Recent methodological advances provided novel insights into the spatial genome organization in mammalian cell nuclei and its functional role. This progress contrasts with the limited knowledge on higher order chromatin folding in mitotic chromosomes. Our preliminary data on chromatin contact frequency at megabase resolution obtained in barley mitotic chromosomes suggest remarkable differences in mitotic chromosome organization between plants and mammals. We will use barley as a model to characterize chromatin folding throughout the cell cycle using chromosome conformation capture sequencing (Hi-C), super-resolution microscopy, fluorescence in situ hybridization and computational polymer modeling. The project will deliver an experimentally validated structural model of mitotic chromosome organization in barley, elucidate conformational changes occurring across the cell cycle and pinpoint differences in mitotic chromatin organization between plants and mammals.