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.
- Keywords
- Cell cycle synchronization, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Metaphase accumulation, Plants,
- MeSH
- Chromosomes, Plant * MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Flow Cytometry methods MeSH
- Plants genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.
- Keywords
- Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
- MeSH
- Chromosomes, Plant * MeSH
- Chromosomes * MeSH
- Cytogenetics MeSH
- Karyotyping MeSH
- Flow Cytometry methods MeSH
- Suspensions MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Suspensions 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.
- Keywords
- Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
- MeSH
- Chromosomes, Plant * genetics MeSH
- Genome, Plant MeSH
- Flow Cytometry methods MeSH
- Restriction Mapping MeSH
- Plants * genetics MeSH
- Sequence Analysis, DNA methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- Keywords
- 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
- Chromosomes, Plant metabolism MeSH
- DNA, Plant genetics MeSH
- In Situ Hybridization, Fluorescence MeSH
- Karyotyping MeSH
- Meristem cytology drug effects MeSH
- Metaphase drug effects MeSH
- Molecular Weight MeSH
- Nitrous Oxide pharmacology MeSH
- Proteomics MeSH
- Flow Cytometry methods MeSH
- Plants genetics MeSH
- Seeds drug effects MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
- Nitrous Oxide MeSH
The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant chemistry genetics MeSH
- Chromosomes ultrastructure MeSH
- Cytogenetics MeSH
- Cytological Techniques methods MeSH
- DNA, Plant genetics MeSH
- Physical Chromosome Mapping MeSH
- Genome MeSH
- Gene Library MeSH
- Flow Cytometry methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DNA, Plant MeSH
Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant * MeSH
- Cytogenetics MeSH
- DNA, Plant genetics MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Karyotyping MeSH
- Meristem cytology MeSH
- Flow Cytometry methods MeSH
- Plant Cells MeSH
- Plants genetics MeSH
- Seeds growth & development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
- MeSH
- Cell Nucleus genetics MeSH
- Interphase genetics MeSH
- Hordeum genetics MeSH
- Proteome genetics MeSH
- Proteomics methods MeSH
- Flow Cytometry methods MeSH
- Plant Proteins genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Proteome MeSH
- Plant Proteins MeSH
Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C approximately 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat-barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H-7H. Thus, the barley genome can be dissected into fractions representing only about 6-12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.
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
- Chromosomes, Plant ultrastructure MeSH
- Genome Size MeSH
- Microscopy, Fluorescence MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hordeum cytology genetics MeSH
- Germination genetics MeSH
- Metaphase genetics MeSH
- Polyploidy MeSH
- Flow Cytometry methods MeSH
- Triticum cytology genetics MeSH
- Sequence Analysis, DNA MeSH
- Seeds genetics MeSH
- Chromosomes, Artificial, Bacterial MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Secale cytology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Chromosomes, Plant genetics MeSH
- Physical Chromosome Mapping MeSH
- In Situ Hybridization, Fluorescence MeSH
- Karyotyping MeSH
- Microsatellite Repeats MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry methods MeSH
- Cell Separation methods MeSH
- Translocation, Genetic MeSH
- Secale genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
