J. Cell Sri. 56, IOI-III (198a)
Printed in Great Britain © Company of Biologists Limited 1982
IOI
THE RELATIONSHIP BETWEEN CHROMOSOME
VOLUME AND DNA CONTENT IN UNSQUASHED
METAPHASE CELLS OF BARLEY, HORDEUM
VULGARE CV. TULEEN 346
M. D. BENNETT, J. B. SMITH, J. P. WARD
AND R. A. FINCH
Plant Breeding Institute, Trumpington, Cambridge CB2 zLQ, England
SUMMARY
The present work used haploid and diploid cells of barley, Hordeum vulgare L. cv. Tuleen 346
(an = 2x — 14), which has three reciprocal translocations. All seven chromosomes of the
haploid set are distinguishable using morphological criteria in Feulgen-stained root-tip squashes
seen in the light microscope, as are five of the bivalents at diakinesis. The relative DNA content
per bivalent was estimated in pollen mother cells at diakinesis. The results showed that all seven
chromosomes or bivalents of Tuleen 346 can be identified using relative DNA content as sole
criterion. The absolute and relative volumes of the seven chromosomes were estimated from
electron micrographs of serial sections of unsquashed root-tip cells of a haploid. The results
show that, using relative chromosome volume as sole criterion, it is highly probable that all
seven chromosomes in single unsquashed cells of Tuleen 346 can be correctly identified.
Consequently, teats for various non-random spatial arrangements of chromosomes in unsquashed cells of Tuleen 346 using this character to identify the chromosomes should be
feasible. There was a very highly significant positive relationship (r>o<)o) between relative
chromosome volume and mean relative DNA content per chromosome for each cell examined
at metaphase of mitosis or meiosis. Thus, some mechanism ensures that the degree of condensation of all seven chromosomes within a cell is usually very similar in Tuleen 346, lespite
its grossly abnormal karyotype.
INTRODUCTION
Using light microscopy, various authors have described close correlations between
chromosome size and the DNA content of chromosomes at metaphase of mitosis in
higher plants. For example, Barlow & Vosa (1969) showed a striking linear relationship (r = 0-99) between the DNA content and the volume of the mitotic metaphase
chromosomes of Puschkinia Ubanotica (syn. Scilla libanotica). Similarly, the results of
Heneen & Caspersson (1973) showed a correlation coefficient of 0-98 when the relative
DNA contents and relative lengths of the seven root-tip metaphase chromosomes of
Secale cereale were compared. However, Nishikawa (1970) reported a smaller correlation coefficient (r = 0-82) between the DNA contents and the lengths of the 21
chromosomes of hexaploid bread wheat (Triticum aestivum cv. Chinese Spring)
measured as univalents at the first division of male meiosis.
Examination of serial electron micrographs of sectioned cells has allowed the
volumes of intranuclear structures to be determined more precisely than was possible
M. D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch
3 7
~
2-6
V
f2
7-3
5-1
1-5
5-1
1-5
O
7-3
6-2
3-7
2-6
Chromosome volume and DNA content in barley cells
103
by light microscopy. Using this technique, it has proved possible to estimate the
volumes of both whole chromosomes (Finch, Smith & Bennett, 1981) and small
chromosome segments such as centromeres (Moens & Church, 1977; Bennett, Smith,
Ward & Jenkins, 1981). It was decided, therefore, to compare the DNA content of
individual chromosomes with their volumes estimated from serial electron micrographs of sectioned cells in a higher plant species. Such a study should show whether
the relationship between DNA content and chromosome size is as precise as some
previous light-microscopic studies have indicated.
MATERIALS AND METHODS
The chosen material was barley {Hordeum vulgare, in = zx = 14), stock 'Tuleen 346*.
This stock, so named by us because it came from a plant numbered 346 by Professor N. A.
Tuleen, is homozygous for the three reciprocal translocations (T), T1-5V, T2-6y and T3-7d
(N. A. Tuleen, 1980, personal communication). Details of the origins and karyotype of this
stock have been published recently (Finch & Bennett, 1982).
Tuleen 346 is notable because all seven chromosomes can be identified in mitotic metaphase
squashes seen in the light microscope using the morphological characters: relative length, arm
ratio, and the presence or absence of a secondary constriction (Fig. 1). Moreover, compared
with normal barley, the range of chromosome sizes is greatly increased. Thus, Tuleen 346
seemed a suitable material for the present study, especially as the increments between adjacent
chromosomes, ranked by size order, was likely to be larger than in most materials including
normal barley.
Unequivocal identification of all the chromosomes in a cell using chromosome volume alone
will depend on there being no overlap between any estimates of the relative volumes of any two
chromosomes. It may be hard to identify homologous chromosomes in diploid material. Any
misidentification would result in an error when calculating the mean relative volume and its
variance for each chromosome. The need to identify homologues does not arise in a haploid.
Consequently, estimates of relative chromosome volume were made using haploid Tuleen 346.
Haploid plants of Tuleen 346 were obtained by pollinating emasculated spikes of diploid
Tuleen 346 with pollen from diploid Hordeum bulbosum clones Ji or L6 (Simpson, Snape &
Finch, 1980). The bulbosum chromosomes were eliminated during early seed development,
as frequently occurs in hybrids between diploid H. vulgare and H. bulbosum (Subrahmanyam &
Kasha, 1973; Bennett, Finch & Barclay, 1976). Haploid embryos were cultured until they
formed plantlets large enough to survive transplantation to soil. When they had grown into
large vegetative plants, one plant was divided into ramets and cultured in hydroponics (Finch
et al. 1981) to produce clean new roots suitable for ultrastructural studies.
An alternative way of avoiding the need to identify homologues with certainty is to study
paired homologues during meiosis in diploid material. Such pairing is very regular in diploid
Tuleen 346. For example, in a plant from a glasshouse a random sample of 20 pollen mother
cells all contained seven bivalents and the mean chiasma frequency per cell was I4'8s. Thus,
plants of diploid Tuleen 346 were grown in a glasshouse until they reached meiosis, when
suitable tillers were sampled for DNA estimations or ultrastructural studies.
A Vickers M86 integrating microdensitometer was used to estimate the relative DNA
contents of the seven bivalents in suitable cells from Feulgen-stained anther squashes. Of many
thousands of pollen mother cells examined, five at diakinesis were identified as most suitable for
Fig. 1. The seven chromosomes of haploid Tuleen 346 at mitotic metaphase
identified using morphological criteria in a Feulgen-stained root-tip squash preparation. Bar, 10 fim.
Fig. 2. The seven bivalents of diploid Tuleen 346 at diakinesis of male meiosis
identified using morphological criteria and relative DNA contents. Bar, 10 fim.
104
M. D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch
Table i. Relative absorption per bivalent expressed as a percentage of the total absorption
per cell in pollen mother cells of diploid Tuleen 346 at diakinesis
Cell
Bivalent
T2-6
Ti-S
T3-7
4
T7-3
T6-2
T5-1
1
1942
18-75
1674
14-21
1279
9-9i #
8-17
2
3
4
1905
1939
19-66
1852
1868
1852
16-96
1521
1239
9-87*
799
16-28*
1554
1227
9-78*
1627
13 95
12-81
10-42*
8-34
806
5
1927
18-13
17-02
1390
1283
10-63*
8-22
Mean
S.E.
Variance
1936
18-52
1665
1456
o-io
005
0-06
013
12-62
IO-I2
816
O-II
0-16
o-34
O'I2
0-17
0-06
058
0-07
0-14
O'O2
• Secondary constriction observed.
microdensitometry because all seven bivalents were clearly separated from one another, and the
five bivalents containing chromosomes T5-1, T6-2, T7-3, 4 and T3-7 were clearly identified
using morphological criteria (e.g. Fig. 2). The other two chromosome pairs containing T1-5
and T2-6 could not be identified by this means at this stage of meiosis. Diakinesis rather than
first metaphase was chosen as the optimal stage, because the degree of chromosome condensation at diakinesis allows cells with seven fully separated bivalents to be found, and both nucleolar
organizers are sometimes clearly visible. In each cell the DNA content of each bivalent was
estimated as the mean of three readings, and these means were expressed as the relative
proportion of the total DNA content of the pollen mother cell.
A root-tip about 1 cm in length was cut from a haploid Tuleen 346 plant and pretreated in
ice-water at about o °C for 24 h. This pretreatment increases the proportion of cells at metaphase (Subrahmanyam & Kasha, 1973).
Root-tip and anther material were fixed, embedded and stained for ultrastructural studies
as previously described (Bennett, Smith, Simpson & Wells, 1979). Sections o-i fim thick were
cut using a Reichert Ultracut 4 microtome with a diamond knife, and collected on Formvarcoated 2 x 1 mm slot-grids, using the technique of Wells (1974). Chromosomes in selected
cells were photographed at x 4500 using a Philips 201 electron microscope, and printed at a
final magnification of x 11 800.
Four cells at metaphase (A, B, C and D, respectively) were identified in sections of a single
haploid root-tip. The total number (n) of serial sections on which chromosomes of these nuclei
appeared were 95, 96, 88 and 83, respectively. All the electron micrographs of chromosomes in a
single cell were arranged in section number sequence in a binder and numbered from 1 to n.
Photographs of all the sections of nuclei A, B, C and D were obtained except section 49 in
nucleus A (which was obscured), and section 75 in B (which was lost).
Two pollen mother cells at first metaphase (E and F, respectively) were identified in sections
of a single anther of diploid material. In these cells bivalents appeared on 68 and 81 successive
sections, respectively. Photographs, arranged as described above for root-tip nuclei, were
available for all sections other than section 64 of nucleus F, which was lost.
For each cell, each chromosome (in haploid material) or bivalent (in diploid material) was
identified in the set of electron micrographs by its number (1-7) written in waterproof ink on
each piece of chromatin belonging to that chromosome or bivalent. Different chromosomes or
bivalents were numbered 1-7 according to the order in which each first appeared in the electron
micrographs. Chromosome or bivalent volume was estimated using a Kontron Videoplan
Digitizer, scaled to give areas in /tm1. The outlines of a single chromosome or bivalent on each
successive photograph where it appeared were traced on the digitizer tablet, and the resulting
areas summed in the memory. The section thickness was o-i fim, so the volume of each
chromosome or bivalent (in fim*) was obtained by multiplying the summed areas by a factor
Chromosome volume and DNA content in barley cells
105
Table 2. Absolute chromosome volume* and the relative chromosome volume expressed
as a percentage of the total chromosome volume per cell in haploid Tuleen 346 root-tip
cells at metaphase
Cell
Mean
Variance
Chromosome
A
B
C
D
T2-6
(11-07)
19-58
(1022)
1806
(1092)
1934
(1025)
1896
(1057)
1903
1925
013
0-07
(10-27)
18-50
18-50
O'l8
0-14
(9-37)
i6-57t
(936)
i7-32f
(1092)
1903
(1059)
18-47
(io-oo)
17-44
(8-41)
14-87
(7-58)
(8-53)
14-01
(7-24)
12-79
12-73
1469
(6-84)
1193
Ti-S
T3-7
4
T7-3
T6-2
Ts-i
(6-88)
(5-59)
9-89f
(4-66)
(5-O9)
(5-77)
942t
(4-44)
10-07
(4-8o)
8-23
8-22
836
S.E.
(885)
16-82
o-35
0-49
iS-94
(810)
I4-59
14-54
0-19
0-14
(7-48)
1273
0-31
o-39
1346
(S-6i)
io-iof
987
0-16
o-io
830
0-04
o-oi
(466)
8-39
• Expressed as /*m' (in parenthesis).
t Secondary constriction observed.
of o-i. Where a section was lost or obscured (as noted above), the unknown area of a chromosome was estimated as the mean of the areas for the same chromosome on the two adjacent
sections.
RESULTS
Relative DNA contents of the seven chromosomes
Table 1 presents the relative absorption of the seven bivalents in five cells of
diploid Tuleen 346 at male diakinesis expressed as a percentage of the total absorption
for each cell. Examination of the estimates for bivalents containing the five chromosomes unequivocally identified using morphological criteria shows that they ranked in
the same order in all five cells. Thus, estimates for T3-7, 4, T7-3, T6-2 and T5-1
ranked 3rd, 4th, 5th, 6th and 7th, respectively. Moreover, there was no overlap
between any estimates of relative absorption for any two chromosomes with adjacent
ranking. Thus, it is reasonable to assign estimates ranked first in each cell to the
longest chromosome (T2-6) and those ranked second to the second longest chromosome (T1-5). It is then possible to estimate the mean relative DNA contents of the
seven chromosomes in diploid Tuleen 346 (Table 1). These values show a range of
about 2-4-fold from 8-16% in T5-1 to 1936% in T2-6.
Microdensitometry of Feulgen-stained mitotic prophase nuclei of diploid Tuleen
346 and Sultan barley showed that their 4C DNA amounts were identical (22-2 pg)
io6
M. D. Bennett, jf. B. Smith, J. P. Ward and R. A. Finch
Table 3. Absolute bivalent volume* and the relative bivalent volume expressed as a
percentage of the total bivalent volume per cell in diploid Tuleen 346 cells at first metaphase
of male meiosis
Cell
Bivalent
E
F
T2-6
(2466)
1873
Ti-S
(2408)
1829
(2913)
1959
(2865)
T3-7
(2313)
I7-S7
(2008)
15-25
(1630)
12-38
(1288)
979
(10-51)
799
4
T7-3
T6-2
Ts-i
1927
(25-52)
17-16
Mean
19-16
1878
17-37
(21-15)
14-22
14-74
(17-73)
11-92
(14-52)
976
(1201)
808
12-15
978
8-04
• Expressed as fim3 (in parentheses).
20
15
8 10
•-
5
5
10
15
20
Mean relative chromosome DNA content
Fig. 3. Relationship between mean relative chromosome volume and mean relative
chromosome DNA content in haploid Tuleen 346 root-tip metaphase cells.
Chromosome volume and DNA content in barley cells
10
15
20
5
10
15
Mean relative chromosome DNA content
107
20
Fig. 4. Relationship between relative chromosome volume and mean relative chromosome DNA content in single cells of haploid Tuleen 346 cells A, B, C and D.
(Finch & Bennett, 1982). Thus, in 4C cells of haploid Tuleen 346 the absolute DNA
contents of the chromosomes were as follows: T2-6, 2-15 pg; T1-5, 2-06 pg; T3-7,
1-85 pg; 4, 162 pg; T7-3, 1-40 pg; T6-2, 1-12 pg; and, T5-1, 091 pg.
A one-way analysis of variance showed very highly significant differences between
relative absorption values for all chromosome pairs. Analysis also showed that the
variance for estimates of the relative absorption for the same chromosome in different
cells was small compared with the difference between pairs of chromosomes of
most-similar DNA content. The results indicate that all seven bivalents in a cell
would be identified correctly with a high probability using relative DNA content as
sole criterion. A Bartlett test showed no significant differences between the variances
for different chromosomes.
Relative volumes of the seven chromosomes
Table 2 presents the absolute volumes of the seven chromosomes in four cells of
haploid Tuleen 346, together with the relative volumes expressed as a percentage
within each cell. Estimates of the absolute volume of homologues in the different
cells are remarkably similar. Examination of estimates of the relative volumes of the
five unequivocally identified chromosomes (T3-7, 4, T7-3, T6-2 and T5-1) shows that
they ranked in the same order and position in each cell. Moreover, there was no
overlap between any estimates of relative volume for any two chromosomes adjacent
in the ranking. It is reasonable, therefore, to identify estimates ranked first with the
longest chromosome (T2-6) and those ranked second with the second longest chromosome (T1-5). It is, therefore, possible to calculate the mean relative volume for all
seven chromosomes. These show a 2-3-fold range from 8-30% to 19-25%. These
M. D. Bennett, jf. B. Smith, J. P. Ward and R. A. Finch
io8
20 i—
E
15
O
>
I
CD
i
10
5
5
10
15
20
Mean relative chromosome DNA content
Fig.
Relationship between mean relative bivalent volume at diakinesis and the
mean relative chromosome DNA content in diploid Tuleen 346.
results are therefore in close agreement with estimates of relative chromosome length
in light micrographs of haploid root-tip cells of Tuleen 346, which gave a 2-2i-fold
range from 1868% for chromosome T2-6 to 8-44% for T5-1 (Finch & Bennett,
1982).
A one-way analysis of variance showed a very highly significant difference
(P<o-ooi) between estimates of relative chromosome volume for all pairs of chromosomes, with the exception of T2-6 and T1-5 where the difference was highly significant (P< o-oi). Analysis also showed that the between-cell variances for estimates
of the relative volume of homologues were small compared with the differences
between the mean relative volumes of any two chromosomes with adjacent ranking.
A Bartlett test showed no significant difference among the variances for different
chromosomes. These results indicate that all seven chromosomes in a cell would be
identified correctly with a high probability using relative volume as sole criterion.
However, chromosomes T2-6 and T1-5 would be confused more often than any other
pair of chromosomes because the size difference between them is smaller than that
between any other pair of chromosomes.
Table 3 presents estimates of the absolute volumes of the seven bivalents in two
pollen mother cells at first metaphase of meiosis from a single anther of diploid
Tuleen 346. The relative bivalent volumes expressed as a percentage of the total
volume of bivalents in each cell are also given, and showed ranges of 2'34-fold and
242-fold, respectively. Bivalents were not identified using morphological criteria at
this stage since, for example, constrictions at nucleolar organizers are poorly expressed
at metaphase. However, the mean relative volumes for bivalents (Table 3) are not
significantly different from the mean relative volume of mitotic metaphase chromosomes ranked in corresponding positions. Moreover, the mean relative volume of
each bivalent, except the two largest, is significantly different (P<o-O5) from the
Chromosome volume and DNA content in barley cells
109
mean relative volume of any mitotic chromosome, except that ranked the same as the
bivalent being compared. Thus, it is reasonable to assume that metaphase chromosomes of Tuleen 346 retain the same relative volumes at meiosis as at mitosis. If so,
it should usually be possible to identify all seven metaphase bivalents in a single cell
using relative volume as sole criterion.
Relationship between chromosome volume and DNA content
Fig. 3 illustrates the very highly significant (P<o-ooi) relationship between the
mean relative chromosome volume (values from Table 2) and the mean relative
chromosome DNA content (values from Table 1) for Tuleen 346. The regression
coefficient and slope are i-ooo, and the intercept of the regression line with the
abscissa (—0-02) is not significantly different from the origin.
Fig. 4A-D shows plots of the relative volume on relative DNA content for the
seven chromosomes in each of the four haploid cells studied. In each case the relationship is very highly significant, the regression is not significantly different from unity,
and the intercept is not significantly different from the origin.
Fig. 5. illustrates the very highly significant relationship (P<o-ooi) between mean
relative bivalent volume and mean relative chromosome DNA content (r = 0997;
b = 1-009; a ~ — O-I 3)- Similar separate plots for cells E and F also showed very
highly significant relationships; P<o-ooi; r>0-099; b = 1-04 (E) and 1-08 (F);
a = —0-13 (E) and —1-14 (F).
DISCUSSION
The precise relationship between chromosome volume and DNA content
Estimates of chromosome volume in single cells at mitotic metaphase obtained by
light microscopy are meaningful, but are subject to unavoidable errors; for example,
the need to assume that chromatids are circular in cross-section and of constant
diameter (Bennett & Rees, 1969). Photographs of the present serially sectioned cells
show that chromatids are often not circular in cross-section (although in general they
approximate to this configuration), and that the diameter of a chromatid varies
considerably along its length. Nevertheless, striking correlations have been shown
between chromosome DNA content and chromosome volume estimated as mean
values for many cells using light microscopy (for references, see Introduction).
Using serial sections of nuclei and electron microscopy, chromosome volume can
be estimated more precisely in single cells than by light microscopy, since the abovementioned assumptions are unnecessary. The present results for Tuleen 346 barley,
using chromosome volumes for four cells studied by electron microscopy, confirm
the existence of a precise general relationship between chromosome volume and DNA
content (Fig. 3) in a diploid angiosperm as described for Puschkinia Ubanotica (Barlow
& Vosa, 1969). They also show that the precise relationship was displayed by each
individual cell studied (Fig. 4). Thus, the present results indicate a precise intracellular control, which ensures that the relative condensation of all seven metaphase
chromosomes in the haploid complement of Tuleen 346 is very similar. Tuleen 346
no
M.D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch
is abnormal in having three translocations, which involve six of the seven chromosomes and increase the range of chromosome sizes to from i to 2-4 compared with the
range from 1 to 1-3 in normal barley. It is interesting to note that the precise control
of chromosome condensation is unaffected by the large rearrangement of segments in
the complement of Tuleen 346 due to the three reciprocal translocations. The
advantages of highly synchronized chromatin condensation among chromosomes
within a cell in facilitating balanced and efficient mitosis are obvious. Thus, it seems
reasonable to expect that precisely synchronized condensation among chromosomes,
as seen in Tuleen 346, is normal and that examples of gross allocycly among chromosomes within a genome are exceptions.
Use of chromosome volume to identify chromosomes
Experiments to test for various types of non-random spatial distribution of chromosomes have mainly been done with squashes, although it has been suspected that
considerable rearrangement of relative chromosome disposition may be unavoidable
in their preparation (Feldman, Mello-Sampayo & Sears, 1966; Stack & Brown, 1969;
Wagenaar, 1969). It is obviously preferable to make such tests using undistorted cells,
but it has rarely been possible to identify all the chromosomes in unsquashed cells of
organisms with more than a few (i.e. zn > 6) chromosomes, using either light or
electron microscopy.
The present results show that using serial sections of nuclei of suitable materials,
it is possible to identify most or all of the chromosomes at both mitotic and meiotic
metaphase in single cells undistorted by squashing, using relative chromosome
volume alone as a criterion. Moreover, by comparing the dispositions of chromosomes
identified in this way it should be possible to test for various non-random chromosome
arrangements in unsquashed dividing nuclei, including somatic association (see
Avivi & Feldman, 1980), secondary association (Kempanna & Riley, 1964), affinity
(Wallace & Gunn, 1965) and specific end-to-end arrangements (Ashley, 1979).
Preliminary studies indicate that such tests are quite feasible using Tuleen 346, and
show that useful results should be obtainable from small numbers of replicate cells.
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(Received 27 January 1982)