Ribosomal RNA gene loci (rDNA) have been among extensively studied components of eukaryotic genomes. They were shown to be associated with nucleolus, the largest functional domain of the nucleus and the site of ribosomal biogenesis. Despite the biological importance, detailed information on molecular organization of rDNA regions is missing, especially in plants. This is mainly due to structure of rDNA loci, which are organized as long arrays of tandem repeats difficult to assemble and thus are heavily underrepresented in genome assemblies. In the project we will focus on studies of 45S rDNA loci in bread wheat and related cereals. To assemble rDNA regions, we will use a new approach using optical mapping and third-generation sequencing technology producing reads as long as 100 kb. We will focus on minor rDNA loci and elucidate their role in ribosomal DNA evolution in wheat and also perform comparative analyses of major and minor loci in wheat, barley and rye. The project will provide novel insights into organization of plant rDNA and its evolution in the tribe Triticeae.