Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 88-93). Also available via the Internet.

BACKGROUND:Sugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n=80 chromosomes while S. spontaneum has 2n=40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80%S. officinaram and 5-20%S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome.RESULTS:A BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760-930 Mb, the library exhibits 5-6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs.CONCLUSION:The SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome.

No / Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.

Hematopoietic stem cell (HSC) development is accompanied by dynamic changes in the transcriptional program. How the corresponding transcriptional programs are related to the epigenetic mechanism is poorly understood. To fill this gap, we first profiled the transcriptomes and epigenomes using RNA-Seq and ChIP-Seq for five key developmental stages of HSC emergence in the mouse embryo. Using epigenetic markers, we identified novel 12,000~17,000 enhancers for each developmental stage. We applied a computational tool to link those enhancers to their target genes. Systematical analysis of enhancer-promoter (EP) pairs using network-based strategy reveals multiple novel key transcription factors for early specification of HSC in the mouse embryo. Second, we compared the 3D genome organization, epigenomes, and transcriptome of fetal and adult HSCs in the mouse. We found that higher-order genome structures are largely conserved between fetal and adult HSCs, including chromosomal compartments and topologically associating domains (TADs). However, chromatin interactions within TADs exhibit substantial differences. We found that promoters within 23% (242/1039) of TADs undergo interaction changes. Transcription factor motif analysis of HSC-specific enhancer-promoter loops suggests a role of KLF1 in mediating condition-specific enhancer looping and regulation of genes involved in cell cycle. Our result provides a comprehensive view of the differences in 3D genome organization, epigenome, and transcriptome between fetal and adult HSCs.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13566号 / 論医博第2293号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柊, 卓志, 教授 篠原, 隆司, 教授 後藤, 慎平 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

TAD-Grenzen sind genomische Regionen mit Isolatorpotenzial, die zwischen benachbarten Chromatindomänen liegen und deren Unterbrechung zu einer pathologischen Genexpression führen kann. Die meisten TAD-Grenzen werden durch das CTCF gebunden, ein Architekturprotein, das Chromatinschleifen bevorzugt zwischen distalen Paaren von CTCF-Bindungsstellen (CBS) mit einer konvergenten Motivausrichtung bildet. An TAD-Grenzen sind die CBS häufig geclustert, wobei die Motive eine divergente Ausrichtung aufweisen und Chromatinschleifen in Richtung der stromaufwärts und stromabwärts gelegenen Regionen projizieren. Wie die CTCF-Besetzung die Isolierung an TAD-Grenzen moduliert, ist immer noch nicht ganz klar. Hier habe ich die regulatorische Logik von CTCF-geclusterten TAD-Grenzen untersucht, indem ich genomweite Analysen und in vivo-Mausexperimente an der Epha4-Pax3-TAD-Grenze kombiniert habe. Analysen einzelner Deletionen zeigten einen deutlichen hierarchischen Beitrag von CBS zur Grenzfunktion. Im Gegensatz dazu zeigten kombinierte CBS-Deletionen ein gewisses Maß an funktioneller Redundanz und Kooperativität zwischen den Stellen. Diese Analysen zeigten auch, dass die abweichende Konfiguration der CBS, die immer wieder an TAD-Grenzen zu finden ist, für eine robuste Isolierung nicht unbedingt erforderlich ist. Genomweite Analysen haben gezeigt, dass es eine Untergruppe von CBS gibt, die unabhängig von der konvergenten Ausrichtung Chromatinschleifen bilden, wofür ich einen Mechanismus der "Schleifeninterferenz" vorschlage. Weitere Vergleiche ergaben, dass das Niveau der Genexpression von den Abständen zwischen Enhancer und Promoter im linearen Genom abhängen könnte. Durch die Quantifizierung der Isolierung der Grenzen, der Pax3-Fehlexpression und der Schwere der Gliedmaßenfehlbildungen konnte ich schließlich zeigen, dass die TAD-Grenzen die Genexpression und den Phänotyp quantitativ beeinflussen. / TAD boundaries are genomic regions with insulator potential located between adjacent chromatin domains, which disruption can cause pathological gene expression. Most TAD boundaries are bound by the CTCF, an architectural protein that forms chromatin loops preferentially between distal pairs of CTCF binding sites (CBSs) with a convergent motif orientation. At TAD boundaries, CBSs are frequently clustered, with motifs displaying a divergent orientation and projecting chromatin loops towards up and downstream regions. How CTCF occupancy modulates insulation at TAD boundaries still remains elusive. Here, I dissected the regulatory logic of CTCF-clustered TAD boundaries by combining genome-wide analysis and in vivo mouse experiments at the Epha4-Pax3 TAD boundary. Analyses of individual deletions revealed a distinct hierarchical contribution of CBS to boundary function. In contrast, combined CBSs deletions revealed a certain degree of functional redundancy and cooperativity between sites. These analyses also demonstrated that the divergent configuration of CBSs, recurrently found at TAD boundaries, is not strictly required for robust insulation. Genome-wide analysis highlighted the existence of a subset of CBSs that establish chromatin loops independently of the convergent orientation bias, for which I propose a mechanism of “loop interference”. This mechanism suggests that CBS forming a robust convergent loop can simultaneously form a non-convergent loop, by stalling Cohesin complexes extruded from both sides. Further comparisons revealed that gene expression levels might depend on enhancer-promoter distances in the linear genome. Finally, by quantifying boundary insulation, Pax3 misexpression and the severity of limb malformation, I demonstrate that TAD boundaries are quantitative modulators of gene expression and phenotypes. Overall, I highlight that TAD boundary composition and strength constitute a fundamental regulatory layer in developmental processes and disease.

No / The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.

Tesis por compendio / [EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CA] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / Compendio

This thesis focuses on the genomic study of symbionts of two different groups of hymenopterans: bees and ants. Both groups of insects have major ecological impact, and investigating their microbiomes increases our understanding of their health, diversity and evolution. The study of the bee gut microbiome, including members of Lactobacillus and Bifidobacterium, revealed genomic processes related to the adaptation to the gut environment, such as the expansion of genes for carbohydrate metabolism and the acquisition of genes for interaction with the host. A broader genomic study of these genera demonstrated that some lineages evolve under strong and opposite substitution biases, leading to extreme GC content values. A comparison of codon usage patterns in these groups revealed ongoing shifts of optimal codons. In a separate study we analysed the genomes of several strains of Lactobacillus kunkeei, which inhabits the honey stomach of bees but is not found in their gut. We observed signatures of genome reduction and suggested candidate genes for host-interaction processes. We discovered a novel type of genome architecture where genes for metabolic functions are located in one half of the genome, whereas genes for information processes are located in the other half. This genome organization was also found in other Lactobacillus species, indicating that it was an ancestral feature that has since been retained. We suggest mechanisms and selective forces that may cause the observed organization, and describe processes leading to its loss in several lineages independently. We also studied the genome of a species of Rhizobiales bacteria found in ants. We discuss its metabolic capabilities and suggest scenarios for how it may affect the ants’ lifestyle. This genome contained a region with homology to the Bartonella gene transfer agent (GTA), which is a domesticated bacteriophage used to transfer bacterial DNA between cells. We propose that its unique behaviour as a specialist GTA, preferentially transferring host-interaction factors, originated from a generalist GTA that transferred random segments of chromosomal DNA. These bioinformatic analyses of previously uncharacterized bacterial lineages have increased our understanding of their physiology and evolution and provided answers to old and new questions in fundamental microbiology.

Les habitats sur le substrat de serpentine représentent un environnement hostile pour le développement des plantes. Ils sont caractérisés par un faible nombre d’espèces mais un haut niveau d’endémisme. Cette étude présente pour la première fois une série des données sur la taille du génome, du nombre chromosomique, du niveau de ploïdie, de l’affinité pour le substrat, du cycle de vie, du type et de la forme de croissance des serpentinophytes dans l’extrême nord-ouest de la zone de serpentine dans les Balkans. Les 308 taxons des plantes étudiées comprennent appartenant à 213 genres, dont la taille du génome est donnée pour la première fois pour 28 genres et 99 espèces. En utilisant les critères de Leitch, plus de la moitié des taxons (55.63%) appartiennent au groupe des très petits génomes, 22.19% aux petits, 18.75% aux moyens, 3.13% aux grands, et seulement 0.31% aux très grands génomes. Concernant l'affinité au substrat, la majorité d’espèces (171) sont indifférentes ou des serpentinophytes facultatives (103). Selon le type de cycle de vie, ~ 4% des espèces sont annuelles, 88.31% pérennes, dont 57% possèdent de très petits génomes. Les hémicryptophytes représentent une forme de vie dominante (48.38%), tandis que les phanérophytes représentent 17%, les chaméphytes 15%, les thérophyte 9% et les géophytes 9%. Il est évident que le stress hydrique, les températures élevées et la présence de métaux lourds dans les habitats sur la serpentine jouent une haute pression sélective et favorisent des espèces pérennes à très petits génomes.Le Narcissus poeticus (Amaryllidaceae), serpentinophyte facultative, est l’ancêtre des narcisses cultivés. C'est la première étude de N. poeticus et de sa rhizosphère dans les populations naturelles. Il montre une tolérance au pH du sol qui varie du 4.64 à 7.85. Les concentrations totales de nickel, de cobalt et de magnésium sont plus élevées dans les sols sur serpentine que dans ceux sur calcaires. Narcissus poeticus est caractérisé par une plus grande accumulation de manganèse, de nickel et de magnésium dans ses parties aériennes. Le cobalt, par contre, a une concentration totale uniforme dans toutes les parties de la plante. Une autre caractéristique inhabituelle de N. poeticus est son plus grand rapport molaire Ca/Mg dans les parties souterraines, probablement dû à sa forme de vie (géophytes) et une dormance estivale. Il est évident que, même si N. poeticus accumule certaines quantités de métaux lourds estimés (Mn, Ni, Co, Fe), il n'est pas pour autant un hyperaccumulateur.Une partie importante de ce travail concerne la variabilité de la structure chromosomique, la taille du génome, le niveau de ploïdie et la présence de chromosomes B dans 13 populations naturelles de N. poeticus poussant sur différents substrats géologiques et dans différentes conditions environnementales. La technique de la cytométrie en flux a été utilisée pour estimer la taille du génome, l’hybridation in situ fluorescente (FISH) pour la cartographie physique de l'ADNr, le fluorochrome banding pour l'organisation de l’hétérochromatine et la coloration au nitrate d’argent pour estimer l'activité des gènes ribosomiques. L’organisation des gènes ribosomiques et l’existence des triploïdes naturels ont été rapportés ici pour la première fois. Présence des individus portant de chromosomes B (dans 9 populations sur 13) et de translocations chromosomiques a été détectée. Un système particulier de chromosomes B présente trois différents morphotypes. Le submétacentrique type, le plus fréquent, possède quatre paternes différents dans l’organisation de l’hétérochromatine et de l'ADNr. La coloration à l’AgNO3 a montré que le nombre de nucléoles formés augmente en présence des chromosomes B portant des gènes ribosomiques, dont l’activité est ainsi prouvée. Les résultats obtenus démontrent que N. poeticus possède un génome dynamique avec la quantité d’ADN variable en raison de la présence de polyploïdie, de chromosomes B et de réarrangements chromosomiques. Il semble que les modifications observées reflètent la réponse du génome à différentes conditions environnementales où les individus portant les chromosomes B pourraient avoir des avantages sélectifs. / Habitats on serpentine substrate present a hostile environment for the plants development. They are characterized by a small number of species, but high levels of endemism. This study shows for the first time a series of data on genome size, chromosome number, ploidy level, the affinity to the substrate, the life cycle, the type and form of growth in the extreme northwest region of serpentine area in the Balkans. The sample includes 308 taxa belonging to 213 genera, with new values recorded for 28 genera and 99 species. Using Leitch’s criteria, more than half of estimated taxa (55.63%) belong to the group of very small genomes, 22.19% small, 18.75% intermediary, 3.13% large and only 0.31% to very large genomes. Regarding the affinity to the substrate, the majority of species (171) were indifferent or facultative serpentinophytes (103). Concerning the life cycle, ~ 4% of species are annuals and 88.31% perennials, and 57% had very small genomes. Hemicryptophytes represent a dominant life form (48.38%), phanerophytes 17%, 1chamaephytes5%, therophytes 9% and geophytes 9%. It is clear that the water stress, high temperatures and presence of heavy metals in serpentine habitats have the high selective pressure and favor perennial species with very small genome.The Narcissus poeticus (Amaryllidaceae), facultative serpentinophyte, is the ancestor of cultivated daffodils. This is the first study of N. poeticus and its rhizosphere in natural populations. It shows tolerance to soil pH ranging from 4.64 to 7.85. Serpentine soils have total concentrations of nickel, cobalt and magnesium highest, compared with calcareous soils. Narcissus poeticus is characterized by the greater accumulation of manganese, nickel and magnesium in the aerial parts of plant. Against the cobalt has a uniform total concentration in all parts of the plant. Another unusual feature of N. poeticus is the highest molar ratio Ca / Mg in the underground parts, probably du to his life form (geophytes) and summer dormancy. It is obvious that although N. poeticus accumulate certain amounts of estimated heavy metals (Mn, Ni, Co, Fe), it does not a hyperaccumulator.An important part of this work concerns the variability of the chromosome structure, genome size, the ploidy level and the presence of B chromosomes in 13 natural populations growing on different soils and under different environmental conditions. The technique of flow cytometry was used to estimate the genome size, fluorescent in situ hybridization (FISH) for the physical mapping of rDNA, the fluorochrome banding for the organization of heterochromatin and silver staining to estimate the activity of ribosomal genes. Organization of ribosomal genes and natural triploids have been reported here for the first time. Presence of individuals carrying B chromosomes (in 9 / 13 populations) and chromosomal translocations were detected. A particular system of B chromosomes presents three different morphotypes. The most common submetacentric type shows four different patterns in the organization of heterochromatin and rDNA. The AgNO3 staining showed that the number of nucleoli formed increases in the presence of B chromosomes carrying ribosomal genes, which proved their activity. The obtained results show that N. poeticus has a dynamic genome with the variable amount of DNA due to the presence of polyploidy, B chromosomes and chromosomal rearrangements. It seems that the observed changes reflect the response of the genome to different environmental conditions in which individuals carrying B chromosomes may have some selective advantages / Habitati na serpentinskim substratima predstavljaju nepovoljne uvjete za razvoj biljaka. Karakteriziraju se sa malim brojem vrsta, ali prisustvom velikog broja endema. U ovoj studiji se po prvi put prezentira serija podataka o veličini genoma serpentinofita, njihovom hromosomskom broju, nivou ploidije, sklonosti ka supstratu, tipu životnog ciklusa i životne forme na krajnjem sjeverozapadnom dijelu serpentinskog područja Balkanskog poluostrva. Uzorak je obuhvatao 308 svojti iz 213 rodova, sa novim vrijednostima za 28 rodova i 99 vrsta. Prema Leitch-evim kriterijima, više od polovine analiziranih svojti (55.63%) pripadale su grupi vrlo malih genoma, 22.19% malim, 18.75% srednjim, 3.13% velikim i samo 0.31% vrlo velikim genomima. U odnosu na sklonost ka supstratu, glavnina vrsta (171) su bile indiferentne ili fakultativne serpentinofite (103). U zavisnosti od životnog ciklusa, ~ 4% vrsta su bile jednogodišnje, a 88.31% višegodišnje, od kojih je 57% imalo vrlo male genome. Hemikriptofite su predstavljale dominantnu životnu formu (48.38%), koju slijede fanerofite (17%), hamefite (15%), terofite (9%) i geofite (9%). Iz dobivenih rezultata proizilazi da vodni stres, visoke temperature i prisustvo teških metala u serpentinskim habitatima imaju visok selektivni pritisak i favoriziraju višegodišnje vrste sa vrlo malim genomom.Narcissus poeticus (Amaryllidaceae), fakultativna serpentinofita, je predak kultiviranih narcisa. Ovo je prva studija o N. poeticus i njegovoj rizosferi u prirodnim populacijama. Ova vrsta pokazuje toleranciju na promjene pH vrijednosti u dijapazonu od 4.64 do 7.85. Totalne koncentracije nikla, kobalta i magnezija u serpenitnskim tlima su bile veće nego u krečnjačkim. Narcissus poeticus se karakterizirao većom akumulacijom mangana, nikla i magnezija u nadzemnim dijelovima biljke. Suprotno, kobalt je imao skoro istu totalnu koncentraciju u svim dijelovima biljke. Druga neuobičajena karakteristika N. poeticus je najveći iskazani molarni odnos Ca/Mg u podzemnim dijelovima, vjerovatno zbog njegove životne forme (geofita) i ljetne dormancije. Očito je da iako N. poeticus akumulira određene količine istraživanih teških metala (Mn, Ni, Co, Fe) on se ne moze smatrati nije njihovim hiperakumulatorom.Važan dio ove studije se odnosi na varijabilnost hromosomske strukture, veličine genoma, nivoa ploidije i prisustva B-hromosoma u 13 prirodnih populacija N. poeticus koje rastu na različitim geološkim supstratima i pod različitim okolišnim uslovima. Korištena je tehnika protočne citometrije za određivanje veličine genoma, fluorescentna in situ hibridizacija (FISH) za fizicko mapiranje rDNK, fluorohrom banding za organizaciju heterohromatina i bojenje srebrenim nitratom za utvrđivanje aktivnosti ribozomalnih gena. Organizacija ribosomalnih gena i prisustvo prirodnih triploida su u ovoj studiji saopćeni po prvi put. Uočeno je prisustvo individua koje nose B-hromosome (u 9 od 13 populacija) i hromosomske translokacije. Poseban sistem B-hromosoma je predstavljen sa tri različita morfotipa. Najčešći submetacentrični tip pokazuje četiri različita obrasca u organizaciji heterohromatina i rDNK. Bojenje s AgNO3 je pokazalo da se formirani broj nukleolusa povećava u prisustvu B-hromosoma koji nose ribosomalne gene, što potvrđuje njihovu aktivnost. Dobiveni rezultati pokazuju da N. poeticus ima dinamičan genom sa različitom količinom DNK usljed prisustva poliploidije, B-hromosoma i hromosomskih rearanžmana. Uočene promjene najvjerovatnije odražavaju odgovor genoma na različite okolišne uslove u kojima individue koje posjeduju B-hromosome imaju izvjesnu selektivnu prednost.