The identification of causal genomic loci and their interactions underlying various traits in plants has been greatly aided by progress in understanding the organization of the nuclear genome. This provides clues to the responses of plants to environmental stimuli at the molecular level. Apart from other uses, these insights are needed to fully explore the potential of new breeding techniques that rely on genome editing. However, genome analysis and sequencing is not straightforward in the many agricultural crops and their wild relatives that possess large and complex genomes. Chromosome genomics streamlines this task by dissecting the genome to single chromosomes whose DNA is then used instead of nuclear DNA. This results in a massive and lossless reduction in DNA sample complexity, reduces the time and cost of the experiment, and simplifies data interpretation. Flow cytometric sorting of condensed mitotic chromosomes makes it possible to purify single chromosomes in large quantities, and as the DNA remains intact this process can be coupled successfully with many techniques in molecular biology and genomics. Since the first experiments with flow cytometric sorting in the late 1980s, numerous applications have been developed, and chromosome genomics has been having a significant impact in many areas of research, including the sequencing of complex genomes of important crops and gene cloning. This review discusses these applications, describes their contribution to advancements in plant genome analysis and gene cloning, and outlines future directions.
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.
- MeSH
- Alleles MeSH
- Cell Cycle drug effects genetics MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant metabolism MeSH
- Fluorescence MeSH
- Genome, Plant * MeSH
- Hydroxyurea pharmacology MeSH
- Karyotype MeSH
- Kinetics MeSH
- Plant Roots drug effects MeSH
- Polyploidy * MeSH
- Flow Cytometry methods MeSH
- Saccharum drug effects genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Jediná tři lysosomální střádavé onemocnění s dědičností vázanou na X chromosom a provázená významným srdečním postižením jsou mukopolysacharidóza typu II (MPSII, deficit iduronát-2-sulfatázy), Danonova (DD, deficit LAMP2) a Fabryho nemoc (FD, deficit ?-galaktosidázy A). V projektu navrhujeme založení a charakterizaci kolekce buněčných modelů na bázi indukovaných pluripotentních buněk od dříve diagnostikovaných pacientů a pacientek. Modely plánujeme následně využít pro testování nových terapeutických a diagnostických postupů stejně jako pro další studium patogeneze zmíněných onemocnění. Jedním z našich hlavních cílů je, v liniích DD a FD pacientek, vývoj průtokově cytometrické metody pro separaci diferencovaných buněk (především kardiomyocytů) dle parentálního stavu jejich X-inaktivace. Separované buňky s příznivou X-inaktivací mutované alely lze, bez dalších genetických manipulací, považovat za vhodné pro další testování autologní buněčné terapie. V případě DD hodláme testovat terapeutický vliv kandidátních malých molekul interferujících s autofagií nebo lysosomální exocytózou.; The only lysosomal storage disorders with X-linked inheritance are mucopolysaccharidosis type II (MPSII, iduronate-2-sulfatase deficit), Danon (DD, LAMP2 deficit) and Fabry (FD, ?-galactosidase deficit) diseases. All express cardiac pathology of either primary cardiomyocytic (DD, FD) or secondary (due valvular dysfunction - MPSII) origin. In the project, we propose to establish and characterize induced pluripotent stem (iPS) cell based models from previously diagnosed male and female patients. The models will be used for testing novel diagnostic and therapeutic approaches as well as for further pathogenesis studies. One of our main goals, in cultures collected from DD and FD female patients, is to develop a protocol for flow cytometric sorting of the differentiated cells (mainly cardiomyocytes) based on their parental X-chromosome inactivation status. Supposedly, the favorably inactivated clones can be consequently utilized for autologous cell therapy testing. In DD, cellular impacts of candidate small molecules interfering with autophagy and/or lysosomal exocytosis will be tested.
- MeSH
- Models, Biological MeSH
- Fabry Disease diagnosis therapy MeSH
- Glycogen Storage Disease Type IIb diagnosis therapy MeSH
- X Chromosome Inactivation MeSH
- Induced Pluripotent Stem Cells MeSH
- Cardiomyopathies MeSH
- Lysosomal Storage Diseases diagnosis therapy MeSH
- Mucopolysaccharidosis II diagnosis therapy MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- genetika, lékařská genetika
- vnitřní lékařství
- cytologie, klinická cytologie
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Fruit of Terminalia chebula Retz. (Combretaceae) has male contraceptive folk medicine reputation but its molecular aspect regarding hypotesticular activity is still in dark. The study focused the hypotesticular efficacy of the most potent fraction out of n-hexane, chloroform and ethyl acetate fractions of hydro-methanolic (3:2) extract of Terminalia chebula in connection with male herbal contraceptive development. Treatment with above fractions of Terminalia chebula showed a significant diminution in the activities of androgenic key enzymes (Δ5, 3β-HSD, 17β-HSD) and inhibition in serum testosterone level in compare to the control. Significant up regulation of testicular Bax gene and down regulation of Bcl-2 gene indicated the hypotesticular activity of these fractions. Flow-cytometric study focused a significant diminution in sperm viability and sperm mitochondrial status after the treatment with different fractions. Out of these, ethyl acetate fraction showed most promising hypotesticular effect without impairing any toxicity in general which highlighted that the fraction may contains antitesticular agent(s) in threshold levels compare to other fractions as it decreases spermiological, testicular genomic sensors and elevates sperm apoptotic sensors that may lead to male contraception.
- MeSH
- Spermatogenesis-Blocking Agents * chemistry isolation & purification MeSH
- Models, Animal MeSH
- Rats, Wistar MeSH
- Flow Cytometry MeSH
- Plant Extracts MeSH
- Spermatozoa drug effects MeSH
- Terminalia chemistry MeSH
- Testis enzymology chemistry drug effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
MutChromSeq is an approach for isolation of genes and DNA sequences controlling gene expression in plants with complex and polyploid genomes. It involves a lossless complexity reduction by flow cytometric chromosome sorting and shotgun sequencing DNA from isolated chromosomes. Comparison of sequences from wild-type parental chromosome with chromosomes from multiple independently derived mutants identifies causative mutations in a single candidate gene or a noncoding sequence. MutChromSeq does not rely on recombination-based genetic mapping and does not exclude any DNA sequence from being targeted.
- MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant genetics MeSH
- Genome, Plant MeSH
- Genomics methods MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Mutation * MeSH
- Polyploidy MeSH
- Flow Cytometry methods MeSH
- Gene Expression Regulation, Plant MeSH
- Plants genetics MeSH
- Sequence Analysis, DNA methods MeSH
- Publication type
- Journal Article MeSH
BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
- MeSH
- Benzophenanthridines analysis MeSH
- Cell Culture Techniques MeSH
- Cell Cycle physiology MeSH
- DNA analysis MeSH
- Fluorescent Dyes analysis MeSH
- Microscopy, Fluorescence methods MeSH
- Humans MeSH
- Flow Cytometry * methods MeSH
- Cell Separation methods MeSH
- Cell Survival MeSH
- Green Fluorescent Proteins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.
- MeSH
- Chromosomes, Plant genetics MeSH
- In Situ Hybridization MeSH
- Flow Cytometry MeSH
- Triticum genetics MeSH
- Publication type
- Journal Article MeSH
Nuclear genomes of many important plant species are tremendously complicated to map and sequence. The ability to isolate single chromosomes, which represent small units of nuclear genome, is priceless in many areas of plant research including cytogenetics, genomics, and proteomics. Flow cytometry is the only technique which can provide large quantities of pure chromosome fractions suitable for downstream applications including physical mapping, preparation of chromosome-specific BAC libraries, sequencing, and optical mapping. Here, we describe step-by-step procedure of preparation of liquid suspensions of intact mitotic metaphase chromosomes and their flow cytometric analysis and sorting.
Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.
