The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

• MeSH
• chromozomy rostlin genetika   MeSH
• Festuca genetika   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) genetika   MeSH
• karyotypizace metody   MeSH
• mapování chromozomů MeSH
• molekulární sekvence - údaje MeSH
• pořadí genů MeSH
• reprodukovatelnost výsledků MeSH
• rýže (rod) MeSH
• sekvenční analýza DNA metody   MeSH
• Sorghum genetika   MeSH
• Southernův blotting MeSH
• syntenie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.

• MeSH
• chromozomy rostlin ultrastruktura   MeSH
• délka genomu MeSH
• fluorescenční mikroskopie MeSH
• genom rostlinný * MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) cytologie   genetika   MeSH
• klíčení genetika   MeSH
• metafáze genetika   MeSH
• polyploidie MeSH
• průtoková cytometrie metody   MeSH
• pšenice cytologie   genetika   MeSH
• sekvenční analýza DNA MeSH
• semena rostlinná genetika   MeSH
• umělé bakteriální chromozomy MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• žito cytologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• Cell cycle synchronization, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Metaphase accumulation, Plants,
• MeSH
• chromozomy rostlin * MeSH
• hybridizace in situ fluorescenční metody   MeSH
• průtoková cytometrie metody   MeSH
• rostliny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin * genetika   MeSH
• metafáze MeSH
• průtoková cytometrie MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.

• Klíčová slova
• cell cycle synchronization, chromosome genomics, chromosome isolation, chromosome sorting, chromosome-specific DNA libraries, flow cytometry, genome sequencing, high-molecular-weight DNA, physical genome mapping, plant chromosomes,
• MeSH
• chromozomy rostlin metabolismus   MeSH
• DNA rostlinná genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• meristém cytologie   účinky léků   MeSH
• metafáze účinky léků   MeSH
• molekulová hmotnost MeSH
• oxid dusný farmakologie   MeSH
• proteomika MeSH
• průtoková cytometrie metody   MeSH
• rostliny genetika   MeSH
• semena rostlinná účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• oxid dusný MeSH

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

• Klíčová slova
• Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
• MeSH
• chromozomy rostlin * genetika   MeSH
• genom rostlinný MeSH
• průtoková cytometrie metody   MeSH
• restrikční mapování MeSH
• rostliny * genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.

• MeSH
• chromozomy genetika   MeSH
• DNA primery MeSH
• elektroforéza v agarovém gelu MeSH
• fyzikální mapování chromozomů MeSH
• hybridizace in situ fluorescenční MeSH
• hydroxymočovina farmakologie   MeSH
• insekticidy farmakologie   MeSH
• ječmen (rod) genetika   MeSH
• karyotypizace MeSH
• kořeny rostlin genetika   MeSH
• metafáze MeSH
• mikrosatelitní repetice genetika   MeSH
• mitóza genetika   MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny farmakologie   MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amiprophos MeSH Prohlížeč
• DNA primery MeSH
• hydroxymočovina MeSH
• insekticidy MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH

A protocol is described for production of micrograms of DNA from single copies of flow-sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome-derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single-chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome-specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole-genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.

• Klíčová slova
• Triticum aestivum L. (bread wheat), chromosome sorting, flow cytometry, next-generation sequencing, sequence assembly, single-chromosome genomics, whole-genome amplification,
• MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná chemie   genetika   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• kontigové mapování metody   MeSH
• kořeny rostlin genetika   MeSH
• průtoková cytometrie MeSH
• pšenice genetika   MeSH
• reprodukovatelnost výsledků MeSH
• rostlinné geny genetika   MeSH
• sekvenční analýza DNA MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.

• MeSH
• chromozomy rostlin genetika   MeSH
• fyzikální mapování chromozomů MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• mikrosatelitní repetice MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie metody   MeSH
• separace buněk metody   MeSH
• translokace genetická MeSH
• žito genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Haynaldia villosa (H. villosa) has been recognized as a species potentially useful for wheat improvement. The availability of its genomic sequences will boost its research and application. RESULTS: In this work, the short arm of H. villosa chromosome 4V (4VS) was sorted by flow cytometry and sequenced using Illumina platform. About 170.6 Mb assembled sequences were obtained. Further analysis showed that repetitive elements accounted for about 64.6% of 4VS, while the coding fraction, which is corresponding to 1977 annotated genes, represented 1.5% of the arm. The syntenic regions of the 4VS were searched and identified on wheat group 4 chromosomes 4AL, 4BS, 4DS, Brachypodium chromosomes 1 and 4, rice chromosomes 3 and 11, and sorghum chromosomes 1, 5 and 8. Based on genome-zipper analysis, a virtual gene order comprising 735 gene loci on 4VS genome was built by referring to the Brachypodium genome, which was relatively consistent with the scaffold order determined for Ae. tauschii chromosome 4D. The homologous alleles of several cloned genes on wheat group 4 chromosomes including Rht-1 gene were identified. CONCLUSIONS: The sequences provided valuable information for mapping and positional-cloning genes located on 4VS, such as the wheat yellow mosaic virus resistance gene Wss1. The work on 4VS provided detailed insights into the genome of H. villosa, and may also serve as a model for sequencing the remaining parts of H. villosa genome.

• Klíčová slova
• Chromosome arm 4VS, Flow sorting, Genome zipper, Haynaldia villosa, Scaffold,
• MeSH
• chromozomy rostlin genetika   MeSH
• druhová specificita MeSH
• genomika MeSH
• lipnicovité genetika   MeSH
• mapování chromozomů MeSH
• pořadí genů genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• sekvenční analýza DNA * MeSH
• Publikační typ
• časopisecké články MeSH