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
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
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
BACKGROUND: Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches. RESULTS: We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template. CONCLUSIONS: Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.
- Keywords
- Flow cytometric sorting, Hordeum vulgare, HvSUT1, Single chromosome amplification, Transgene localization, Triticum aestivum,
- Publication type
- Journal Article MeSH
Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.
- Keywords
- PLZF, fluorescence-activated cell sorting, germ stem cell, gonad, spermatogonia, sturgeon,
- Publication type
- Journal Article MeSH
Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.
- Keywords
- flow cytometry, immunophenotyping:, leukemia, pediatric, standardization,
- MeSH
- Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis MeSH
- Acute Disease MeSH
- Child MeSH
- Phenotype MeSH
- Immunophenotyping standards MeSH
- Consensus MeSH
- Humans MeSH
- Flow Cytometry standards MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
A new computational framework for FLow cytometric Analysis of Rare Events (FLARE) has been developed specifically for fast and automatic identification of rare cell populations in very large samples generated by platforms like multi-parametric flow cytometry. Using a hierarchical Bayesian model and information-sharing via parallel computation, FLARE rapidly explores the high-dimensional marker-space to detect highly rare populations that are consistent across multiple samples. Further it can focus within specified regions of interest in marker-space to detect subpopulations with desired precision.
- MeSH
- Automation, Laboratory methods MeSH
- Probability MeSH
- Flow Cytometry methods MeSH
- Models, Theoretical * MeSH
- Publication type
- Journal Article MeSH
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
- Names of Substances
- DNA, Plant MeSH
- Hydroxyurea MeSH
BACKGROUND AND AIMS: Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. METHODS: The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. KEY RESULTS: FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. CONCLUSIONS: The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.
- Keywords
- Ae. cylindrica, Ae. triuncialis, Aegilops markgrafii, COS, FISH, GISH, Poaceae, Triticum aestivum, conserved orthologous set markers, flow cytometric chromosome sorting, fluorescence in situ hybridization, genomic in situ hybridization, goatgrass, physical mapping, wheat,
- MeSH
- Chromosomes, Plant genetics MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Indoles MeSH
- Karyotype MeSH
- Karyotyping MeSH
- Conserved Sequence genetics MeSH
- Poaceae genetics MeSH
- Metaphase MeSH
- Flow Cytometry methods MeSH
- Triticum genetics MeSH
- Sequence Homology, Nucleic Acid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DAPI MeSH Browser
- Indoles 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
- 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
