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Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome
size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking
discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was
taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of
instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C
l approx. 0n330 pg DNA). Each of the four laboratories involved in this study used a different buffer and\or
procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based
instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with
those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding
preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but
statistically significant, differences were found between data obtained with individual instruments. Differences
between the same type of instruments were negligible, while larger differences were observed between lamp- and laserbased instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by
lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow
cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the
study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between
different types of flow cytometers, analysis of very small differences in genome size should be made in the same
laboratory and using the same instrument.
# 1998 Annals of Botany Company
Key words : DAPI, Feulgen densitometry, flow cytometry, plant genome size, nuclear DNA content, propidium
iodide, standardization.
INTRODUCTION
Most of the genetic information of an organism is coded by
DNA localized in cell nuclei. However, it has been noted
that most or all eukaryotic organisms contain more DNA
than is needed for coding and regulatory sequences, and
that the quantity of DNA in a nucleus (genome size) is not
correlated with organismic complexity. This inconsistency
has been called the C-value paradox (Thomas, 1971). It
has been suggested that the DNA amount itself has
phenotypic effects via its influence on cell size and mitotic
cycle time ; these effects have been termed nucleotypic , the
term nucleotype denoting the physico-mechanical properties of the nucleus (Bennett, 1972). The best known
nucleotypic effect is that of genome size on life-cycle time in
herbaceous angiosperms, where annuals, and in particular
ephemerals, have smaller genomes than perennials (Bennett,
* Some results of this study were presented in a poster session during the Angiosperm Genome Size Workshop and Discussion Meeting,
Jodrell Laboratory, Royal Botanic Gardens, Kew, 1997.
For correspondence at : Institute of Experimental Botany, Laboratory of Molecular Cytogenetics and Cytometry, Sokolovska! 6, CZ77200 Olomouc, Czech Republic. E-mail : dolezel!risc.upol.cz
0305-7364\98\0A0017j10 $30.00\0
18
Light source
L1
L2
L3
L4
Propidium iodide
DAPI
Emission filters
Propidium iodide
DAPI
514 nm
500 mW
535 nm BP
351n1363n8 nm
100 mW
630 nm BP
400 nm LP
590 nm LP
535 nm BP
UG1
590 nm LP
435 nm LP
514 nm
200 mW
630 nm BP
reported many times from the late sixties to the present day
(Miksche, 1968 ; Laurie and Bennett, 1985 ; Rayburn et al.,
1985, 1997 ; Graham, Nickell and Rayburn, 1994 ; to quote
only the earliest and some more recent references). Certain
results even seem to support the concepts of a plastic
genome , i.e. quantitative changes in genomic DNA of an
organism in response to environmental or developmental
stimuli (e.g. Evans, Durrant and Rees, 1966 ; Price and
Johnston, 1996).
In contrast to interspecific comparisons when genome size
differences are not unexpected, technical variation becomes
a much more obvious problem when analysing intraspecific
variation. Clearly, rapid but at the same time reliable
methods for genome size estimation are needed. At present,
the two most widely used methods are Feulgen densitometry
and flow cytometry. The former has been by far the most
frequently used method and is still providing the highest
number of estimates (Bennett and Leitch, 1997). Flow
cytometry is more convenient and rapid, and hence is
becoming increasingly popular. However, the simplicity of
flow cytometry may be deceiving and may lead to the
generation of flawed data. For instance, it has been shown
that fluorochromes which bind preferentially at AT- or GCrich regions of DNA are not suitable for genome size
estimation in plants (Michaelson et al., 1991, Dolez) el,
Sgorbati and Lucretti, 1992, Godelle et al., 1993). However,
some authors prefer to use DAPI as the fluorochrome for
genome size estimation and find propidium iodide (PI) less
reliable (e.g. Rayburn, Auger and McMurphy, 1992), a
finding that contradicts the results mentioned above. In
addition, attention should be paid also to the proper
fluorochrome concentration (Dolez) el, 1991).
The choice and correct use of reference standards is
another critical factor which has been largely neglected ;
practically each major laboratory uses different standards
(animal or plant). As a result, the quality of genome size
data obtained using flow cytometry compared with Feulgen
densitometry does not generally seem to have been improved
(Bennett and Leitch, 1995). Estimates obtained by flow
cytometry in the same species may vary up to 100 % as in
maize (see Bennett and Leitch, 1997 : Table 1). This is
especially concerning given that the technique has the
potential to detect differences in genome size as small as 1 %
(Lysa! k, Dolez) elova and Dolez) el, 1997).
19
L1
Chopping
L2
Chopping
L3
Chopping
L4
Chopping
Isolation buffer
DAPI
Propidium iodide
Mg#+ buffer
1 g ml"
(Galbraith et al., 1983)
LB01 buffer
800
50 g ml"
j50 g ml" RNase
50 g ml"
j50 g ml" RNase
50 g ml"
j150 g ml" RNase
50 g ml"
j50 g ml" RNase
A
G. max
600
Z. mays
400
Number of nuclei
Laboratory
Method of nuclei
isolation
200
0
400
B
Z. mays
300
G. max
200
100
50
200
100
150
Relative nuclear DNA content
250
Feulgen densitometry
Root and shoot tips were fixed either in neutral
formaldehyde or 3 : 1 fixative and stored in ethanol. After
hydrolysis in 5 HCl at 20 mC, samples were stained in
Feulgen reagent, washed in SO -water and squashed.
#
Integrated extinction was determined with a scanning
densitometer at 570 nm using a square diaphragm (0n5 m)
and step size of 0n5 or 1n0 m. Early prophase or telophase
nuclei were measured in plants with large genomes (Z. mays
20
Linear regression analysis indicated a strong and statistically highly significant correlation between the ratios of
DNA content estimated by Feulgen densitometry and by
flow cytometry of PI-stained samples (r l 0n999, n l 8,
P 0n0001 ; Fig. 2 A). On the other hand, ratios estimated
by flow cytometry of DAPI-stained samples were different to
those obtained after PI staining for most of species pairs
(Table 7) ; the difference reached 58 % in the case of
G. max\Z. mays (see also Fig. 1). Correlation between
ratios of DNA content estimated by flow cytometry
of DAPI-stained samples and by Feulgen densitometry
(Fig. 2 B) was not significant at P l 0n025 (r l 0n769, n l 8).
In these calculations, we analysed overall means calculated
for DNA content ratios obtained in all four or two
laboratories for PI-stained and DAPI-stained samples,
respectively. The same results were obtained when the
analysis was performed for each laboratory separately (data
not shown).
Ratios of DNA content determined by Feulgen densitometry (Table 3) and by flow cytometry after PI staining
(Table 4) were used to calculate relative DNA content of
individual species. In these calculations, the relative DNA
content of A. cepa was arbitrarily set to 100 arbitrary
units (A.U.). Linear regression analysis between the relative
DNA content estimated by Feulgen densitometry and
relative DNA content estimated by flow cytometry of
PI-stained samples (Fig. 3) showed a strong and
T 3. Ratios of nuclear DNA content estimated for pairs of species using Feulgen densitometry
Ratio of relative DNA content (meanps.d.)
V. faba\
A. cepa
S. cereale\
V. faba
H. ulgare\
S. cereale
P. satium\
H. ulgare
Z. mays\
P. satium
G. max\
Z. mays
R. satius\
G. max
A. thaliana\
R. satius
0n790p0n001
0n737p0n004
0n762p0n042
0n757p0n003
0n762p0n022
0n608p0n001
0n569p0n005
0n599p0n009
0n564p0n013
0n585p0n022
0n635p0n001
0n659p0n007
0n626p0n008
0n659p0n008
0n645p0n017
0n876p0n001
0n936p0n016
0n886p0n009
0n920p0n004
0n905p0n028
0n605p0n008
0n577p0n025
0n582p0n013
0n594p0n011
0n590p0n013
0n439p0n006
0n451p0n004
0n464p0n006
0n457p0n001
0n453p0n011
0n416p0n004
0n521p0n001
0n452p0n007
0n469p0n007
0n465p0n044
0n282p0n009
0n298p0n011
0n312p0n004
0n311p0n020
0n301p0n014
Tissue\fixation*
Root\3 : 1
Root\F
Shoot\3 : 1
Shoot\F
Mean ratio
T 4. Ratios of nuclear DNA content estimated for pairs of species using flow cytometry after propidium iodide staining
Ratio of relative DNA content (meanps.d.)
Laboratory
L1 (laser)
L2 (lamp)
L3 (lamp)
L4 (laser)
Mean ratio
Largest difference (%)
Difference between
laser cytometers (%)
Difference between
lamp cytometers (%)
V. faba\
A. cepa
S. cereale\
V. faba
H. ulgare\
S. cereale
P. satium\
H. ulgare
Z. mays\
P. satium
G. max\
Z. mays
R. satius\
G. max
A. thaliana\
R. satius
0n778p0n007
0n776p0n010
0n752p0n016
0n792p0n028
0n774p0n017
5n1
1n8
0n613p0n006
0n595p0n005
0n586p0n008
0n606p0n017
0n600p0n012
4n4
1n1
0n647p0n004
0n638p0n005
0n632p0n007
0n661p0n005
0n645p0n013
4n4
2n1
0n874p0n009
0n863p0n007
0n879p0n004
0n869p0n008
0n870p0n007
1n8
0n6
0n639p0n021
0n609p0n008
0n586p0n003
0n658p0n019
0n623p0n032
10n9
2n9
0n469p0n031
0n441p0n007
0n438p0n005
0n519p0n004
0n467p0n038
15n6
9n6
0n506p0n006
0n462p0n008
0n465p0n009
0n464p0n003
0n474p0n021
8n7
8n3
0n310p0n003
0n300p0n002
0n313p0n011
0n302p0n001
0n306p0n006
4n2
2n6
3n1
1n5
0n9
1n8
3n8
0n7
0n6
4n2
21
120
0.75
0.5
8
0.25
90
60
30
y = 0.71 + 1.00x; r = 0.999
0.5
0.75
1.0
30
60
90
120
1.3
B
4
1.0
6
3
0.7
1
5
0.3
2
y = 0.07 + 1.14x; r = 0.769
0.3
0.7
1.0
1.3
22
4
0.75
0.5
Parameters of regression
line (y l ajbx)
L3 (lamp)
L2 (lamp)
L1 (laser)
L4 (laser)
8
0.25
k0n004
j0n032
j0n033
Slope (b)
Correlation
coefficient
1n015
0n985
0n990
0n997
0n994
0n981
0n0001
0n0001
0n0001
1.0
0.5
1.0
0.75
5
2
0.5
7
8
0.25
y = 0.06 + 0.94x; r = 0.989
0
0.25
0.5
1.0
0.75
T 5. Estimates of 2C nuclear DNA amounts using flow cytometry after propidium iodide staining
Standard : A. cepa*
Standard : P. satium
Species
L1
(laser)
L2
(lamp)
L3
(lamp)
L4
(laser)
Max
difference
(%)
A. cepa
V. faba
S. cereale
H. ulgare
P. satium
Z. mays
G. max
R. satius
A. thaliana
33n50
26n06
15n98
10n34
9n03
5n77
2n71
1n37
0n42
33n50
26n00
15n47
9n87
8n52
5n19
2n29
1n06
0n32
33n50
25n19
14n76
9n33
8n20
4n81
2n10
0n98
0n31
33n50
26n53
16n08
10n63
9n24
6n08
3n15
1n46
0n44
5n3
8n9
13n9
12n7
26n4
50n0
48n9
41n9
Mean
L1
(laser)
L2
(lamp)
L3
(lamp)
L4
(laser)
Max
difference
(%)
Mean
33n50
25n95
15n57
10n04
8n75
5n46
2n56
1n22
0n37
33n71
26n22
16n07
10n40
9n09
5n81
2n72
1n38
0n43
35n76
27n75
16n51
10n53
9n09
5n54
2n44
1n13
0n34
37n13
27n92
16n36
10n34
9n09
5n33
2n33
1n08
0n34
32n97
26n11
15n82
10n46
9n09
5n98
3n10
1n44
0n43
12n6
6n3
4n4
1n8
12n1
33n0
33n3
26n5
34n89
27n00
16n19
10n43
9n09
5n67
2n65
1n26
0n39
* The value of 33n5 pg DNA (2C) was assigned to A. cepa (Bennett and Leitch, 1997) which was used as a primary standard. Nuclear DNA
content of other species was then calculated using DNA content ratios given in Table 4.
The value of 9n09 pg DNA (2C) was assigned to P. satium which was used as a primary standard. This value was determined in a preliminary
experiment in Laboratory 2 using human leukocytes (2C l 7n0 pg DNA, Tiersch et al., 1989) as a reference standard. DNA contents of Z. mays,
G. max, R. satius and A. thaliana were then calculated using DNA content ratios given in Table 4. DNA contents of other species were calculated
using inverted ratios.
23
T 7. The effect of the type of flow cytometer on the ratio of relatie nuclear DNA content
Mean ratio of relative DNA content
Type of the flow
cytometer
Propidium iodide staining
Lamp (L2, L3)
Laser (L1, L4)
Difference
(laser lamp)
Mean difference
(laser lamp)
DAPI staining
Lamp (L3)
Laser (L1)
Difference
(laser lamp)
Mean difference
(laser lamp)
H. ulgare\
S. cereale
V. faba\
A. cepa
S. cereale\
V. faba
0n764
0n785
j0n021
0n591
0n609
j0n018
0n635
0n654
j0n019
0n423
0n435
j0n012
0n641
0n661
j0n002
P. satium\
H. ulgare
Z. mays\
P. satium
G. max\
Z. mays
R. satius\
G. max
A. thaliana\
R. satius
0n871
0n872
j0n001
0n598
0n649
j0n051
0n439
0n494
j0n055
0n464
0n485
j0n021
0n307
0n306
k0n001
1n192
1n183
j0n006*
0n408
0n441
j0n033
0n728
0n750
j0n022
0n429
0n443
j0n014
0n292
0n308
j0n016
j0n023
0n629
0n658
j0n029
j0n017
* In this special case, G nuclei of standard (P. satium) had apparently higher relative DNA content than the G nuclei of a sample (H. ulgare).
"
"
To permit a comparison with the other species pairs, the difference was calculated using reciprocal values of DNA content ratios.
T 8. Comparison of ratios of nuclear DNA content obtained with flow cytometry after propidium iodide staining and
Feulgen densitometry
Mean ratio of relative DNA content
H. ulgare\
S. cereale
Type of
cytometer
V. faba\
A. cepa
S. cereale\
V. faba
0n764
0n785
0n762
j0n023
0n591
0n609
0n585
j0n024
0n635
0n654
0n645
j0n009
j0n006
k0n010
P. satium\
H. ulgare
Z. mays\
P. satium
G. max\
Z. mays
R. satius\
G. max
A. thaliana\
R. satius
0n871
0n872
0n905
k0n033
0n598
0n649
0n590
j0n059
0n439
0n494
0n453
j0n041
0n464
0n485
0n465
j0n020
0n307
0n306
0n301
j0n005
k0n034
j0n008
k0n014
k0n001
j0n006
j0n019
j0n002
k0n005
24
25
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