The combination of a flow cytometric seed screen and genotyping of each single seed offers a cost-effective approach to detecting complex reproductive pathways in flowering plants. Reproduction may be seen as one of the driving forces of evolution. Flow cytometric seed screen and genotyping of parents and progeny are commonly employed techniques to discern various modes of reproduction in flowering plants. Nevertheless, both methods possess limitations constraining their individual capacity to investigate reproductive modes thoroughly. We implemented both methods in a novel manner to analyse reproduction pathways using a carefully selected material of parental individuals and their seed progeny. The significant advantage of this approach lies in its ability to apply both methods to a single seed. The introduced methodology provides valuable insights into discerning the levels of apomixis, sexuality, and selfing in complex Rubus taxa. The results may be explained by the occurrence of automixis in Rubus, which warrants further investigation. The approach showcased its effectiveness in a different apomictic system, specifically in Taraxacum. Our study presents a comprehensive methodological approach for determining the mode of reproduction where flow cytometry loses its potential. It provides a reliable and cost-effective method with significant potential in biosystematics, population genetics, and crop breeding.

• Klíčová slova
• Rubus, Taraxacum, Apomixis, Automixis, FCSS, SSR-seq,
• MeSH
• apomixie * genetika   fyziologie   MeSH
• genotyp MeSH
• průtoková cytometrie * MeSH
• rozmnožování MeSH
• semena rostlinná * genetika   fyziologie   MeSH
• Taraxacum fyziologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

• Klíčová slova
• Best practices, DAPI, DNA-ploidy level, Desiccated tissues, Flow cytometry, Genome size, Plant nuclei isolation, Plant tissues, Propidium iodide, Seeds,
• MeSH
• buněčné jádro * genetika   chemie   MeSH
• délka genomu MeSH
• DNA rostlinná genetika   analýza   MeSH
• genom rostlinný MeSH
• ploidie MeSH
• průtoková cytometrie metody   MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

• MeSH
• cukry metabolismus   MeSH
• cytokininy * metabolismus   MeSH
• ječmen (rod) * genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sacharosa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cukry MeSH
• cytokininy * MeSH
• rostlinné proteiny MeSH
• sacharosa MeSH

Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

• Klíčová slova
• CENPC-k, cenh3-4, centromere, haploid induction, kinetochore null 2, temperature stress,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• centromera genetika   MeSH
• haploidie MeSH
• kinetochory MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AND AIMS: It is unclear how widespread polyploidy is throughout the largest holocentric plant family - the Cyperaceae. Because of the prevalence of chromosomal fusions and fissions, which affect chromosome number but not genome size, it can be impossible to distinguish if individual plants are polyploids in holocentric lineages based on chromosome count data alone. Furthermore, it is unclear how differences in genome size and ploidy levels relate to environmental correlates within holocentric lineages, such as the Cyperaceae. METHODS: We focus our analyses on tribe Schoeneae, and more specifically the southern African clade of Schoenus. We examine broad-scale patterns of genome size evolution in tribe Schoeneae and focus more intensely on determining the prevalence of polyploidy across the southern African Schoenus by inferring ploidy level with the program ChromEvol, as well as interpreting chromosome number and genome size data. We further investigate whether there are relationships between genome size/ploidy level and environmental variables across the nutrient-poor and summer-arid Cape biodiversity hotspot. KEY RESULTS: Our results show a large increase in genome size, but not chromosome number, within Schoenus compared to other species in tribe Schoeneae. Across Schoenus, there is a positive relationship between chromosome number and genome size, and our results suggest that polyploidy is a relatively common process throughout the southern African Schoenus. At the regional scale of the Cape, we show that polyploids are more often associated with drier locations that have more variation in precipitation between dry and wet months, but these results are sensitive to the classification of ploidy level. CONCLUSIONS: Polyploidy is relatively common in the southern African Schoenus, where a positive relationship is observed between chromosome number and genome size. Thus, there may be a high incidence of polyploidy in holocentric plants, whose cell division properties differ from monocentrics.

• Klíčová slova
• Schoenus, Aneuploidy, Cape Floristic Region, Cyperaceae, Schoeneae, chromosome fission, chromosome fusion, climate, genome size, holocentric chromosomes, polyploidy, soil chemistry,
• MeSH
• biodiverzita MeSH
• chromozomy rostlin MeSH
• fylogeneze MeSH
• genom rostlinný MeSH
• ploidie MeSH
• polyploidie MeSH
• šáchorovité * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AND AIMS: While variation in genome size and chromosome numbers and their consequences are often investigated in plants, the biological relevance of variation in chromosome size remains poorly known. Here, we examine genome and mean chromosome size in the cyperid clade (families Cyperaceae, Juncaceae and Thurniaceae), which is the largest vascular plant lineage with predominantly holocentric chromosomes. METHODS: We measured genome size in 436 species of cyperids using flow cytometry, and augment these data with previously published datasets. We then separately compared genome and mean chromosome sizes (2C/2n) amongst the major lineages of cyperids and analysed how these two genomic traits are associated with various environmental factors using phylogenetically informed methods. KEY RESULTS: We show that cyperids have the smallest mean chromosome sizes recorded in seed plants, with a large divergence between the smallest and largest values. We found that cyperid species with smaller chromosomes have larger geographical distributions and that there is a strong inverse association between mean chromosome size and number across this lineage. CONCLUSIONS: The distinct patterns in genome size and mean chromosome size across the cyperids might be explained by holokinetic drive. The numerous small chromosomes might function to increase genetic diversity in this lineage where crossovers are limited during meiosis.

• Klíčová slova
• Chromosome number, Cyperaceae, Juncaceae, Thurniaceae, chromosome size, distribution range size, genome size, holocentric chromosomes, holokinetic drive,
• MeSH
• chromozomy rostlin * genetika   MeSH
• délka genomu MeSH
• fylogeneze MeSH
• genom rostlinný genetika   MeSH
• molekulární evoluce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1 /G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

• Klíčová slova
• C-value, GC content, best practices, flow cytometry, genome size, plant sciences, plant standard species, standardization,
• MeSH
• DNA rostlinná genetika   MeSH
• genom rostlinný * MeSH
• ploidie * MeSH
• průtoková cytometrie metody   MeSH
• referenční standardy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA rostlinná MeSH

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

• Klíčová slova
• allopolyploidy, genome size, genomic shock, marsh orchids, transposable elements,
• MeSH
• diploidie MeSH
• genom rostlinný MeSH
• lidé MeSH
• mokřady MeSH
• Orchidaceae * genetika   MeSH
• polyploidie MeSH
• sourozenci * MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transpozibilní elementy DNA MeSH

Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and other aspects of tick cellular processes. Tick cell cultures are often continuously cultivated, as freezing can affect their viability. However, the long-term cultivation of tick cells can influence their genome stability. In the present study, we investigated karyotype and genome size of tick cell lines. Though 16S rDNA sequencing showed the similarity between Ixodes spp. cell lines at different passages, their karyotypes differed from 2n = 28 chromosomes for parental Ixodes spp. ticks, and both increase and decrease in chromosome numbers were observed. For example, the highly passaged Ixodes scapularis cell line ISE18 and Ixodes ricinus cell lines IRE/CTVM19 and IRE/CTVM20 had modal chromosome numbers 48, 23 and 48, respectively. Also, the Ornithodoros moubata cell line OME/CTVM22 had the modal chromosome number 33 instead of 2n = 20 chromosomes for Ornithodoros spp. ticks. All studied tick cell lines had a larger genome size in comparison to the genomes of the parental ticks. Thus, highly passaged tick cell lines can be used for research purposes, but possible differences in encoded genetic information and downstream cellular processes, between different cell populations, should be taken into account.

• MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• Ixodidae genetika   MeSH
• karyotyp MeSH
• klíšťata genetika   růst a vývoj   MeSH
• Ornithodoros genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.

• MeSH
• biomasa MeSH
• chlorofyl metabolismus   MeSH
• chromozomy rostlin genetika   MeSH
• diploidie MeSH
• dřevo genetika   fyziologie   MeSH
• duplikace chromozomů MeSH
• fenotyp MeSH
• fotosyntéza genetika   fyziologie   MeSH
• genom rostlinný genetika   MeSH
• genotyp MeSH
• karotenoidy metabolismus   MeSH
• konfokální mikroskopie MeSH
• kořeny rostlin genetika   fyziologie   MeSH
• kůra rostlin genetika   fyziologie   MeSH
• listy rostlin genetika   fyziologie   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Salix genetika   fyziologie   MeSH
• stonky rostlin genetika   fyziologie   MeSH
• tetraploidie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• karotenoidy MeSH
• regulátory růstu rostlin MeSH