SOLMAZ KHOSRAVI (Iran) - Live colored imaging of chromatin by CRISPR-imaging
Chromatin has a dynamic structure and spatial positioning of genes have a crucial influence in cellular programing. Many high-tech methods have been established to help us to understand the behavior of biological components. However, all this methods have provided us just a glimpse of what is really going on in the living cells. Although, cell imaging techniques like fluorescent in situ hybridization (FISH) have also been attempted to provide more knowledge about chromatin structure, they do not have suitable resolution for detection of positional alterations of single genes in one hand, and application of fixation steps during this techniques in the other hand perturbs the chromatin structure. To overcome these obstacles and prepare an opportunity to study chromatin structure in its native environment we intend to use CRISPR-imaging.
Deactivated Cas9 (dCas9) which is produced by induction of two point mutations in RuvC1 and HNH nuclease domains, has the ability of recognizing its target region, though not capable of cutting it. Fused with appropriate fluorophores, CRISPR-imaging is a convenient method to have a real time observation of defined genomic regions in the chromatin structure in a more dynamic and quantitative way. However, obtaining of strong signals and reduction of background noise is still challenging in this method. Here, we will use different strategies to improve the efficiency of multi-color CRISPR-FISH imaging system of telomeric and centromeric regions of chromosome in different plant species.
DAIYAN LI (China) - Characterization of mitotic nondisjunction and identification of the B chromosome nondisjunction regulatory region in the goat grass Aegilops speltoides
B chromosomes (Bs) are supernumerary, dispensable, but often preferentially inherited in plants. Aegilops speltoides is an annual diploid Poaceae with a maximum number of eight additional Bs in addition to its inherent seven pairs of standard A chromosomes. In Ae. speltoides, it has shown that the nondisjunction of Bs occurs during the post-meiotic drive and Bs undergo specific elimination in the root tissue during embryogenesis. So far, it still remains to elucidate what and how determines the process of nondisjunction. Furthermore, it is also unknown whether a nondisjunction regulatory region exists in this species as has been reported for the B chromosome in rye. To investigate what and how that controls the nondisjunction of Bs, a set of histone variant-specific antibodies will be employed to immunolabel young embryos of +B Ae. speltoides that are undergoing B elimination for a comparison of the distribution of immunosignals along separated A chromatids and non-separated B chromatids. In the case of identifying the nondisjunction regulatory region, Ae. speltoides plants carrying a mutated B will be selected from a seed library derived from crosses between non-irradiated 0B plants and irradiated +B plants taking advantage of B chromosome specific sequence AesTR-183.
ALEVTINA RUBAN (Russia) - Analysis of a B chromosome undergoing tissue-type specific elimination during embryogenesis of Aegilops speltoides
Programmed elimination of specific DNA is part of the developmental program, associated with the generation of distinct germline and somatic genomes in some eukaryotes. Entire chromosomes as well as certain sequences may be subjected to elimination. In plants, supernumerary B-chromosomes (Bs) are associated with the process of programmed DNA elimination. B-chromosomes are known to be an optional addition to the standard chromosome set. They are dispensable and do not affect significantly on the host phenotype if present in low number. In Aegilops speltoides Bs demonstrate tissue-type-specific elimination during embryogenesis. As it was found, B-chromosome elimination occurs during embryonic root and stem apex differentiation and leads to complete loss of Bs from the root cells.
A key question in the B-chromosome elimination phenomenon is how they are selectively lost in certain tissue. We are assuming that the tissue-type specific elimination of Bs in Ae. speltoides is likely a result of a segregation defect, either by centromere inactivation or nondisjunction of sister chromatids. To test these assumptions we will trace the Bs during mitosis in well-formed B-positive embryos. In order to address whether the elimination of Bs in the root tissue is required for normal embryo development we wish to employ tissue culture to generate somatic embryos. For identification of B-chromosomes in the cells we will use FISH with newly identified B-specific repeat. Analysis of histone modifications possibly involved into elimination process will be performed using Immunocytochemistry.
Besides the B-chromosome elimination we are highly interested in their origin, evolution and molecular composition.