The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Edible banana cultivars are diploid, triploid, or tetraploid hybrids, which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid Musa balbisiana. The participation of two other wild diploid species Musa schizocarpa and Musa textilis was also indicated by molecular studies. The fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far, and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. Fluorescence in situ hybridization (FISH) with chromosome-arm-specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure, discriminating individual accessions. These results permitted the identification of putative progenitors of cultivated clones and clarified the genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in the selection of appropriate parents for cross hybridization.

Summary: Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F₂ generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F₂ generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently eliminated. Genome sequence analysis revealed a number of non‐synonymous mutations between copies of the kinetochore genes from Festuca and Lolium genomes. Furthermore, we found that outer kinetochore proteins NDC80 and NNF1 were exclusively expressed from the Lolium allele. We hypothesize that silencing of Festuca alleles results in improper attachment of Festuca chromosomes to karyokinetic spindle and subsequently their gradual elimination.

Yam (Dioscorea spp.) is a monocotyledonous herbaceous vine plant grown in the tropics and subtropics. It is a multi-species plant with varied intra- and interspecific ploidy levels. Of the 600 species, 11 are cultivated supporting the livelihood of over 300 million people. The paucity of information on ploidy and the genomic constitution is a significant challenge to the crop’s genetic improvement through crossbreeding. The objective of this study was to investigate the ploidy levels of 236 accessions across six cultivated and two wild species using chromosome counting, flow cytometry and genotyping-based ploidy determination methods. Results obtained from chromosome counting and genotyping-based ploidy determination were in agreement. In majority of the accessions, chromosome counting and flow cytometry were congruent, allowing future rapid screening of ploidy levels using flow cytometry. Among cultivated accessions, 168 (71%) were diploid, 50 (21%) were triploid, and 12 (5%) were tetraploid. Two wild species included in the study were diploids. Resolution of ploidy level in yams offers opportunities for implementing successful breeding programmes through intra- and interspecific hybridization.

Summary: The B chromosomes exhibit diverse behaviour compared with conventional genetic models. The capacity of the B chromosome either to accumulate or to be eliminated in a tissue‐specific manner is dependent on biological processes related to aberrant cell division(s), but here yet remains compatible with normal development. We studied B chromosome elimination in Sorghum purpureosericeum embryos through cryo‐sections and demonstrated the B chromosome instability during plant growth using flow cytometry, molecular markers and fluorescent in situ hybridization techniques. Consequently, using B chromosome‐specific probes we revealed the non‐Mendelian inheritance of B chromosomes in developing pollen. We disclosed that the occurrence of the B chromosome is specific to certain tissues or organs. The distribution pattern is mainly caused by an extensive elimination that functions primarily during embryo development and persists throughout plant development. Furthermore, we described that B chromosome accumulation can occur either by nondisjunction at first pollen mitosis (PMI) or the initiation of extra nuclear division(s) during pollen development. Our study demonstrates the existence of a not‐yet‐fully described B chromosome drive process, which is likely under the control of the B chromosome.

Despite the recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains limited. In this study, DNA replication timing and interphase chromosome positioning was analyzed in seven Poaceae species differing in genome size. A multidisciplinary approach combining newly replicated DNA labelling by EdU, nuclei sorting by flow cytometry, three-dimensional immuno-FISH, and confocal microscopy revealed similar replication timing order for telomeres and centromeres as well as for euchromatin and heterochromatin in all seven species. The Rabl configuration of chromosomes that lay parallel to each other and their centromeres and telomeres are localized at opposite nuclear poles, was observed in wheat, oat, rye and barley with large genomes, as well as in Brachypodium with a small genome. On the other hand, chromosomes of rice with a small genome and maize with relatively large genome did not assume proper Rabl configuration. In all species, the interphase chromosome positioning inferred from the location of centromeres and telomeres was stable throughout the interphase. These observations extend earlier studies indicating a more complex relation between genome size and interphase chromosome positioning, which is controlled by factors currently not known. Highlight: Telomere and centromere replication timing and interphase chromosome positioning in seven grass species differing in genome size indicates a more complex relation between genome size and the chromosome positioning.