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Analysis of Plant Genomes Using Flow Cytometry
 

Analysis of Plant Genomes Using Flow Cytometry

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Principles of flow cytometry,Application in plants,Flow cytometry of plant genomes,Analysis and sorting cell nuclei

Principles of flow cytometry,Application in plants,Flow cytometry of plant genomes,Analysis and sorting cell nuclei

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    Analysis of Plant Genomes Using Flow Cytometry Analysis of Plant Genomes Using Flow Cytometry Presentation Transcript

    • Analysis of Plant Genomes Using Flow Cytometry Jaroslav Doležel Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic
    • Our location IEB, Olomouc Czech Republichttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Our weather
    • Our research• Analysis of plant genome structure and evolution - Constitution and evolution of hybrid genomes of Festuca x Lolium hybrids (Festuloliums) - Development and application of Chromosome Genomics to analyze complex genomes of wheat, barley and rye - Evolution of Musa genome at chromosomal and molecular level http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • New facility for plant genomicshttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Musa Genome Resources Centrehttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Outline Principles of flow cytometry Application in plants Flow cytometry of plant genomes Analysis and sorting cell nuclei http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • How does a flow cytometry work?Flow cytometry involves theanalysis of fluorescence and lightscatter properties of particles inflow, moving with respect to thepoint of measurement Excitation Detector light source The sample for flow cytometry should be a suspension of single particles (no clumps allowed) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • A brief history1934: a proposal for a „flow cytometer“ (Moldavan) Hydrodynamic focusing1947: the first working flow Sample Sheath fluid cytometer (Gucker)1953: hydrodynamic focusing (Crossland-Taylor) Hydrodynamic focusing1965: fluidic switch sorter zone (Kamentsky)1965: electrostatic cell sorter Light beam (Fulwyler)1969: fluorescence measurement http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Flow cytometer and sorter Drop-Charging Signal Fluorescence Sheath Fluid Sample detectors Filter Beam Splitter Vibration Transducer Filter Flow Chamber Collecting Lens for Fluorescent Light Light Detector Obscuration Laser Focusing Lens Bar Collecting Lens for Forward-Scattered Light Positively Charged Negatively Charged Electronics Deflection Plate Deflection Plate Console Left Collector Right Collector Wastehttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • And the real thing …BD FACSVantage (two lasers, 8 parameters) Close-uphttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • What can a flow cytometry do? Measurements of: – Light scatter • Particle size • Surface, internal cell structure – Fluorescence detection: in multiple wavelength bands • Total intensity (integral) • Maximum intensity • Polarization • Lifetime – With labeling reagents, provides information about: • Amount of: DNA, RNA, protein, surface molecules,… • Environment within a cell or membrane http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Some cytometers are very sophisticatedHigh-speed flow cytometer and sorter (MoFlo, Cytomation) • System pressure: up to 100 psi • Drop drive: up to 200 kHz • Sort rate: up to 70,000 cells / sec Sorting rare cells (hematopoietic stem cells, fetal cells, circulating dendritic cells) Large-scale sorting (chromosome purification, separation of X- and Y- chromosome bearing sperm) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • And some specialized and compact OptoFlow MICROCYTE Partec CYFLOWhttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Flow cytometry in plants Analysis and sorting of: • Microspores • Protoplasts • Cell nuclei • Chromosomes • Chloroplasts http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Flow cytometry of plant cell nuclei Applications: • Relative DNA content • DNA content in absolute units (genome size) • Nuclear DNA base content (AT/GC ratio) • Gene expression (nuclear-targeted GFP) • Nuclei purification (proteins, DNA, RNA) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Estimation of DNA content – the stone age DNS-Bestimmung an Keimwurzeln von Vicia faba L. mit Hilfe der Impulscytophotometrie Friedrich Otto Heller Institut für Landwirtschaftliche Botanik der Universität Bonn Vorgetragen auf der Botaniker-Tagung in Hannover an 21. September 1972 Ber. Deutsch. Bot. Ges. 86:437-441, 1973• Suspension of intact nuclei prepared by lysis of protoplasts obtained after enzymatic digestion of root tips• DNA stained with ethidium bromidehttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Breakthrough in 1983Rapid Flow Cytometric Analysis of the Cell Cycle in IntactPlant TissuesDavid W. Galbraith, Kristi R. Harkins,Joyce M. Maddox, Nicola M. Ayres,Dharam P. Sharma, Ebrahim FiroozabadyScience 220: 1049-1051, 1983• Suspensions of intact nuclei prepared by chopping small amounts of fresh plant tissues with a sharp razor blade• Nuclei stained in the crude homogenate with mithramycin http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Estimation of relative nuclear DNA contentNuclei isolation Flow cytometric analysis ofbuffer + DNA relative fluorescence intensity ofstain nuclei in suspension 20 mg of fresh leaf tissue Peak representing G1 nuclei with 2C DNA content Isolation of nuclei Peak representing by chopping G2 nuclei with 4C DNA content Removal of large debris by filtration Relative DNA content http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Galbraith et al., Science 220: 1049, 1983
    • Fluorescent dyes for DNAFluorochrome Primary Binding Mode Wavelength (nm)* Excitation EmissionEthidium bromide** Intercalation 530 605Propidium iodide** Intercalation 540 615Hoechst 33258 AT-binding 365 465Hoechst 33342 AT-binding 360 460DAPI AT-binding 365 450DIPI AT-binding 365 450Chromomycin A3 GC-binding 445 570Mihtramycin GC-binding 445 575Olivomycin GC-binding 440 560* Dye-DNA complex**Binds also to double stranded RNA! http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • DNA content and cell cycle 2C 2C - 4C S G1 DNA content G2 2C 4C M 4C DNA content 4Chttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Analysis of nuclear DNA content Distribution of nuclear DNA content in a population of asynchronously growing cells: G1 (2C) Number of nucleiNumber of nuclei G2 (4C) S Nuclear DNA content Nuclear DNA content Ideal distribution as it would The distribution as it is actually be measured in a perfect measured, broadened system. because of imperfections in the staining and measurement http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html procedure.
    • An easy method for ploidy screening? 5 μm  Metaphase spreads are difficult to prepare  Chromosomes are very smallhttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Ploidy screening in Musa using flow cytometry 500 500 500 2C Known ploidy 3C Triploid (3x) Tetraploid (4x)Number of nuclei 400 400 400 (diploid, 2x) 4C 300 300 300 200 200 200 100 100 100 4C 6C 8C 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Relative nuclear DNA content (channel number) Advantages:  Convenient and rapid (>100 samples per working day)  Does not require dividing (mitotic) cells  Non-destructive (only milligram amounts of plant tissues are needed) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Biol. Plant. 36: 351, 1994
    • Ploidy manipulations  An integrated system for production of polyploids has been developed and applied in banana and cassava  The protocol combines in vitro induction of polyploidy and ploidy screening using flow cytometry  The advantage of the protocol is the production of solid (non-mixoploid) polyploidshttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Van Duren et al., Euphytica 88: 25, 1996
    • Identification of mixoploids Standard (2x) Mixoploid (2x + 4x) Mixoploid (4x + >5x) 500 500 500 2C 2C 4CNumber of nuclei 400 400 4C 400 300 300 300 200 200 200 >5C 100 100 100 4C 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Relative nuclear DNA content (channel number)  Plant body consists of three histological layers (L1, L2 and L3), which may differ in ploidy (= chimerism, mixoploidy)  Chromosome counting in roots (only L3 layer) cannot be used for reliable identification of mixoploid individuals  Flow cytometry allows rapid and reliable detection of mixoploidy http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Dissociation of mixoploids A1A A1A 3x 1 3x A1 A1A 2 3x 3x + 6x A1B A1B 3x + 6x 1 3x + 6x A A1B 2 3x 3x + 6x A2A A2A Mixoploid 3x + 6x 1 Shoot-tips treated selected 3x + 6x A2A2 with colchicine shoot A2 3x A2B1 3x + 6x A2B 6x 6x A2B2 6x M1V0 M1V1 M1V2 M1V3 M1V4http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Roux et al., PCTOC 66: 189, 2001
    • Germplasm characterization 500 400 Pisang Kluai Tiparot A Pisang Balonkawe B Mas Mas 300 200 3x, NOT 4x 3x, NOT 4x Number of nuclei 100 0 400 Pisang Pisang Jambe C (Kluai) Ngoen D Mas Pisang 300 Mas 200 3x, NOT 4x 4x, NOT 3x 100 0 0 100 200 300 400 0 100 200 300 400 500 Relative nuclear DNA contenthttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Horry et al., Infomusa 7: 5, 1998
    • Characterization of Musa germplasm (ploidy)Flow cytometry was used to verify the classification of Musa germplasmheld at the INIBAP Transit Centre (KU Leuven, Belgium) Ploidy analysis of 1150 out of 1175 accessions Confirmed (83.3%) Determined for Mixoploidy the first time (0.79%) (7.04%) Mixed ploidy Other ploidy (1.22%) (7.65%) ITC, KU Leuven http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doleželová et al., Infomusa 14: 34, 2005
    • Population biology Ploidy screening of large populations (cytotype distribution, hybrid zones, …) 2x Empetrum 2x + 3x + 4x 4x 3xDistribution of Empetrum cytotypes in the Giant Mountains(Czech Republic) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Identification of hybrids F1 hybrids may be conveniently detected based on intermediate DNA content L. multiflorum (2n = 14) F. arundinacea (2n = 42) G1 G1 CRBC CRBC X G2 F1 hybrid CRBC G1 G2 http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Aneuploidy 120 A E A E THE USE OF EUPLOID PLANT OF 90 THE SAME SPECIES AS AN INTERNAL STANDARD 60 CV = 1.2% CV = 1.0% Discrimination is possible when the coefficient of variation of G1 peaks is 30 lower than half of the difference in DNA content (2.4% in this example) 0 0 100 200 300 40 0 0 100 200 300 400 500 RELATIVE NUCLEAR DNA CONTENT 120 G1 Peak Ratio 1 G1 Peak Ratio 2 THE USE OF A DIFFERENT SPECIES 90 AS AN INTERNAL STANDARD E S A S 60 Relative difference in DNA content (D): G1 Peak Ratio 2 - G1 Peak Ratio 1 30 D= * 100 [%] G1 Peak Ratio 1 0 0 100 200 300 400 0 100 200 300 400 500 RELATIVE NUCLEAR DNA CONTENTE = euploid, A = aneuploid, S = standardhttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Aneuploidy in MusaTriploid (2n = 3x = 33) 300 3x CRBC 250 Number of nuclei 200 Peak DI CV% 3x 0.79 1.58 150 CRBC 1.00 1.74 100 50 0 1 21 41 61 81 101 121 141 161 181 201 221 241 Relative DNA content 300 CRBC 250 3x-1 Number of nuclei 200 Peak DI CV% 150 3x-1 0.76 0.99 CRBC 1.00 1.25 100 50 2n = 33 - 1 0 1 21 41 61 81 101 121 141 161 181 201 221 241 Relative DNA content 300 3x-2 CRBC 250 Number of nuclei 200 Peak DI CV% 150 3x-2 0.74 1.02 CRBC 1.00 1.26 100 50 0 2n = 33 - 2 1 21 41 61 81 101 121 141 161 Relative DNA content 181 201 221 241http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Roux et al., Plant Cell Rep. 21: 483, 2003
    • Reproduction mode (FCSS)C-values of unreplicated embryo and endosperm nuclei depend onwhether the female and/or male gametes were reduced or unreduced,and whether the embryo and/or endosperm developed autonomouslyor after fertilization: a: antipodals; c: central cell with two polar nuclei; e: egg apparatus with egg cell and two synergids. http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Matzk et al., Plant J. 21: 97, 2000
    • Cell cycle G1 G1 = 47.4% S = 30.5% G2 = 22.1% G2 S Relative DNA contentDistribution of DNA content of nuclei isolated from field bean meristemroot tip cells. A non-parametric curve-fitting method was used forhistogram deconvolution for cell cycle phases.http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Endoreduplication (polysomaty) Flow cytometry allows to analyse the ENDOREDUPLICATION degree of endopolyploiy and the frequency of endopolyploid cells M 1000 2C Mammillaria san angelensis G2 800 (parenchym) Number of nuclei ER G1 600 S 400 8C 4C 8C 16C 4C 200 2C 32C 0 G1 S G2 G1 S G2 0 50 100 150 200 250 Nuclear DNA contenthttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Palomino et al., Plant Sci. 19: 191, 1999
    • Flow cytometry of plant cell nuclei Applications: • Relative DNA content • DNA content in absolute units (genome size) • Nuclear DNA base content (AT/GC ratio) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • The size of nuclear genome H. sapiens 12000065 16400 2700 1200 4400 7800 Nuclear genome Musa acuminata (591 - 615) size (Mb) Musa balbisiana (534 - 540)http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • The method SAMPLE STANDARD Musa G1 Glycine G1 nuclei nuclei 5 mg of G. max20 mg of Musa leaf (2C = 2.50 pg DNA) Number of nucleitissue leaf tissueSimultaneousisolation and Nuclei isolationstaining of buffer + propidiumnuclei iodide Relative nuclear DNA content Ratio of G1 peak positions Glycine Removal of large to Musa is 1.984 => 2C nuclear debris by filtration DNA content of M. acuminata errans is 2.50 / 1.984 = 1.26 pg DNA (or 608 Mbp / 1C*) *) 1pg DNA = 0.978 Mbp http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Biol. Plant. 36: 351, 1994
    • Germplasm characterization for genome size Accession name Section Genome size Musa genomes (Mbp/1C)* Calcutta 4 Eumusa 627 differ by size: Galeo 626 - A ~ 630 Mbp Pisang Mas 635 - B ~ 580 Mbp M. acuminata ssp. banksii 646 Guyod 647 - S ~ 700 Mbp M. balbisiana type Cameroun 578 - T ~ 730 Mbp Honduras 579 - C ~ 790 Mbp M. schizocarpa 704 M. laterita Rhodochlamys 624*) 1pg DNA = 0.978 Mbp M. velutina 635 M. mannii 649 Kluai Bou 609 M. ornata 664 M. beccarii Callimusa 798 M. peekelii ssp. peekelii Australimusa 791Bartoš et al., Cytogenet. M. textilis 734Genome Res. 109: 50, 2005 M. maclay type Hung Si 755 Kawaputa 766 http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Ensete gilletii Related genus 619
    • DNA base contentHistograms of relative nuclear DNA content of maize and humanleukocytes obtained using fluorescent dyes with different DNA basepreferences (FR = ratio of DNA peaks - maize / leukocytes) Propidium iodide DAPI Mithramycin 1000 leukocytes maize leukocytes maize Number of nuclei maize 800 leukocytes 600 400 FR = 0.817 FR = 0.601 FR = 1.083 200 0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 250 Relative nuclear DNA content (channel number)Due to different AT/GC ratio of human and maize, peak ratios are differentfor each DNA fluorochrome http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Physiol. Plant. 85: 625, 1992
    • AcknowledgementsDavid Galbraith (Tucson)Jan Suda (Prague)Fritz Matzk (Gatersleben)Nicolas Roux (Montpellier)Pietro Pifanelli (Genoa)Rony Swennen (Leuven)Jean-Pierre Horry (Montpellier) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • The firstbookon plantflowcytometry
    • Purification of cell nuclei • Musa balbisiana Intact cells Nuclei Cellular debris cv. Pisang Klutuk Wulung • Genome size: 530 Mbp • Scientific interest: BSV, B genome structure Nuclei isolation G1 nuclei Nuclei sorting Sort window Debris G2 nuclei Relative DNA contenthttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Purification of cell nucleiBAC library from Library screening with a cp probe Musa balbisiana PKW Standard procedure Flow sorting- Number of clones: 36,864- Average insert size: 135 kb- Genome equivalents: 9x- Clones with cp DNA: 3%- Clones with mt DNA: 0.004% 8.27% 0.09%The use of flow-sorted nuclei avoids problems with secondarymetabolites and eliminates cytoplasmic DNA contamination. IsolatedDNA of high quality and ideal for cloning. http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Gene expression Complex interactions of many genes Gene expression patterns specific for particular tissues RNA isolation from heterogeneous organs: - Difficult interpretation Solution - Isolation of particular cell types • Microdissection • Cell sorting using flow cytometry http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
    • Gene expression in root of Arabidopsis thaliana Simultaneous analysis of GFP (FL1) a DAPI (FL4) (A) Wild-type plant (B) - (F) Transgenic plants expressing nuclear GFP regulated by: (B) p35S (C) pRPL16B (D) pSHR (E) pSCR (F) pSultr2-1 http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html