Compare the spatial distribution and chromatin composition of standard A chromosomes (As) and supernumerary B chromosomes (Bs) of rye. To determine if supernumerary chromosomes have a preferential or nonrandom distribution, (Obj1) we will analyse the relative position of As and Bs in intact root nuclei of tissue sections labelled using chromosome painting and imaged using 3D super-resolution (SIM) and Imaris-based image analysis (S1). To determine whether B chromosomes have a different epigenetic setup than As, (Obj2) we will quantify the levels of three different histone modifications marking euchromatin and heterochromatin states on Bs versus As (SIM imaging) and the number of RNA Pol II molecules (PALM imaging). To determine the transcriptional status of As versus Bs, (Obj3) we will quantify the amount of nascent RNA from four A- and B-encoded paralogous genes using allele-specific 3D smRNA-FISH and single molecule imaging (PALM). This quantitative, cytogenetic work will complement ongoing molecular profiling of B versus A chromosomes’ epigenetic and transcriptional state.