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

1. Analysis of Plant Genomes Using
Flow Cytometry
Jaroslav Doležel
Laboratory of Molecular Cytogenetics and Cytometry,
Institute of Experimental Botany, Olomouc, Czech Republic

2. Our location
IEB, Olomouc
Czech Republic
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

3. Our weather

5. 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

6. New facility for plant genomics
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

7. Musa Genome Resources Centre
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

8. 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

9. How does a flow cytometry work?
Flow cytometry involves the
analysis of fluorescence and light
scatter properties of particles in
flow, moving with respect to the
point 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

10. A brief history
1934: a proposal for a „flow
cytometer“ (Moldavan) Hydrodynamic focusing
1947: the first working flow
Sample Sheath fluid
cytometer (Gucker)
1953: hydrodynamic focusing
(Crossland-Taylor)
Hydrodynamic
focusing
1965: fluidic switch sorter
zone
(Kamentsky)
1965: electrostatic cell sorter Light beam
(Fulwyler)
1969: fluorescence measurement
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

11. 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
Waste
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

12. And the real thing …
BD FACSVantage (two lasers, 8 parameters) Close-up
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

13. 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

14. Some cytometers are very sophisticated
High-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

15. And some specialized and compact
OptoFlow MICROCYTE Partec CYFLOW
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

16. Flow cytometry in plants
 Analysis and
sorting of:
• Microspores
• Protoplasts
• Cell nuclei
• Chromosomes
• Chloroplasts
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

17. 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

18. 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 bromide
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

19. Breakthrough in 1983
Rapid Flow Cytometric Analysis of the Cell Cycle in Intact
Plant Tissues
David W. Galbraith, Kristi R. Harkins,
Joyce M. Maddox, Nicola M. Ayres,
Dharam P. Sharma, Ebrahim Firoozabady
Science 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

20. Estimation of relative nuclear DNA content
Nuclei isolation Flow cytometric analysis of
buffer + DNA relative fluorescence intensity of
stain 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

21. Fluorescent dyes for DNA
Fluorochrome Primary Binding Mode Wavelength (nm)*
Excitation Emission
Ethidium bromide** Intercalation 530 605
Propidium iodide** Intercalation 540 615
Hoechst 33258 AT-binding 365 465
Hoechst 33342 AT-binding 360 460
DAPI AT-binding 365 450
DIPI AT-binding 365 450
Chromomycin A3 GC-binding 445 570
Mihtramycin GC-binding 445 575
Olivomycin GC-binding 440 560
* Dye-DNA complex
**Binds also to double stranded RNA!
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

22. DNA content and cell cycle
2C 2C -
4C
S
G1
DNA content
G2
2C 4C M 4C
DNA content
4C
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

23. Analysis of nuclear DNA content
Distribution of nuclear DNA content in a population of
asynchronously growing cells:
G1 (2C)
Number of nuclei
Number 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.

24. An easy method for ploidy screening?
5 μm
 Metaphase spreads
are difficult to prepare
 Chromosomes are
very small
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

25. 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

26. 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)
polyploids
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Van Duren et al., Euphytica 88: 25, 1996

27. Identification of mixoploids
Standard (2x) Mixoploid (2x + 4x) Mixoploid (4x + >5x)
500 500 500
2C 2C 4C
Number 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

28. 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 M1V4
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Roux et al., PCTOC 66: 189, 2001

29. 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 content
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Horry et al., Infomusa 7: 5, 1998

30. Characterization of Musa germplasm (ploidy)
Flow cytometry was used to verify the classification of Musa germplasm
held 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

31. Population biology
 Ploidy screening of large populations (cytotype distribution,
hybrid zones, …)
2x
Empetrum
2x + 3x + 4x 4x
3x
Distribution of Empetrum cytotypes in the Giant Mountains
(Czech Republic)
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

32. 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

33. 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 CONTENT
E = euploid, A = aneuploid, S = standard
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

34. Aneuploidy in Musa
Triploid (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
181 201 221 241
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Roux et al., Plant Cell Rep. 21: 483, 2003

35. Reproduction mode (FCSS)
C-values of unreplicated embryo and endosperm nuclei depend on
whether the female and/or male gametes were reduced or unreduced,
and whether the embryo and/or endosperm developed autonomously
or 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

36. Cell cycle
G1 G1 = 47.4%
S = 30.5%
G2 = 22.1%
G2
S
Relative DNA content
Distribution of DNA content of nuclei isolated from field bean meristem
root tip cells. A non-parametric curve-fitting method was used for
histogram deconvolution for cell cycle phases.
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

37. 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 content
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Palomino et al., Plant Sci. 19: 191, 1999

38. 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

39. The size of nuclear genome
H. sapiens
120000
65
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

40. The method
SAMPLE STANDARD Musa G1 Glycine G1
nuclei nuclei
5 mg of G. max
20 mg of Musa leaf (2C = 2.50 pg DNA)
Number of nuclei
tissue leaf tissue
Simultaneous
isolation and Nuclei isolation
staining of buffer + propidium
nuclei 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

41. 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 791
Bartoš et al., Cytogenet. M. textilis 734
Genome 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

42. DNA base content
Histograms of relative nuclear DNA content of maize and human
leukocytes obtained using fluorescent dyes with different DNA base
preferences (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 different
for each DNA fluorochrome
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Physiol. Plant. 85: 625, 1992

43. Acknowledgements
David 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

44. The first
book
on plant
flow
cytometry

46. 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 content
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

47. Purification of cell nuclei
BAC 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 secondary
metabolites and eliminates cytoplasmic DNA contamination. Isolated
DNA of high quality and ideal for cloning.
http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

48. 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

49. 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