Most cited article - PubMed ID 7495553
Primer-induced labeling of pea and field bean chromosomes in situ and in suspension
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.
- Keywords
- 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
- Cell Cycle MeSH
- Chromosomes, Plant * genetics MeSH
- Metaphase MeSH
- Flow Cytometry MeSH
- Plants * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.
- MeSH
- Chromosomes chemistry genetics MeSH
- Physical Chromosome Mapping methods MeSH
- Genome, Human MeSH
- Genomics methods MeSH
- Gene Library MeSH
- Karyotype MeSH
- Humans MeSH
- Chromosome Painting methods MeSH
- Mitosis MeSH
- Flow Cytometry methods MeSH
- Plants chemistry genetics MeSH
- Oligonucleotide Array Sequence Analysis methods MeSH
- Chromosome Structures chemistry genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The cereals are of enormous importance to mankind. Many of the major cereal species - specifically, wheat, barley, oat, rye, and maize - have large genomes. Early cytogenetics, genome analysis and genetic mapping in the cereals benefited greatly from their large chromosomes, and the allopolyploidy of wheat and oats that has allowed for the development of many precise cytogenetic stocks. In the genomics era, however, large genomes are disadvantageous. Sequencing large and complex genomes is expensive, and the assembly of genome sequence is hampered by a significant content of repetitive DNA and, in allopolyploids, by the presence of homoeologous genomes. Dissection of the genome into its component chromosomes and chromosome arms provides an elegant solution to these problems. In this review we illustrate how this can be achieved by flow cytometric sorting. We describe the development of methods for the preparation of intact chromosome suspensions from the major cereals, and their analysis and sorting using flow cytometry. We explain how difficulties in the discrimination of specific chromosomes and their arms can be overcome by exploiting extant cytogenetic stocks of polyploid wheat and oats, in particular chromosome deletion and alien addition lines. Finally, we discuss some of the applications of flow-sorted chromosomes, and present some examples demonstrating that a chromosome-based approach is advantageous for the analysis of the complex genomes of cereals, and that it can offer significant potential for the delivery of genome sequencing and gene cloning in these crops.
- MeSH
- Chromosomes, Plant genetics MeSH
- Cytogenetics MeSH
- Genomics methods MeSH
- Gene Library MeSH
- Edible Grain cytology genetics MeSH
- Flow Cytometry methods MeSH
- Sequence Analysis, DNA MeSH
- Chromosomes, Artificial, Bacterial genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.
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
- Chromosomes genetics MeSH
- DNA Primers MeSH
- Electrophoresis, Agar Gel MeSH
- Physical Chromosome Mapping MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hydroxyurea pharmacology MeSH
- Insecticides pharmacology MeSH
- Hordeum genetics MeSH
- Karyotyping MeSH
- Plant Roots genetics MeSH
- Metaphase MeSH
- Microsatellite Repeats genetics MeSH
- Mitosis genetics MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds pharmacology MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- amiprophos MeSH Browser
- DNA Primers MeSH
- Hydroxyurea MeSH
- Insecticides MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
