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
Methods in molecular biology ; 91
x, 274 s.. : il.
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
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.
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.
Despite remarkable recent progress in the analysis of plant genome organization and chromosome structure, there is a need for methods enabling DNA sequences to be mapped by fluorescence in situ hybridization (FISH) at high spatial resolution. We sorted mitotic metaphase chromosomes of wheat by flow cytometry and observed the occurrence of hyperexpanded chromosomes among them. However, this phenomenon was not reproducible in subsequent experiments. An investigation into the procedures of flow cytometry revealed that the hyperexpansion of chromosomes became reproducible when the concentration of formaldehyde used in sample fixation was reduced. We conducted FISH analysis with 45S rDNA, 5S rDNA and wheat centromeric repeat sequences as probes on flow-sorted chromosomes and also on chromosomes from squash preparations. We measured the length of chromosomes 1B and 6B, identified by FISH. On average, the hyperexpanded 1B and 6B chromosomes were 7.26 and 7.53 times longer, respectively, than the same chromosomes from the squash preparations. The most stretched 1B and 6B chromosomes both exceeded 100 micrometers.
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.
- 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
Průtoková cytometrie našla uplatnění jako analytická i diagnostická metoda v mnoha oblastech praktické medicíny i v základním a aplikovaném biomedicínském výzkumu. S postupujícím technickým pokrokem se její možnosti stále rozšiřují a jednoduché dvojbarevné a trojbarevné průtokové cytometry jsou stále častěji nahrazovány vysoce citlivými polychromatickými analyzátory či vysokorychlostními průtokovými třídiči buněk, které se používají při multiparametrické charakterizaci a třídění dobře definovaných buněčných subpopulací, včetně prekurzorů a progenitorů s minimálním zastoupením, a buněk kmenových. Polychromatická FCM se stala nenahraditelnou v klinické medicíně a v současné době se zvažuje její využití i v oblasti polního zdravotnického systému. Zde se tato metoda jeví jako velmi perspektivní zejména z biodozimetrického hlediska, a to např. pro stanovení zpětného odečtu obdržené dávky ionizujícího záření. Článek podává přehled nejběžnějších aplikací používaných v klinickém výzkumu i praxi, s možným rozšířením do oblasti vojenské medicíny.
Flow cytometry has proved a useful analytical and therapeutic technique in many fields of practical medicine as well as in basic and applied biomedical research. With ongoing technological development its potential permanently increases and simple, two- and three-color flow cytometers are being replaced by highly sensitive polychromatic analyzers and high speed sorters that are used for multiparametric characterization and sorting of well-defined cell subpopulations including precursors, progenitors and stem cells. Polychromatic flow cytometry has thus become an essential tool in clinical medicine and its use in the battlefield medical system is under consideration. In military healthcare this technique appears quite promising namely for biodosimetric purposes, e.g. for retrospective determination of the received dose of ionizing radiation. The paper brings an overview of most frequent applications used in clinical research and practice with possible ramifications to military medicine.
- MeSH
- Chromosome Aberrations radiation effects MeSH
- Financing, Organized MeSH
- Hematology methods trends MeSH
- Immunophenotyping methods utilization MeSH
- Medical Oncology methods trends MeSH
- Humans MeSH
- Flow Cytometry methods instrumentation trends MeSH
- Radiobiology methods trends MeSH
- Radiometry methods trends utilization MeSH
- Transplantation methods utilization MeSH
- Military Medicine methods instrumentation trends MeSH
- Check Tag
- Humans MeSH
Hlavní stanovisko: Vyšetření spermiogramem neposkytuje dostatek informací o kvalitě spermií. Pomocí průtokové cytometrie je možné stanovit zastoupení apoptotických spermií v ejakulátu, fragmentaci jejich DNA a integritu akrozomu. Tyto testy umožňují diagnostikovat pacienty s rizikem nižší fertility, kteří by měli být vyšetřeni andrologem. Cíl: Přehled výsledků vyšetření kvality spermií pomocí průtokové cytometrie Soubor pacientů a metody: Vyšetření ejakulátu bylo provedeno u 800 mužů (ve věku 21-66 let) léčených v Centrech asistované reprodukce GENNET. Soubor zahrnoval 366 normozoospermiků a 434 pacientů s patologickým spermiogramem. Pomocí průtokové cytometrie byl u všech pacientů stanoven počet živých a apoptotických spermií, u 213 pacientů byl stanoven počet spermií s nízkou integritou akrozomu a u 65 pacientů byl stanoven počet spermií s fragmentací DNA metodou TUNEL. Pacienti byli rozděleni do skupiny ApoHigh s vysokým zastoupením apoptotických spermií (>50 % apoptotických spermií) a ApoLow s nízkým zastoupením apoptotických spermií (<50 % apoptotických spermií). Výsledky: U normozoospermiků bylo zjištěno 16,9 % ApoHigh vzorků, u pacientů s patologickým spermiogramem 53,9 % ApoHigh vzorků. Čím více je apoptotických spermií v ejakulátu, tím vyšší je výskyt fragmentace DNA a nízké integrity akrozomu. Průměrná hodnota fragmentace DNA byla vyšší u vzorků ApoHigh než u ApoLow jak u normozoospermiků (24,0 % vs. 13,5 %), tak u pacientů s patologickým spermiogramem (33,3 % vs. 18,0 %). Vyšší průměrný počet spermií s nízkou integritou akrozomu byl zjištěn u ApoHigh vzorků než u ApoLow jak u normozoospermiků (40,2 % vs. 23,9 %), tak u pacientů s patologickým spermiogramem (53,1 % vs. 35,2 %). Závěr: Vyšší zastoupení apoptotických spermií v ejakulátu snižuje jeho kvalitu. Test apoptózy spermií, fragmentace DNA a stanovení integrity akrozomu doplňují informace získané ze spermiogramu a umožňují vytipovat rizikové subfertilní pacienty, pro které je vhodné podrobné andrologické vyšetření.
Semen analysis does not provide enough information about sperm quality. Flow cytometry tests allow us to analyse percentage of apoptotic sperm, DNA fragmentation and acrosome integrity. Sub‑fertile patients are better diagnosed using these tests. Objective: Overview of sperm quality data measured by flow cytometry. Material and method: 800 sub‑fertile men (aged 21–66 years) were treated at GENNET – assisted reproduction centres. 366 patients had normal results and 434 patients had pathological parameters of semen analysis. Flow cytometry analyses of apoptotic sperm were done in all patients. DNA fragmentation rate was measured by TUNEL assay in 65 samples. The acrosome integrity was measured in 213 samples. Two groups of patients were set according to the number of apoptotic sperm in the semen samples – ApoHigh group included samples with > 50 % of apoptotic sperm and ApoLow group included samples with < 50 % of apoptotic sperm. Results: 16.9 % of normal semen samples were ApoHigh as well as 53.9 % pathological semen. Higher average values of DNA fragmentation were detected in the ApoHigh group than in the ApoLow group both in normal semen (24.0 % vs. 13.5 %) and in pathologic semen too (33.3 % vs. 18.0 %). Higher average values of the acrosome integrity were detected in the ApoHigh group than in the ApoLow group both in normal semen (40.2 % vs. 23.9 %) and in the pathologic samples as well (53.1 % vs. 35.2 %). Conclusion: The presence of apoptotic sperm decreases the semen quality. The examination of apoptosis, DNA fragmentation and acrosome integrity together with microscopic semen analyses provide a better diagnostic tool how to identify sub‑fertile men, than simple semen analysis. These tests might be helpful for selection of patients for further uro‑andrological examination and treatment.
- MeSH
- Fertility * MeSH
- Humans MeSH
- Infertility, Male genetics prevention & control MeSH
- Flow Cytometry statistics & numerical data MeSH
- Spermatozoa abnormalities cytology pathology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Observational Study MeSH
