Analysis of Plant Genomes Using Flow Cytometry

Laboratory of Molecular Cytogenetics and Cytometry,Institute of Experimental Botany, Olomouc, Czech Republic

Analysis of Plant Genomes UsingFlow Cytometry

Jaroslav Doležel

IEB, Olomouc

Czech Republic

Our location

http://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


New facility for plant genomics

Musa Genome Resources Centre


Principles of flow cytometry

Application in plants

Flow cytometry of plant genomes

Analysis and sorting cell nuclei

Outline


DetectorExcitation

light source

Flow cytometry involves the

analysis of fluorescence and light

scatter properties of particles in

flow, moving with respect to the

point of measurement

The sample for flow cytometry

should be a suspension of single

particles (no clumps allowed)

How does a flow cytometry work?


1934: a proposal for a „flow

cytometer“ (Moldavan)

1947: the first working flow

cytometer (Gucker)

1953: hydrodynamic focusing

(Crossland-Taylor)

1965: fluidic switch sorter

(Kamentsky)

1965: electrostatic cell sorter

(Fulwyler)

1969: fluorescence measurement

Hydrodynamic

focusing

zone

Sample Sheath fluid

Light beam

Hydrodynamic focusing

A brief history


Sheath Fluid Sample

Laser

Fluorescencedetectors

BeamSplitter

Drop-Charging Signal

Filter

Filter

Collecting Lens forFluorescent Light

LightDetector

Collecting Lens forForward-ScatteredLight

Vibration Transducer

Flow Chamber

Focusing Lens

Positively ChargedDeflection Plate

Negatively ChargedDeflection Plate

Waste

Right CollectorLeft Collector

ObscurationBar

ElectronicsConsole

Flow cytometer and sorter


BD FACSVantage (two lasers, 8 parameters) Close-up

And the real thing …


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

What can a flow cytometry do?


• 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)

Some cytometers are very sophisticated


High-speed flow cytometer and sorter (MoFlo, Cytomation)

OptoFlow MICROCYTE Partec CYFLOW

And some specialized and compact


Analysis and

sorting of:

• Microspores

• Protoplasts

• Cell nuclei

• Chromosomes

• Chloroplasts

Flow cytometry in plants


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)

Flow cytometry of plant cell nuclei


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


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


Flow cytometric analysis of

relative fluorescence intensity of

nuclei in suspension

Peak representing

G1 nuclei with 2C

DNA content

Peak representing

G2 nuclei with 4C

DNA content

Relative DNA content

Nuclei isolation

buffer + DNA

stain

20 mg of fresh

leaf tissue

Isolation of nuclei

by chopping

Removal of large

debris by filtration

Estimation of relative nuclear DNA content

http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Galbraith et al., Science 220: 1049, 1983

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!

Fluorescent dyes for DNA


G1

S

G2M

DNA content

DNA content and cell cycle


2C 4C

DNA content

2C

4C

2C -

4C

4C

The distribution as it is actually

measured, broadened

because of imperfections in

the staining and measurement

procedure.

G1 (2C)

G2 (4C)

S

Nuclear DNA content

Num

ber

of

nucle

i

Nuclear DNA contentN

um

ber

of

nucle

i

Analysis of nuclear DNA content

Distribution of nuclear DNA content in a population of

asynchronously growing cells:

Ideal distribution as it would

be measured in a perfect

system.


5 μm

Metaphase spreads

are difficult to prepare

Chromosomes are

very small

An easy method for ploidy screening?


0 50 100 150 200 250

Known ploidy

(diploid, 2x)2C

0

100

200

300

400

500

4C0

100

200

300

400

500

0 50 100 150 200 250

Tetraploid (4x)

4C

8C0

100

200

300

400

500

0 50 100 150 200 250

3C

6C

Triploid (3x)

Relative nuclear DNA content (channel number)

Nu

mb

er

of n

ucle

i

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)

Ploidy screening in Musa using flow cytometry

http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Biol. Plant. 36: 351, 1994


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

Ploidy manipulations

Van Duren et al., Euphytica 88: 25, 1996

0

100

200

300

400

500

0 50 100 150 200 250

Mixoploid (2x + 4x)

2C 4C

0

100

200

300

400

500

0 50 100 150 200 250

Mixoploid (4x + >5x)

4C

>5C

0 50 100 150 200 250

Standard (2x)

2C

0

100

200

300

400

500

4C


Nu

mb

er

of n

ucle

i

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

Identification of mixoploids


Shoot-tips treated

with colchicine

Mixoploid

selected

shoot

M1V1 M1V2 M1V3 M1V4

3x + 6x

3x + 6x

3x + 6x

3x + 6x

3x + 6x

3x + 6x

3x + 6x

3x

3x

3x

3x

3x

6x

6x

6x

M1V0

A1B

A

A1

A1A

A2B

2A

2B

1A

2A

2A

1B

2

A2A

1

A1B

1

A1A

2

A1A

1

A2

A2A

A2B


Dissociation of mixoploids

Roux et al., PCTOC 66: 189, 2001

0

100

200

300

400

500

0

100

200

300

400

0 100 200 300 400 0 100 200 300 400 500

Relative nuclear DNA content

Nu

mb

er

of

nu

cle

i

A B

C D

PisangMas

PisangMas

PisangMas

PisangMas

Kluai Tiparot

Pisang Jambe

Balonkawe

(Kluai) Ngoen

3x, NOT 4x 3x, NOT 4x

3x, NOT 4x 4x, NOT 3x

Germplasm characterization

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

ITC, KU Leuven

Characterization of Musa germplasm (ploidy)

Mixoploidy

(0.79%)

Other ploidy

(7.65%)

Determined for

the first time

(7.04%)

Confirmed

(83.3%)

Mixed ploidy

(1.22%)

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

http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doleželová et al., Infomusa 14: 34, 2005

4x

2x

3x

Empetrum

2x + 3x + 4x


Population biology

Distribution of Empetrum cytotypes in the Giant Mountains

(Czech Republic)

Ploidy screening of large populations (cytotype distribution,

hybrid zones, …)

F1 hybrids may be conveniently detected based on

intermediate DNA content

XCR

BC

G1

G2

L. multiflorum (2n = 14)

CR

BC G1

G2

F1 hybrid C

RB

C

G1

F. arundinacea (2n = 42)


Identification of hybrids

THE USE OF EUPLOID PLANT OF

THE SAME SPECIES AS AN INTERNALSTANDARD

Discrimination is possible when the coefficient of variation of G1 peaks islower than half of the difference in DNA content (2.4% in this example)

THE USE OF A DIFFERENT SPECIESAS AN INTERNAL STANDARD

Relative difference in DNA content (D):

G1 Peak Ratio 1

G1 Peak Ratio 2D = * 100 [%]

0 100 200 300 400 5000

30

60

90

120

0 100 200 300 400

SE S A

G1 Peak Ratio 1 G1 Peak Ratio 2

RELATIVE NUCLEAR DNA CONTENT

0 100 200 300 400 5000

30

60

90

120

0 100 200 300 40 0

CV = 1.2% CV = 1.0%

E EA A

RELATIVE NUCLEAR DNA CONTENT

- G1 Peak Ratio 1

E = euploid, A = aneuploid, S = standard


Aneuploidy

0

50

100

150

200

250

300

1 21 41 61 81 101 121 141 161 181 201 221 241


Nu

mb

er

of

nu

cle

i

Peak DI CV%

3x 0.79 1.58

CRBC 1.00 1.74

CRBC3x

0

50

100

150

200

250

300

1 21 41 61 81 101 121 141 161 181 201 221 241


Nu

mb

er

of

nu

cle

i

Peak DI CV%

3x-1 0.76 0.99

CRBC 1.00 1.25

CRBC

3x-1

0

50

100

150

200

250

300

1 21 41 61 81 101 121 141 161 181 201 221 241


Nu

mb

er

of

nu

cle

i

Peak DI CV%

3x-2 0.74 1.02

CRBC 1.00 1.26

CRBC3x-2

Triploid (2n = 3x = 33)

2n = 33 - 1

2n = 33 - 2


Aneuploidy in Musa

Roux et al., Plant Cell Rep. 21: 483, 2003

a: antipodals; c: central cell with two polar nuclei; e: egg apparatus with egg cell

and two synergids.


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:

Matzk et al., Plant J. 21: 97, 2000

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.


G1 = 47.4%

S = 30.5%

G2 = 22.1%


Cell cycle

G1

G2

S

Flow cytometry allows to analyse the

degree of endopolyploiy and the

frequency of endopolyploid cells

G2

M

G1

S

ER

4C

8C

2C

S S G2G2 G1G1

ENDOREDUPLICATION

00

200

400

600

800

1000

50 100 150 200 250

2C

4C8C

16C

32C

Mammillaria san angelensis

(parenchym)

Nuclear DNA content

Nu

mbe

r of

nu

cle

i

Endoreduplication (polysomaty)

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

Applications:

• Relative DNA content

• DNA content in absolute units (genome size)

• Nuclear DNA base content (AT/GC ratio)

Flow cytometry of plant cell nuclei


Nuclear genome

size (Mb)Musa acuminata (591 - 615)Musa balbisiana (534 - 540)

65

1200

4400

7800

16400

120000

2700

H. sapiens

The size of nuclear genome


The method

Ratio of G1 peak positions Glycine

to Musa is 1.984 => 2C nuclear

DNA content of M. acuminata

errans is 2.50 / 1.984 = 1.26 pg

DNA (or 608 Mbp / 1C*)

SAMPLE

20 mg of Musa leaf

tissue

STANDARD

5 mg of G. max

(2C = 2.50 pg DNA)

leaf tissue

Simultaneous

isolation and

staining of

nuclei

Nuclei isolation

buffer + propidium

iodide

Relative nuclear DNA content

Num

ber

of

nucle

i

Musa G1

nuclei

Glycine G1

nuclei

Removal of large

debris by filtration


*) 1pg DNA = 0.978 Mbp

Doležel et al., Biol. Plant. 36: 351, 1994


Germplasm characterization for genome size

Accession name Section Genome size

(Mbp/1C)*

Calcutta 4 Eumusa 627

Galeo 626

Pisang Mas 635

M. acuminata ssp. banksii 646

Guyod 647

M. balbisiana type Cameroun 578

Honduras 579

M. schizocarpa 704

M. laterita Rhodochlamys 624

M. velutina 635

M. mannii 649

Kluai Bou 609

M. ornata 664

M. beccarii Callimusa 798

M. peekelii ssp. peekelii Australimusa 791

M. textilis 734

M. maclay type Hung Si 755

Kawaputa 766

Ensete gilletii Related genus 619

Musa genomes

differ by size:

- A ~ 630 Mbp

- B ~ 580 Mbp

- S ~ 700 Mbp

- T ~ 730 Mbp

- C ~ 790 Mbp

*) 1pg DNA = 0.978 Mbp

Bartoš et al., Cytogenet.

Genome Res. 109: 50, 2005

0 50 100 150 200 2500 50 100 150 2000 50 100 150 200

Propidium iodide

maizeleukocytes

leukocytes

DAPI Mithramycin

FR = 0.817 FR = 0.601 FR = 1.083


0

200

400

600

800

1000

Num

ber

of

nucle

i

leu

ko

cyte

s

maiz

e

maiz

e

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)

Due to different AT/GC ratio of human and maize, peak ratios are different

for each DNA fluorochromehttp://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html

DNA base content

Doležel et al., Physiol. Plant. 85: 625, 1992

Acknowledgements


David Galbraith (Tucson)

Jan Suda (Prague)

Fritz Matzk (Gatersleben)

Nicolas Roux (Montpellier)

Pietro Pifanelli (Genoa)

Rony Swennen (Leuven)

Jean-Pierre Horry (Montpellier)

The first

book

on plant

flow

cytometry

• Musa balbisiana

cv. Pisang Klutuk Wulung

• Genome size: 530 Mbp

• Scientific interest: BSV,

B genome structure

NucleiIntact cells Cellular debris

Nuclei isolation

Nuclei sorting


G1 nuclei

G2 nucleiDebris

Sort window

Purification of cell nuclei


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.

Library screening with a cp probe

0.09%8.27%

Standard procedure Flow sorting

BAC library from

Musa balbisiana PKW

- Number of clones: 36,864

- Average insert size: 135 kb

- Genome equivalents: 9x

- Clones with cp DNA: 3%

- Clones with mt DNA: 0.004%

Purification of cell nuclei


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


Gene expression

(A) Wild-type plant

(B) - (F) Transgenic

plants expressing

nuclear GFP

regulated by:

(B) p35S

(C) pRPL16B

(D) pSHR

(E) pSCR

(F) pSultr2-1

Simultaneous analysis of GFP (FL1) a DAPI (FL4)


Gene expression in root of Arabidopsis thaliana

Analysis of Plant Genomes Using Flow Cytometry

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