Dark matter in plant cell nuclei – characterization of nuclear proteins.

Most of genetic information in eukaryotes is stored in cell nuclei, which are highly organized and dynamic organelles. Various proteins were described to play essential role in packing DNA, its replication and transcription in the nuclei of human and some model species. However, there is almost no information about the majority of these proteins in plants. This reminds dark matter in the universe, whose existence is generally accepted, but its nature remains obscure. In order to reveal the secrets of this component of plant cell nuclei, and to discover as yet uncharacterized plant nuclear proteins and their possible interactors, we will employ multidisciplinary approaches, including proteomics, flow cytometry and immunohistochemistry. In addition to identifying new proteins, we will perform localization analysis of some of them and follow changes in nuclear protein composition during cell cycle. The results will shed light on the nature and function of the vital component of plant cell nuclei.

Sex chromosome evolution - chromosome-specific genomics in genus Silene

In contrast to animals with mostly separated two sexes, a number of reproductive strategies coexist in higher plants. Surprisingly, the presence of well-established sex chromosomes in dioecious plants is rare. We intend to investigate structure and evolution of sex chromosomes in dioecious plant Silene latifolia. Since only limited sequence information on sex chromosomes is available in this species we focus on characterization of X chromosome as the fundamental step towards understanding the evolution of sex chromosomes. Our first goal is to construct X chromosome-specific BAC library using flow sorting approach. Subsequently, X chromosome specic physical map will be assembled and individual BACs will be sequenced. In parallel, massive sequencing using sorted X and Y chromosomes and sex chromosome-corresponding BAC clones in gynodioecious S. vulgaris will be carried out. Expression data for individual tissues, sexes and species will be generated based on RNA-seq approach. Comparative analyses of the data obtained will improve our understanding of processes forming sex chromosomes.

Evolution and function of complex plant genomes

The project aims at combining experience, skills and advanced methodologies of five research teams in order to accelerate scientific competitiveness of the Czech Republic in the filed of plant genomics. Since modern biology is increasingly driven by technology, the project will greatly benefit by tight integration of state-of-the-art methods and instruments provided by the participating teams. The proposed research will bring new knowledge on the structure, evolution and function of complex plant genomes, mainly focusing on (1) consequences of hybridization and polyploidization, (2) evolution and function of specialized chromoso- mes, (3) role of repetitive sequences in shaping plant genomes and their compartments, (4) evolutionary significance of epigenetic information, and (5) karyotype changes accompanying evolution and speciation. Although the project has no ambitions of immediate application outputs, its results will provide a basis for future applications of genomics tools in breeding improved cultivars of agricultural crops.

Physical map of wheat chromosome arm 7DS and its use to clone a Russian wheat aphid resistance gene

Map-based cloning in bread wheat is a daunting task due to enormous size (~17 Gbp) and polyploid nature of its genome. One of the strategies facilitating accomplishment of this goal is use of genomic resources created from smaller segments of the genome - chromosomes or chromosome arms. Here we propose assembling clones of a BAC library previously constructed from the short arm of wheat 7D chromosome into a physical map. The ordered clones will be sequenced by Illumina technology and assembled. The resulting BAC clone sequences will enable landing of the physical map on the chromosome. The integrated map as well as clone-by-clone sequence of the 7DS will signicantly speed up cloning of all genes from this part of the wheat genome. Within the project, we assume map-based cloning of a gene conferring resistance to one of the most devastating wheat pests, Russian wheat aphid (Diuraphis noxia) located on 7DS. Besides of elucidating molecular basis of the trait, the knowledge of the gene enables host-pathogen interaction studies and development of markers for wheat breeding.

Will orchids reshape our understanding of genome-wide processes? Solving the enigma of progressively partial endoreduplication

The project aims at investigating the phenomenon of progressively partial endoreduplication (PPE) that has recently been discovered in the genome of some orchids. Whereas both the generative and somatic polyploidy have been intensively researched, nothing is known about the patterns and processes behind the PPE. With the aid of advanced molecular cytogenetic techniques (flow cytometry and sorting, Illumina next-generation sequencing, FISH), we will address the incidence of PPE across the family Orchidaceae and search for correlations with phylogeny and species traits. Ludisia discolor, a taxon with a small genome size and ca 50% PPE, will serve as a model system to elucidate differences in sequence composition between normal (2C) and partially reduplicated genomes. To localize structural changes in the genome and assess their phylogenetic stability, repetitive elements showing different quantities will be mapped to interphase nuclei and somatic chromosomes of Ludisia and related species. The results will provide a new level of understanding of genome-wide processes in eukaryotes.

Genome interactions in interspecific hybrids xFestulolium

Polyploidy is a key force in the evolution of flowering plants. Allopolyploidy involves a merge of distanc genomes followed by whole-genome duplication. Recently formed allopolyploids retain duplicated copies of most genes on homoeologous chromosomes. However, it appears that the contribution of parentel genomes need not to be equal and that altered gene expression is common. Such a divergence is believed to develop in two phases. The first takes place immediately after the hybrid formation, the second is a gradual evolution of gene expression mediated by diversification of duplicated genes. Here we propose to sequence transcriptome of two grass species (Festuca pratensis and Lolium multiflorum). The sequences will be used to design a Nimblegene chip, which will be used to analyze gene expression in three successive generations of newly synthesized Festuca × Lolium hybrids. This will provide novel data on genome interactions and silencing of genes belonging to the parental genomes. Moreover, komparative genomic hybridization will be used to identify the loss of particular gene loci as a consequence of the merge of two distinct genomes.
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