Nejvíce citovaný článek - PubMed ID 7743889
Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry
Phytic acid is the main storage of phosphate in grains of staple crops. As phytic acid is hardly digestible for non-ruminants microbial phytases are used to supplement animal feed to enhance phosphate digestibility. A fungal phytase gene was introduced into barley with the aim of enhancing phosphate digestibility. Transgenic homozygous barley over-expressing fungal phytase phyA showed a 3.3fold increase in mature grain phytase activity. Field trials at two locations in the Czech Republic were conducted in a five-year experiment to test transgene stability and activity under field conditions. Increased phytase activity gradually decreased over the generations showing the most significant drop in the initial years of field trials. Molecular analysis revealed methylation in the coding sequence of the phyA transgene, suggesting transcription gene silencing. On the other hand, herbicide resistance used for selection of transgenic plants was functional over all generations. The feasibility of crossing the transgene into the feeding cultivar Azit was demonstrated with subsequent stabilization of hybrid progeny through androgenesis. Our results indicate that the Azit genetic background tended to reduce phytase activity in mature grains of hybrids. Grain-specific over-expression of fungal phytase driven by an amylase promoter improved phosphate levels during germination. Unfortunately, a malting experiment revealed that phytase over-expression did not significantly improve malting parameters. In fact, the higher nitrogen content in unmalted grain negatively affected the quality of the malt produced from them.
- Klíčová slova
- Transgenic barley, androgenesis, field trials, hybridization, phytase,
- MeSH
- 6-fytasa * genetika metabolismus MeSH
- Aspergillus niger * enzymologie genetika MeSH
- fosfáty metabolismus MeSH
- fungální proteiny * genetika metabolismus MeSH
- geneticky modifikované rostliny * genetika metabolismus MeSH
- ječmen (rod) * genetika enzymologie metabolismus MeSH
- kyselina fytová metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 6-fytasa * MeSH
- fosfáty MeSH
- fungální proteiny * MeSH
- kyselina fytová MeSH
The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.
- Klíčová slova
- Vigna, DNA content, FISH, Karyotype evolution, Molecular cytogenetics, rDNA sites,
- MeSH
- chromozomy rostlin genetika MeSH
- DNA rostlinná genetika MeSH
- Fabaceae genetika MeSH
- fylogeneze MeSH
- genetická variace MeSH
- karyotyp MeSH
- ribozomální DNA genetika MeSH
- vigna * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA rostlinná MeSH
- ribozomální DNA MeSH
Exploring the fitness consequences of whole-genome multiplication (WGM) is essential for understanding the establishment of autopolyploids in diploid parental populations, but suitable model systems are rare. We examined the impact of WGM on reproductive traits in three major cytotypes (2x, 3x, 4x) of Pilosella rhodopea, a species with recurrent formation of neo-autopolyploids in mixed-ploidy populations. We found that diploids had normal female sporogenesis and gametogenesis, high fertility, and produced predominantly euploid seed progeny. By contrast, autopolyploids had highly disturbed developmental programs that resulted in significantly lower seed set and a high frequency of aneuploid progeny. All cytotypes, but particularly triploids, produced gametes of varying ploidy, including unreduced ones, that participated in frequent intercytotype mating. Noteworthy, the reduced investment in sexual reproduction in autopolyploids was compensated by increased production of axillary rosettes and the novel expression of two clonal traits: adventitious rosettes on roots (root-sprouting), and aposporous initial cells in ovules which, however, do not result in functional apomixis. The combination of increased vegetative clonal growth in autopolyploids and frequent intercytotype mating are key mechanisms involved in the formation and maintenance of the largest diploid-autopolyploid primary contact zone ever recorded in angiosperms.
- Klíčová slova
- aneuploidy, apospory, clonality, mixed-ploidy, root-sprouting, unreduced gametes,
- MeSH
- diploidie MeSH
- fertilita * MeSH
- ploidie * MeSH
- polyploidie MeSH
- rozmnožování MeSH
- semena rostlinná 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
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
BACKGROUND AND AIMS: Dioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY1Y2 sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes. METHODS: We flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons. KEY RESULTS: We identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes. CONCLUSIONS: The X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.
- Klíčová slova
- Rumex acetosa, genome dynamics, satellites, sex chromosomes, transposable elements,
- MeSH
- chromozomy rostlin MeSH
- hybridizace in situ fluorescenční MeSH
- molekulární evoluce MeSH
- pohlavní chromozomy MeSH
- retroelementy MeSH
- Rumex * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- retroelementy MeSH
Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.
- Klíčová slova
- androgenesis barley, double haploid, embryo culture, transformation,
- Publikační typ
- časopisecké články MeSH
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
BACKGROUND AND AIMS: Apomixis or asexual seed reproduction is a key evolutionary mechanism in certain angiosperms providing them with reproductive assurance and isolation. Nevertheless, the frequency of apomixis is largely unknown, especially in groups with autonomous apomixis such as the diploid-polyploid genus Hieracium. METHODS: Using flow cytometric analyses, we determined the ploidy level and reproductive pathways (sexual vs. apomictic) for 7616 seeds originating from 946 plants belonging to >50 taxa sampled at 130 sites across Europe. KEY RESULTS: Diploid seeds produced by diploids were formed exclusively by the sexual pathway after double fertilization of reduced embryo sacs. An absolute majority of tri- and tetraploid seeds (99.6 %) produced by tri- and tetrapolyploid taxa were formed by autonomous apomixis. Only 20 polyploid seeds (0.4 %) were formed sexually. These seeds, which originated on seven polyploid accessions of four taxa, were formed after fertilization of either unreduced embryo sacs through a so-called triploid bridge or reduced embryo sacs, and frequently resulted in progeny with an increased ploidy. In addition, the formation of seedlings with increased ploidy (4x and 6x) was found in two triploid plants. This is the first firm evidence on functional facultative apomixis in polyploid members of Hieracium sensu stricto (s.s.). CONCLUSIONS: The mode of reproduction in Hieracium s.s. is tightly associated with ploidy. While diploids produce seeds exclusively sexually, polyploids produce seeds by obligate or almost obligate apomixis. Strict apomixis can increase the reproductive assurance and this in turn can increase the colonization ability of apomicts. Nevertheless, our data clearly show that certain polyploid plants are still able to reproduce sexually and contribute to the formation of new cytotypes and genotypes. The finding of functional facultative apomicts is essential for future studies focused on evolution, inheritance and ecological significance of apomixis in this genus.
- MeSH
- DNA analýza MeSH
- falešně pozitivní reakce MeSH
- imunofenotypizace MeSH
- imunologické techniky * MeSH
- lidé MeSH
- proliferace buněk MeSH
- průtoková cytometrie MeSH
- řízení kvality MeSH
- RNA analýza MeSH
- separace buněk MeSH
- směrnice jako téma * MeSH
- software MeSH
- T-lymfocyty cytologie MeSH
- výzkumný projekt MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- DNA MeSH
- RNA MeSH
