the position of a particular multi-ploidy complex between these two extremes and co-determine the type of cytotype co-existence in its mixed- ploidy populations. Despite the fact that the number of studies revealing cytotype co-existence has been increasing rapidly, evolutionary background and consequences of such co-...

Although genomes are perhaps the single most important element of living systems, why they feature such striking variation and how this variation is maintained within and across natural populations remains unclear. One of the most common and important means by which genomic variation is generated is ploidy elevation. While polyploidy has been implicated in the remarkably successful radiations of angiosperms, teleost fish, and amphibians, the phenotypic consequences of changes in ploidy level are poorly understood, especially in animals. I use a large, multi-year common garden experiment to identify potential life history costs and benefits of polyploidy and asexual reproduction, a trait often associated with polyploidy, in Potamopyrgus antipodarum. This snail is well suited for studying ploidy variation and sex because diploid sexuals and triploid and tetraploid asexuals frequently coexist, allowing us to use comparisons of sexuals to asexuals and triploid to tetraploid asexuals to study both the effects of ploidy elevation and sex. I detected a strong negative correlation between growth rate and time to maturity and found that sexual P. antipodarum grew and matured significantly more slowly than the polyploid asexuals. Sexual P. antipodarum were also more likely to die before achieving reproductive maturity than their asexual counterparts. By contrast, there were no apparent life history differences between triploid and tetraploid asexuals, indicating that direct phenotypic benefits of ploidy elevation are unlikely to explain the relatively rapid growth and maturation of asexuals. My results suggest that ploidy elevation does not inevitably confer phenotypic consequences, that reproductive mode influences life history trait expression, and that sexual P. antipodarum persist in many natural populations in spite of substantial life history disadvantages.

Peculiarities of edible onions (Allium cepa L.) gynogenesis investigated in the dissertation work. Conditions for creation of dihaploid plants evaluated. Biological assumptions for preparation of methodical suggestions, that would enable improving of gynogenesis, determined. Stimulation effect of using TDZ and NAR in media on the formation of edible onion embryogenic tissue has been revealed for the first time. Characteristic higher frequency of gynogenesis of unfertilized flower of edible onion isolated from the flower stems soaked in 2,4-D solution has been evaluated. By using experimental method it has been established that 3 day length gynogenesis induction period, using media containing 2,4-D and BA, is enough to induce gynogenesis of edible onion. High heterogeneity of edible onion variety population ‘Lietuvos didieji’ has been demonstrated from the point of organogenesis in isolated unfertilized flower culture. It has been established that the efficiency of edible onion gynogenesis can be increased by decreasing plant donor growing temperature in the final phases of flower development and using of exogenous growth regulators TDZ and NAA in plant regeneration media as well as by flower stems soaking in 2,4-D solution for 14 days. Plants with characteristic high gynogenesis frequency have been determined according to evaluation of edible onion variety population ‘Lietuvos didieji’ organogenetic response. 10 homozygous lines of edible onion have been created. 2 lines with... [to full text]

The Master's thesis studied the Odontites vernus group in the Czech Republic. The group was presented by two different taxa based on the seasonal types and the ploidy levels: an early flowering tetraploid Odontites vernus subsp. vernus (2n = 4x = 40) and a late-flowering diploid Odontites vernus subsp. serotinus (2n = 2x = 18). Plants from 33 populations were sampled for measuring the ploidy level and 27 morphological characters for morphological analysis. Two ploidy levels were confirmed in the Czech Republic and a new late flowering tetraploid taxon (2n = 4x = 40) was found. The three taxa were separated from each other based on the seasonal variation, ploidy level, morphology and ecology.