Genotypic control of centromere positions of parental genomes in
Hordeum x Secale hybrid metaphases
TRUDE SCHWARZACHER-ROBINSON, R. A. FINCH, J. B. SMITH and M. D. BENNETT
Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, England
Summary
The spatial disposition at metaphase of centromeres from Hordeum and Secale in root tip cells of
H. chilense X S. africanum is described and compared with corresponding results for H. vulgare X
S. africanum obtained previously. In both of these
Fj sexual hybrids (2n = 2x = 14) each of the seven
chromosome types from Secale was easily distinguished by its large size from any of the seven
from Hordeum.
In H. chilense X S. africanum, centromeres of
Secale chromosomes tended to be nearer the
centre of the metaphase plate than did centromeres of Hordeum chromosomes in both squash
preparations seen by light microscopy and
unsquashed cells examined using electron microscope three-dimensional serial thin section reconstructions. This difference was significant in
some individual cells, and highly significant for
pooled data for reconstructed cells and separately for squashed metaphases. In no cell were
Hordeum centromeres on average significantly
nearer the centre of the metaphase plate than
Secale centromeres.
These results agreed with those previously
obtained for H. vulgare X S. africanum in that:
(1) centromeres of the two parental haploid sets
tended to be spatially separate; and (2) centromeres from one particular parent usually tended
to be in the peripheral region of the metaphase
plate that surrounded the central region containing the centromeres from the other parent. However, these results contrasted completely with
those obtained previously in that Secale centromeres tended to be more central than Hordeum
centromeres in H. chilense X S. africanum, but
more peripheral than Hordeum centromeres in
H. vulgare x S. africanum.
As centromeres of the parental set with the
larger chromosomes (i.e. Secale) can be either
inside, or outside, centromeres from the parental
genome with the smaller chromosomes (i.e. Hordeum), then clearly, a tendency for a concentric
separation of parental genomes is not a packing
phenomenon determined by chromosome' size
perse, but is presumably under genotypic control.
Introduction
1984a). Concentric parental genome separation
(CPGS) of centromeres (Bennett, 1984a) occurs in a
cell when: (1) centromeres from one parent are on
average significantly farther from the MCP than are
centromeres from the other parent; and (2) all centromeres from one parent lie within a circle drawn in the
plane of the metaphase plate, which excludes all other
centromeres (e.g. see Figs 4, 5; and Finch & Bennett,
1983).
In H. vulgare X S. africanum and H. vulgare X
5. cereale, all Secale chromosomes were larger than
any Hordeum chromosome. (N.B. In this paper,
chromosomes from Secale in a hybrid will be called
Recent quantitative studies of metaphase chromosome
positions in reconstructed root tip cells of Hordeum
vulgare X Secale africanum and H. vulgare X 5.
cereale showed a significant tendency for the centromeres of the genome from H. vulgare to be nearer the
cell mean centromere position (MCP) than were the
centromeres of the genome from Secale (Finch et al.
1981; Finch & Bennett, 1981).
Spatial separation of centromeres into two groups
according to parental origins was termed parental
genome separation (Finch & Bennett, 1981; Bennett,
Journal of Cell Science 87, 291-304 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Key words: genome separation, centromere position,
Hordeum X Secale.
291
'Secale chromosomes' when this aids brevity without
confusion with chromosomes in non-hybrids; and
similarly for chromosomes from other parents.)It was
therefore questioned whether a tendency towards
CPGS was caused by genetic control or was a packing
phenomenon due to some mechanical constraint that
preferentially brought smaller chromosomes near the
centre of the metaphase plate. However, Finch et al.
(1981) found negative correlations between chromosome volume and distance from the MCP for centromeres of either parent separately, in the same hybrid
cell where these two characters were positively correlated for the cell as a whole. This suggested that
the tendency towards CPGS was under genotypic
control, since a mechanical effect on chromosome size
should act similarly both within and between parental
genomes in hybrid cells (Finch et al. 1981; Bennett,
1982).
This paper describes the karyotype, and the relative
positions within cells of centromeres from either
parent, in the diploid hybrid, H. chilense X S. africanum. All Secale chromosomes were again so much
larger than any Hordeum ones that their identities as
to specific origin were unequivocal in hybrid cells.
This hybrid thus provides another test of whether
centromeres of small chromosomes tend to be relatively more central on a metaphase plate than do those
of large chromosomes. If Hordeum centromeres were
again more central in hybrid metaphases, this might
support the hypothesis that packing constraints acting
on relative chromosome size control centromere position, at least in hybrids whose parents differ greatly in
chromosome size. However, if Hordeum centromeres
were the more peripheral, then this hypothesis must
be abandoned in favour of one invoking genotypic
control of centromere position.
had seven Hordeum and seven Secale chromosomes. Unlike
our H. vulgare X S. africanum hybrids (Finch et al. 1981),
H. chilense X 5. africanum flowered profusely each year.
However, no chiasmata were seen in 22 metaphase I pollen
mother cells from a sample anther and all plants had
indehiscent anthers at maturity and were sterile on open
pollination.
Roots of parent species came from seeds germinated for 2
days at 20 °C in the dark on filter paper moistened with
tapwater. Actively growing hybrid and parental root tips
about 1 cm long were excised and fixed at once or after
pretreatment for 24 h at 0—1 °C in freshly aerated tapwater to
accumulate metaphases (Subrahmanyam & Kasha, 1973).
Electron-microscope study
Roots fixed in 5 % (v/v) glutaraldehyde in O'lmoll" 1
phosphate buffer for 4-24 h were prepared for EM study
according to Bennett et al. (1979). Serial sections of all the
chromosomes of metaphase cells EMI to -9 and two cells
from each parent were cut 0-1 fim thick, photographed and
printed at x l l 800 final magnification. None of the 77 and
98 sections of H. chilense cells was lost, but in nine hybrid
and two 5. africanum cells, prints of one to two of the
105-179 sections were unavailable, a mean overall loss of
0-63% of sections.
The volume (in fun) and arm volume ratio or AVR (long
arm volume -f- short arm volume) of each chromosome, and
the volume of the satellite when nucleolar-organizing regions
(NORs) were visible, in reconstructed cells were estimated
from prints using the MOP Videoplan image analyser as
previously described (Bennett et al. 1982; Heslop-Harrison
& Bennett, 1983). This identified the parental origin of each
chromosome (see Results).
The three-dimensional mean centromere position
(MCP:x,;y,2) of all 14 chromosomes, distance (CD:.v,3>,z)
of the middle of the centromere of each chromosome from
the M C P : * ^ ^ , and, for the genome from each parent
separately, genome mean distance (GMD:.v,v,z) of the
centromeres of its seven chromosomes from the MCP:.v,y,z
of all 14 chromosomes were calculated for each cell as in
Finch et al. (1981) and Heslop-Harrison & Bennett (1983).
Materials and methods
Genotypes and plant culture
Six Fj hybrids (2rt = Zx = 14) were produced by pollinating a spike of Hordeum chilense Roem. & Schult.
(2n = 2x = 14) Plant Breeding Institute line 1 with pollen
from Secale africanum Stapf (2n = 2x = 14) Plant Breeding
Institute line R102 (Finch & Bennett, 1980). Embryos
excised 3 weeks later were kept on agar for 3-24 weeks while
developing into plantlets. After agar culture, plants were
grown for 1-35 months in compost and/or hydroponics
according to Finch et al. (1981), before roots were fixed. In
both electron-microscope (EM) and light-microscope (LM)
studies, most metaphases came from roots of plants grown in
compost for 32-35 months and then hydroponics for 3-10
days.
All H. chilense X S. africanum hybrids were vigorous,
healthy plants showing little or no sign of the mitotic
instability seen in many Hordeum hybrids (Finch, 1983),
and nearly all the many somatic mitoses seen and all used
292
T. Schwarzacher-Robinson
et al.
Light-microscope study
Roots fixed in acetic acid—chloroform—ethanol (8:25:50, by
vol.) for 24 h were stained by the Feulgen method. A squash
preparation of the meristematic tip of each root was ma<je,
on a separate slide, sometimes irrigating with propionic
orcein to deepen stain.
Metaphase cells chosen solely because each had seven
Hordeum and seven Secale chromosomes in focus in one
field of view under the X100 objective were photographed
and printed at X1350-2250 final magnification. Rectangular
coordinates in the plane of the print were measured for each
centromere middle directly or with the digitizer, and the
two-dimensional mean centromere position (MCP:.vj>) of
all 14 chromosomes, distance (CD:.x,;y) of the centromere
middle of each chromosome from the MCP:x,_y and, for the
genome from each parent separately, genome mean distance
(GMD:xj>) of the centromeres of its seven chromosomes
from the MCP:x v y of all 14 chromosomes were calculated
for each metaphase according to Finch et al. (1981).
Hordeum vulgare L. cv. Sultan X 5. africanum Stapf
Results for H. chilense X S. africanum are compared in the
present discussion with results previously obtained for
reconstructed root tip metaphase cells in H. vulgare cv.
Sultan X S. africanum. The methods used to obtain the
latter data were substantially the same as those used in the
present work and have been described (Finch et ai. 1981).
Results
Electron-microscope reconstructions
The volume and arm volume ratio of each chromosome were measured in reconstructed metaphases of
two cells from each parent and in hybrid cells EMI
to-9.
Data from parents indicated that most or all
chromosomes in hybrid cells should be identifiable as
to parental origin by size. Thus in the parental lines
that gave the present hybrids, the 1C DNA amount
was estimated as 7-4pg in S. africanum and 5-4pg in
H. chilense (Bennett & Smith, 1976). In each reconstructed parental metaphase, the largest chromosome
volume was 1-27 or 1-32 (mean 1-29) times the
smallest, in Secale, and 1-39 or 1-51 (mean 1-45)
times the smallest, in Hordeum, and volume differences between adjacent chromosomes ranked by size
were similar over the whole size range. Therefore,
assuming direct proportionality of volume to DNA
content in a chromosome, most or all Secale chromosomes should exceed Hordeum chromosomes in volume in hybrid cells; and observation confirmed this.
It was obvious from chromosome volumes alone
that, in each of the cells EMI to -9, the seven largest
and seven smallest chromosomes were from Secale
and Hordeum, respectively, with a distinct size gap
between the smallest from Secale and the largest from
Hordeum (Fig. 1). Thus, the largest chromosome
volume in a cell was only 1-19-1-27 (mean 1-21) times
3
4
5
6
7
Relative volume (%)
Fig. 1. Histogram of the relative volume of each
chromosome in cells EMI to -9 as a percentage of the total
volume of chromosomes in its cell. The distinct gap in
relative size was used to distinguish chromosomes of the
respective haploid parental genomes from H. chilense
(open) and S. africanum (filled).
the seventh largest, but 1-64—1-85 (mean 1-72) times
the eighth largest. The eighth largest was only
1-26-1-40 (mean 1-33) times the smallest. However,
the seventh largest was 1-73-2-01 (mean 1-89) times
the smallest, and 1-29-1-52 (mean 1-42) times the
eighth largest. The parental origins of all chromosomes, and the individual identities of 12-14 chromosomes were clear in all nine hybrid cells.
Table 1 gives, for each chromosome in cells EMI to
-9, its volume as a percentage of the total volume for its
parental set within its cell, and its arm volume ratio.
Chromosome identifications in cells EM3, -6 and -9
were based on Fig. 2, which shows the mean hybrid
karyotype from reconstructed mitoses in cells EMI,
-2, -4, -5, -7 and -8 where all chromosome types were
individually identifiable and of accurately known volume. Hordeum types were numbered in descending
order of mean size in cells EMI, -2, -4, -5, -7 and -8
and Secale types as in H. vulgare X S. africanum
(Bennett, 1982). These numbers do not imply interspecific homoeologies.
Expression of the NOR in hybrid cells seen at the
EM level was as reported by Finch & Bennett (1980)
and in the present paper in the LM. Diploid parental
cells had two NOR pairs in H. chilense and one NOR
pair in S. africanum. In all nine hybrid cells, both
Hordeum NORs were visible but the Secale NOR was
not. Arm volume ratios, and volumes as percentages of
the total genome from the same parent in the cell of the
most distinctive chromosome types from Hordeum
(two satellite types) and Secale (chromosome 5) in
hybrid cells (Table 1) showed no significant differences in (-tests from those of parental homologues,
except that the mean AVR of H. chilense chromosome
5 was higher in parent (AVR = 2-26) than in hybrid
(AVR = 2-01) cells (P<0-05 in 2-tailed (-test). However, the total volume of the genome from Hordeum
was consistently and significantly smaller, relative to
that of the genome from Secale, than expected from
parental 1C DNA amounts (Table 2), suggesting that
allocycly may occur in this hybrid.
Table 3 gives, for each parent in each of reconstructed hybrid cells EMI to -9, the genome mean
distance (GMD :x,;y,z) between the seven centromeres in that parental genome and the mean position
of all 14 centromeres in the cell (MCP:xj>,z). In
seven cells, the genome mean distance of Hordeum
centromeres from the cell mean centromere position
exceeded that of Secale centromeres, significantly so
in cells EMI, -5 and -8. In cell EM2, parental genome
mean distances of centromeres from the cell mean
centromere position were almost equal to each other.
The genome mean distance of Secale centromeres
exceeded that of Hordeum centromeres from the cell
mean centromere position by over 1 % in only one cell
(EM7), but not significantly so. Differences between
Genotypic control of centromere positions
293
Hordeum and Secale in mean distance of centromeres
from the cell mean centromere position were highly
significant for separate pooled sets of untreated and
cold-treated hybrid cells, and very highly significant
for all nine cells pooled (Table 3). The ratio of Secale
to Hordeum in genome mean distance of centromeres
from the cell mean centromere position ranged from
1:0-94 to 1:1-75 (mean 1:1-31) in hybrid cells.
Untreated and treated cells did not differ significantly
in this ratio.
Table 4 gives correlation coefficients and their probabilities in linear regressions of distances (CD:x,y,z)
between centromeres and cell mean centromere position on chromosome volumes for each hybrid cell.
Coefficients for the whole hybrid genome were negative in: (1) seven of nine individual reconstructed
cells, significantly so in cells EMI (P< 0-001) and
EM8 (P<0-01); (2) pooled sets of untreated (P<
0-05) and treated (P< 0-001) cells; and (3) all nine
cells pooled (P<0-001). Only the smallest and least
significant such coefficients were positive (cells EM2
and -7) and only two exceeded 0-43 in absolute value
(cells EMI and-8).
Coefficients for genomes from separate parents in
hybrid cells showed no consistent trend even for
pooled data. They were negative in three cells for the
genome from Hordeum and in seven cells for that from
Secale and significantly negative for that from Secale
in cell EMI (P<0-05) and positive for that from
Hordeum in cell EM2 (/ ) <0-01).
Light-microscope study
LM studies agreed unambiguously with the EM result
that in hybrid cells, all Secale chromosomes were
clearly larger than any Hordeum ones (Fig. 3C,D).
Despite the lesser accuracy of LM measurements
compared with the present EM estimates, this finding
also seemed likely beforehand, merely from LM
observations of chromosome lengths, given the parental \C DNA values and that length is proportional to
Table 1. Volumes of complete haploid genomes and individual chromosome types (as percentages of sum of all chromosomes
from same parent in the cell) from each parent and chromosome arm volume ratios in reconstructed metaphases of
H. chilense X S. africanum (cells EMI to -9) and parents (two cells each)
% Volume (V) and arm volume ratio (AVR) of chromosome type
1
Genome
Parent
Cell
H. chilense
EMI
EM2
EM3
V \JIU111C
3
EM4
EM5
EM6
EM7
EM8
EM9
8
S. afneanum
3
2
(Mm )
V
AVR
V
AVR
V
AVR
V
AVR
46-08
46-50
51-07b
45-81
48-13
46-77
43-81
40-89
43-64
16-64
15-59
15-88C
16-48
15-73
1-26
1-53
1-39
1-48
1-40
1-52
1-56
1-63
1-34
1-48
14-56
14-24
1-27
1-16
14-47
14-95
13-12C
13-93
14-52
1-03
1-03
1-03
1-02
1-13
1-48
14-33
b
Mean of
EMI t o - 9 :
45-86
Parent mean:
40-30
EMI
EM2
EM3
EM4
EM5
EM6
EM7
EM8
EM9
83-55
82-29
91-84C
82-26
86-10
84-07
76-34
73-05
75-49
Mean8 of
EMI to -9:
Parent mean:
81-67C
16-25
16-31
15-74
1-20
119
115
14-87
16-06
14-74'
16-20
15-48
16-01
14-24
15-21
14-73
16-08
1-18
15-28
e
15-73
15-69
h
15-00
16-26
15-33
15-72
15-03
15-59
15-54
•19
•12
1-16
1-17
c
1-06
1-06
h
1-04
1-03
105
102
105
1-02
1-04
15-22
15-03
14-48 '
14-55
14-04
15-04
15-00
15-00
15-31
14-85
84-72
115
113
116
1-15
1-18
1-17
1-08
1-11
1-13
1-14
d
14-56
14-36
14-05
14-95
13-57
t
14-77
15-49
14-33b
15-21
14-64
14-47
15-17
15-40
14-98
14-94
d
1-23
1-27
1-30
1-23
1-17
t
1-23
1-81
1-88
1-87
1-91
2-01
1-78
1-81
1-90
1-56
1-84
V
7
6
5
4
AVR
V
AVR
1-07
13-17"
1-07
13-74'
12-18"'c 119
1-07
13-60"
102
13-63"
1-01
12-83'
14-02"
1-01
14-31" 0-94
13-68"
1-08
1-05
13-46
14-06
13-74
14-85
1-08
1-05
1-10
14-02'
13-81"
15-61"'c
13-47'
14-40*
13-73'
13-65'
14-82"
14-60"
14-21
1-06
14-23
2-12
2-23
1-76
2-23
2-05
1-99
1-83
1-81
2-06
2-01
14-09"
2-26
13-87"
1-11
12-81
13-09
14-23b
13-94
13-54
14-42
13-75
13-55
14-23
13-73
2-14
2-31
2-29
2-24
2-28
2-48
2-24
2-32
2-30
2-29
13-33
13-31
12-85b
14-09
13-66
14-09
13-06
13-76
13-47
1-01
1-03
1-01
1-05
1-09
111
1-04
1-01
1-03
1-04
13-19
2-27
c
14-72
14-30
14-39b
14-42
14-48
14-61
14-34
14-28
13-33
14-32
e
1-61
1-50
1-52
1-56
1-63
1-53
1-49
1-48
1-50
1-54
13-51
V
AVR
12-26
11-61
12-7T
11-77
11-88
12-49
12-83
12-03
12-53
1-58
1-70
1-73
1-70
1-64
1-74
1-68
1-62
1-74
12-23
1-68
13-42
13-09
13-96b
12-79
13-38
12-05
12-96
12-98
13-09
13-08
1-38
1-48
1-37
1-42
1-41
1-56
1-43
1-41
1-38
1-43
Data on individual chromosome types in parents are from three replicates of H. chilense chromosome 6, and four replicates each of others.
"NOR visibly expressed. b Maximum estimate. "Minimum estimate. d l h These chromosomes were stuck together in sections 70—80 and so volume and arm
ratio estimates are less exact than usual. Volumes in /im3 are: (d) ^8-05 (AVR = =1-37) and (h) S14-47 (AVR = =»l-04). "These are chromosomes 1 and 4
from H. chilense, but it is unclear which is which. Volumes in /im3 are: 6-58 (AVR = 1-11) and 7-87 (AVR = 1-05). 'This chromosome was off the
metaphasc plate and so its volume (13-86/im3) and AVR (1-45) may be anomalous. g Excludes data not in body of Table.
294
T. Schwarzacher-Robinson et al.
1
3
2
4
6
5
7
1
2
3
4
5
6
7
aa
X
X
X
X
X
X
X
V(%) 9-98 9-68 9-49 9-24 8-67 8-61 8-39 5-81 5-51 5-17 5-13 5-04 4-94 4-34
AVR 1-04 1-89 1-13 1-55 1-04 2-26 1-42 1-20 1-47 1-22 1-06 2-05 1-03 1-65
Fig. 2. H. chilense X S. afncanum idiogram and chromosome type mean volumes (V) and arm volume ratios (AVR) of
serially sectioned reconstructed metaphases in cells EMI, -2, -4, -5, -7 and -8. Chromosomes are in order of mean volume
as % of total hybrid genome volume. Chromosome types from Secale (open) are numbered (top) after Bennett (1982).
Types from Hordeum (filled) are numbered (top) in order of mean volume. NORs are shown by stippling in the type from
Secale (based on four NORs in two serially sectioned reconstructed S. afncanum cells, as this NOR was not seen in hybrid
cells) and indenting in the types from Hordeum.
Table 2. Total chromosome volume and percentages
of it from each parent and x2 tests for differences
between volumes in jxm3 observed and those expected
from parental 1C DNA amounts in H. chilense X
S. africanum cells EMI to -9
Chromosome volume
% Observed from
Cell
EMI
EM2
EM3
EM4
EM5
EM6
EM7
EM8
EM9
Mean of
EMI to -9
Total
(Mm3)
129-63
128-79
142-91
128-07
134-17
130-75
120-15
113-94
119-13
127-50
Hordeum
Secale
35-55
3611
35-74
35-77
35-87
35-77
36-46
35-89
36-63
64-45
63-89
64-26
64-23
64-13
64-23
63-54
64-11
63-37
64-04
35-96
2-345
1-957
2-439
2-163
2-197
2-207
1-615
1-855
1-508
18-287
d.f.
P<
1
1
1
1
1
1
1
1
1
0-2
0-2
0-2
0-2
0-2
0-2
0-3
0-2
0-3
8
0-02*
Expected percentages are 42-19 (Hordeum) and 57-81 (Secale).
DNA content in a chromosome. Thus in samples of
seven unsquashed metaphases from each parental
species, the longest chromosome of the 14 in its cell
was on average l - 3 times as long as the shortest in
S. africanum, while in H. chilense it was on average
1-5 times as long as the shortest (Finch, unpublished)
and within each cell, size differences between adjacent
chromosomes ranked by size were similar over the
whole size range.
Satellite chromosomes and sometimes heterochromatin gave further help in identifying chromosomes.
S. africanum has one satellite pair (Fig. 3B) but its
NOR was seldom visible in hybrid metaphases and
then only on a large chromosome. H. chilense has two
satellite pairs, both distinct in shape from that of
S. africanum (Fig. 3A). Both NORs from H. chilense
were visibly expressed in nearly all hybrid metaphases
(Fig. 3C,D) and these were always among the seven
small chromosomes identified as the Hordeum
genome. In cold-treated parental roots, conspicuous
telomeric heterochromatin was often visible in metaphase chromosomes of S. africanum (Fig. 3B) but not
H. chilense. Significantly, such telomeric heterochromatin was also visible in some hybrid metaphases after
cold treatment (Fig. 3B,C), but only on the seven
large chromosomes identified as from
Secale.
Chromosomes in cells from treated roots tended to be
shorter and thicker than those in cells from untreated
roots. In both treated and untreated hybrid roots,
however, the parental origin of each chromosome in a
cell was clear.
Table 5 gives, for each parent in each of 60
squashed hybrid metaphases, the genome mean distance (GMD:x,y) between the seven centromeres in
that parental genome and the mean position of all 14
centromeres in the cell (MCP ::t,;y). Table 5 also
gives, for each cell, the ratio of the Secale to the
Hordeum GMD:xj>. In 41 (68-3%) of these 60
cells, the mean distance (GMD:x,;y) of Hordeum
centromeres from the cell mean centromere position
(MCP:x,;y) exceeded that of Secale centromeres from
Genotypic control of centromere positions
295
Table 3. Means (GMD: \,y,z) and population standard deviations (s.D.) of the distances between the cell
mean centromere position (MCP: x,y,z) and centromeres of the genome from Hordeum and of that from Secale
and ratio of Secale to Hordeum GMD:x,y,z in H. chilense X S. africanum cells EMI to -9 and results of
one-tailed t-tests of significance of differences in GMD: \,y,z between centromeres from Hordeum and
those from Secale
(-test
GMD:*,y,2
S.D.
Ratio
(
d.f.
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
3-29
1-88
2-48
2-49
2-99
2-39
0-44
0-81
0-84
0-95
0-56
0-63
1:1-75
3-742
12
<0-005"
1:1-00
0-028
12
>0-45
1:1-25
1-753
12
<0-l
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
Hordeum
Secale
8-05
6-24
3-30
2-82
2-91
2-00
3-86
2-83
3-14
3-34
3-43
2-07
3-15
2-67
1-95
2-38
0-89
1-18
0-91
0-60
0-99
1-48
0-87
0-55
0-80
0-78
0-62
0-98
1:1-29
2-627
40
<0-01»*
1:1-17
0-808
12
<0-25
1:1-46
2-051
12
<0-05»
1:1-36
1-423
12
<0-l
1:0-94
0-472
12
<0-35
1:1-66
2-970
12
<0-01"
1:1-18
1-021
12
<0-2
Hordeum
Secale
Hordeum
Secale
8-00
6-29
8-01
6-27
218
2-40
2-11
2-39
1:1-27
3-379
82
<0-005«
1:1-28
4-306
124
Genome from
EMI
EM2
EM 3
Mean of EMI to -3
(untreated)
EM4
EM5
EM 6
EM7
EM8
EM9
Mean of EM4 to -9
(cold-treated)
Mean of EMI to -9
P
Mean
Cell
<0-0005»«
Each GMD :.\\y,z value is in ^im (individual cells) or is the mean of all 42, 84 or 126 percentage distances of centromeres from their cell
MCP:.v,v,2 (pooled data from 3, 6 or 9 cells), where the sum of the 14 distances in a cell = 100%.
the cell mean centromere position, significantly so in
cells 21, 41, 51 and 54 and highly significantly overall.
In the remaining 19 cells, the mean distance of
Hordeum centromeres from the cell mean centromere
position was less than that of Secale centromeres
from the cell mean centromere position, but never
significantly so. Thus although spreading may greatly
disturb chromosome positions in a cell, LM measurements of mean two-dimensional relative positions of
parental genomes in hybrid cells in regard to centromeres agreed with three-dimensional EM reconstructions.
Since far more cells could be studied in the LM than
in the EM, the LM study was used to show that cold
treatment and prolonged and varied cultivation had
little effect on relative genome positions in the
hybrids, as far as centromeres were concerned. Variance analyses and i-tests done on the ratios of Secale
to Hordeum G M D : . V , J I showed no significant differences among hybrids, between compost (cells 7-20)
and hydroponics culture, between fixations 1-2
months after embryo culture (cells 1-20) and those at
32-35 months, between pretreated (cells 1-40) and
296
T. Schwarzacher-Robinson et al.
untreated cells, or between cells from plants treated
with colchicine 32 months before fixation (cells 34-40,
54—60), and others. (Mitoses in roots from colchicinetreated plants seemed no different from normal in any
way.)
Discussion
Concentric parental genome separation in
H. chilense X S. africanum
The genome mean distance (GMD:x,y,z) from the
cell mean centromere position of centromeres from
Hordeum exceeded the GMD:x,y,z of centromeres
from Secale in seven of nine reconstructed mitoses,
significantly so in three cells and overall, and by
an average of 22% of the Hordeum
GMD:x,y,z
(Table 3). LM data similarly showed that in spreads of
most hybrid root tip metaphases, the mean distance
(GMD:x,>>) from the cell mean centromere position
was greater for centromeres from Hordeum than for
those from Secale. This difference was highly significant overall though not significant in most single
Table 4. Correlation coefficients and their
probabilities in linear regressions of distances
(CD: x,y,z) between centromeres and cell mean
centromere position on chromosome volumes for each
of cells EMI toEM9
P
Correlation
coefficient (2-tailed)
Cell
Genome origin
EMI
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
-0-426
-0-818
-0-834
0-919
0-707
0-232
0-297
-0-208
-0-430
<0-4
<0-05»
<0-001«*
<0-01«
<0-l
<0-5
<0-6
<0-7
<0-2
Hordeum
Secale
All 42 chromosomes
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
Whole cell
Hordeum
Secale
All 84 chromosomes
Hordeum
Secale
All 126 chromosomes
0-349
-0-034
-0-341
-0-481
-0-151
-0-291
0-341
0-559
-0-406
-0-206
-0-211
-0-418
0-334
-0-273
0-134
0-001
-0-661
-0-696
0-392
-0-025
-0-253
-0-012
-0-134
-0-358
0-106
-0-100
-0-353
<0-2
<0-9
<0-05»
<0-3
<0-8
<0-4
<0-5
<0-2
<0-2
<0-7
<0-7
<0-2
<0-5
<0-6
<0-7
>0-9
<0-2
<0-01«
<0-4
>0-9
<0-4
>0-9
<0-4
<0-001»«
<0-5
<0-5
<0-001"#
EM2
EM3
EMI to -3
(untreated)
pooled
EM4
EM5
EM6
EM7
EM8
EM9
EM4 to -9
(cold-treated)
pooled
EMI to -9 pooled
A separate coefficient is given for the genome from each parent
and the whole cell. For pooled data from 3, 6 and 9 cells, distance
and volume values used were percentages of total for 14
chromosomes.
cells (Table 5), and averaged only about 8% of
the GMD:jc,y of centromeres from Hordeum. The
squash technique may drastically disturb relative
chromosome positions, but results from nine reconstructed metaphases, where estimates of relative centromere positions differ only negligibly from those in
vivo, agreed with results from squashes. As the mean
distance from the cell mean centromere position was
greater for centromeres from Hordeum than for those
from Secale, as a consistent tendency in treated and
untreated roots of several genotypes grown in a variety
of conditions over a 3-year period, it is concluded that
H. chilense X S. africanum showed a real significant
tendency towards a concentric parental genome separation for centromeres. Thus, it is like H. vulgare X
S. africanum and H. vulgare XS. cereale, the two
other Hordeum X Secale hybrids examined using reconstructed metaphases (Finch et a!. 1981; Finch &
Bennett, 1981).
When testing for concentric separation of parental
genomes, it is worth examining separately first the
tendency towards CPGS in the population of cells,
and second the proportion of individual cells showing
CPGS (as defined in the Introduction).
In a simulation, two sets of seven points were placed
at random on an approximately circular metaphase
plate and this was repeated 10000 times. These
showed that CPGS as defined in the Introduction,
would occur by chance in not more than 1-23% of
metaphases in diploid hybrids where 2w = 14. However, as Figs 4 and 5 show, CPGS occurred in four of
18 (i.e. 22-2%) of reconstructed hybrid metaphases,
including one of nine (i.e. 11-1%) in H. chilense X
S. africanum (Fig. 4), and three of nine (i.e. 33 %) in
H. vulgare X 5. africanum (Fig. 5). The frequency of
concentric parental genome separation obtained approached significance for H. chilense X S. africanum
(P = 0-105) and was significantly greater than expectation (i.e. 1-23%) both for the pooled data
(P <* 0-001), and for H. vulgare XS. africanum
(P <S 0-001). Cell 3 in Fig. 5 had only 13 chromosomes
and has a slightly larger expectation than 1-23 %, but
even omitting this cell leaves the probabilities as <0-01
for both pooled data and H. vulgare X S. africanum
data.
The significant overall tendency for CPGS found
in H. chilense X 5. africanum and H. vulgare X S.
africanum using pooled data for reconstructed cells
might reflect the occurrence of CPGS in a minority of
cells while the majority of cells show no such tendency. To investigate this possibility, tests were
repeated on pooled data, but excluding cells with
CPGS. Thus, when data for only eight cells of
H. chilense X S. africanum (i.e. omitting cell EMI;
Fig. 4) were pooled, Hordeum centromeres were still
significantly farther (P<0-01) from the MCP than
those of Secale were. Similarly, when data for only six
cells of H. vulgare X S. africanum (omitting cells 1, 3
and 7 in Fig. 5) were pooled, Hordeum centromeres
were still significantly nearer (P< 0-005) to the MCP
than those of Secale were. Thus, cells without CPGS
nevertheless still display a strong tendency towards it.
Comparing the mean distance of centromeres of
either parental genome from the cell MCP permits a
relatively simple yet highly meaningful test for a
tendency towards CPGS in a population of cells from a
hybrid. However, it involves calculation and does not
provide a ready visual impression of the degree and
Genotypic control of centromere positions
297
type of separation. On the other hand, the test for
CPGS in single qualifying cells, using a circle drawn
in the plane of the metaphase plate, does give an
immediate visual impression of the separation but is
biologically meaningful only in qualifying cells with
circular, or nearly circular, metaphase plates. However, the shape of a metaphase plate is restricted by,
and often closely reflects, the shape of the bounding
cell wall. Thus, the test becomes increasingly artificial
as cells, and the shape of the constrained metaphase
plate, become more elongated. For example, the
distributions of centromeres in Fig. 6A and B are
identical except for the foreshortening in B due to the
reduction in the scale of the vertical axis. The mean
distance from the MCP of points for parent 1 is
significantly less than for parent 2 in both Fig. 6A and
B. Consequently, an attempt is being made to develop
an alternative test for arrangements essentially similar
to CPGS that is biologically meaningful in elongated
and non-elongated metaphase plates.
Preliminary work indicates that drawing the polygons of minimum perimeter that contain all the
centromeres from a parental genome in a cell may
provide a simple but meaningful general test for a
range of types of parental genome separation, including the concentric form. Figs 4 and 5 show for each
cell such a polygon drawn for the parental genome,
which for that hybrid was on average nearer the MCP.
This test has the advantage that it is simple to
perform, involves little calculation once basic simulations like those below have been done, and provides
an immediate visual impression of the degree and type
of parental genome separation.
In a simulation, two sets of seven points were again
placed at random on an approximately circular metaphase plate, and polygons of minimum perimeter were
then drawn round each. This was repeated 10000
times, and the mean number of points from one set
included in the polygon for the other set (range 0-7)
was 2-1932, and 10-64% of cells had a polygon that
contained no point from the other set. These values
were adopted as random expectations in the present
work.
Using this test, genome separation is indicated by
the number of centromeres of either parent contained
in polygons for the other parent deviating significantly
from expectation (i.e. 2-1932 per cell when 2n = 14).
Scores tending significantly to zero for both parents
indicate a significant tendency towards a side-by-side
arrangement of parental genomes, while scores tending significantly towards zero for one parent but
towards seven for the other parent, indicate a significant tendency towards CPGS.
The total number of Hordeum centromeres contained in polygons for Secale in the nine reconstructed
cells of H. chilense X 5. africanum (Fig. 4) was five,
Fig. 3. Squashed metaphases from cold-treated roots of//, chilense (A), S. africanum (B) and H. chilense X S. africanum
(C,D) stained in Feulgen with (A) or without (B-D) propionic orcein. Satellites (s) by visible NORs and Secale telomeric
heterochromatin (arrows) are marked. Secale centromeres (arrowheads) are marked in (C,D). Bar, 10 fjm.
298
T. Schwarzacher-Robinson et al.
Table 5. Mean distances (GMD:x,y) in jim of centromeres of genomes from H. chilense (H.C.) and
S. africanum (SA.) from cell mean centromere positions (MCP:x,y), and ratios of SA. GMD:x,y to H.C.
GMD:x,y, in 60 hybrid cells
Cold-treated
Untreated
Genome mean <listance
Cell
1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
Genome mean distance
Genome mean distance
Plant
H.C.
S.A.
Ratio
Cell
Plant
H.C.
S.A.
Ratio
Cell
A
A
A
A
A
A
10-04
9-27
8-75
8-47
7-52
8-75
11-58
9-14
11-68
8-98
9-76
8-92
7-95
7-54
7-75
9-30
12-13
11-55
8-28
5-47
7-67
7-26
7-91
7-76 •
7-24
11-32
10-62
9-09
10-18
6-41
7-04
7-44
7-68
7-57
8-91
6-38
9-18
10-39
7-60
7-33
1:1-31
1:1-28
1:1-11
1:1-09
1:1-04
1:0-77
1:1-09
1:1-01
1:1-15
1:1-40
1:1-39
1:1-20
1:1-04
1:1-00
1:0-87
1:1-46
1:1-32
1:1-11
1:1-09
1:0-75
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
B
B
B
B
B
B
B
B
B
B
B
B
B
D
D
D
D
D
D
D
10-22
8-76
8-64
8-89
8-20
6-57
7-13
7-11
5-83
5-71
6-95
5-99
5-19
6-51
7-05
6-93
8-80
6-61
7-91
5-70
7-15
6-30
6-92
7-47
7-58
6-13
6-72
6-93
6-06
7-08
8-73
7-60
8-56
4-86
5-67
6-20
8-69
7-08
8-75
7-24
1:1-43*
1:1-39
1:1-25
1:1-19
1:1-08
1:1-07
1:1-06
1:1-03
1:0-96
1:0-81
1:0-80
1:0-79
1:0-61
1:1-34
1:1-24
1:1-12
1:1-01
1:0-93
1:0-90
1:0-79
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Mean of
B
C
D
E
E
E
E
E
E
F
F
F
F
F
H.C.
S.A.
Ratio
17-52
7-20
7-10
6-84
9-21
B
8-52
B
B
10-33
B
5-23
4-34
B
B
6-77
C
7-95
6-50
C
5-12
C
D
7-86
10-30
D
7-72
D
D
7-30
D
6-59
D
6-35
D
6-20
1-60: 8-04
11-35
5-41
5-45
5-30
7-30
8-37
10-99
6-35
5-43
8-54
4-78
6-62
6-54
5-30
8-54
6-69
6-44
6-01
6-25
6-88
7-42
1:1-54°
1:1-33
1:1-30
1:1-29
1:1-26
1:1-02
l:-094
1:0-82
1:0-80
1:0-79
1:1-66'
1:0-98
1:0-78
1:1-48*
1:1-21
1:1-15
1:1-13
1:1-10
1:1-02
1:0-90
l:l-08 b
Plant
B
B
B
B
•H. chilense GMD:*,.v>S. africanum GMD:x,y (P<0 •OS, 1-tail) in t-test (d.f. = 12).
h
H. chilense GMD:x,j>>S. africanum GMD:x,y (P<0 •005) in Mest (d.f. = 59).
while the total number of Secale centromeres contained in polygons for Hordeum (omitted for clarity
from Fig. 4) was 34. Compared with random expectation (19-74) these numbers were, respectively,
significantly lower (P<0-01) and higher (P<0-01).
Similarly, in the nine cells of H. vulgare X 5. africanum (Fig. 5) the total number of Secale centromeres
contained in polygons for Hordeum was three, while
the total number of Hordeum centromeres contained
in polygons for Secale (omitted for clarity in Fig. 5)
was 41. Compared with random expectation (19-74)
these numbers were, respectively, significantly lower
(P< 0-001) and higher(P < 0-001). Further, the
probabilities that, in samples of nine cells, as in Figs 4
and 5, and 18 as in both pooled, the numbers of
polygons drawn there that contain no centromere from
the other genome would be as many as the observed 5,
6 and 11 are <0-01, < 0 0 0 1 and <*0-001, respectively.
Thus, all three types of test applied to both hybrids
agree in showing that there is both a significant
number of individual cells with CPGS for centromeres, and a significant tendency towards CPGS for
pooled results both for all cells, and excluding those
cells not showing CPGS. Clearly, therefore the
phenomenon is real in metaphase cells of these
hybrids, which raises two important questions concerning how widely it occurs and how it is controlled.
TTie occurrence of concentric parental genome
separation
Besides the above mentioned work on H. chilense X
S. africanum, H. vulgare xS. africanum and H.
vulgare X S. cereale, EM serial thin section reconstructions of three of four serially sectioned metaphases in H. vulgare X H. bulbosum premeiotic anther
archesporium (Bennett, unpublished) and one of two
in H. marinum X H. vulgare embryos showed CPGS
(Finch & Bennett, 1983). Significant differences between parental genomes in mean distance of centromeres from the MCP at metaphase were shown in
H. vulgare XH. bulbosum embryos and roots (Finch
& Bennett, 1983) and similar differences were noted
but not quantified for centromeres in H. vulgare X
H. bulbosum and H. marinum X H. vulgare endosperms (Finch & Bennett, 1983), and chromosomes in
H. vulgare X Psathyrostachys fragilis >roots (LindeLaursen & von Bothmer, 1984). All these cases were
diploid hybrids, but Linde-Laursen & Jensen (1984)
showed quantitatively in four polyploid hybrids (H.
roshevitzii X H. vulgare, H. jubatum X H. vulgare,
H. vulgare X Triticum aestivum and H. vulgare X
Genotypic control of centromere positions
299
H. brevisubulatum ssp. turkestanicum) that "H. vulgare chromosomes were located nearer the centre of
the metaphase plate than the non-H. vulgare chromosomes" (using centromere positions as chromosome
positions). Thus in all 10 interspecific Hordeum
hybrid combinations, involving four genera in all,
where chromosome or centromere dispositions were
described, concentric separation of centromeres of
parental genomes was found and was often clear and
significant and in several tissues. This strongly
suggests that the phenomenon is widespread in
Hordeum hybrids. Indeed there is no report of it
having been sought but not found in such hybrids.
Partial or complete genome separation has been
reported, claimed or strongly suggested in various
materials including: (1) non-embryonic somatic cells
of naturally occurring plant and animal species (e.g.
Triticum aestivum, Avivi et al. 1982; Chlorophytum
elatum, Storey, 1968; Caledia captiva, Coates &
Smith, 1984) and hybrids (e.g. Rubus procerus X
R. laciniatus, Bammi, 1965; and chicken X quail,
Bammi et al. 1966); (2) zygotes (e.g. Polychoerus
carmelensis, Costello, 1970; and Tigriopus, ArRushdi, 1963); and (3) nuclei of man-made intergeneric hybrid fusion cells of plants (e.g. soybean-pea,
Constabel et al. 1975, and soybean-V»«'<2 hajastana,
Constabel et al. 1977), and animals (e.g. humanmouse, Rechsteiner & Parsons, 1976). However, as far
as the present authors are aware, CPGS has not been
reported in other materials. Consequently, whether it
has a wider taxonomic distribution is unknown. As
CPGS is a special form of genome separation, its
occurrence seems unlikely to be restricted to hybrids
involving the genus Hordeum.
The control of concentric parental genome separation
Packing phenomenon or genetic control. When concentric parental genome separation was first noted in
H. vulgare X S. africanum, where all chromosomes
from Hordeum are smaller than those from Secale, it
o
6>
o
o
Fig. 4. Polar views of positions of Hordeum (O) and Secale ( • ) centromeres on metaphase plates, and polygons of least
perimeter including all Secale centromeres, in serially sectioned reconstructed cells EMI to -9 (1 to 9, respectively) of
H. chilense X 5. africanum. The broken circle in EMI reveals concentric parental genome separation (CPGS). Bar,
300
T. Schwarzacher-Robinson et al.
was questioned whether the phenomenon resulted
from a genotypic control of centromere position, or
whether it was due to some kind of mechanical
constraint acting preferentially to bring smaller
chromosomes toward the centre of the metaphase
plate. In other words, do Hordeum centromeres tend
to cluster nearer the centre of the metaphase plate than
Secale centromeres because Hordeum chromosomes
are smaller than those of Secale ? This concern was
increased because many examples are known where
within a species (e.g. Yucca, Miiller, 1909; and man,
e.g. Ockey, 1969; Rohlfefa/. 1980; Hensef al. 1982)
or a hybrid (e.g. chicken-quail, Bammi et al. 1966)
small chromosomes tend to lie nearer the centre of the
metaphase plate. However, this tendency is by no
means general, since Albertson & Thomson (1982)
reported a tendency for large chromosomes to be
central on the meiotic metaphase plate in Caenorhabditis elegans, while Wollenberg et al. (1982)
reported chromosome 1 (the largest chromosome in
man) to be more central than expected in females, but
not in males.
The \C DNA amounts of H. vulgare cv. Sultan
(5-5 pg) and H. chilense PBI line 1 (5-4pg) are very
similar to each other, but markedly smaller than that
of 5. africanum line R102 (7-4pg) (Bennett & Smith,
1976). The same accession of S. africanum (line
R102) was used for DNA measurement and as male
parent to make the hybrids H. vulgare X 5. africanum
and H. chilense X S. africanum. As already noted, all
Hordeum chromosomes were markedly smaller than
any Secale chromosomes and there was a significant
tendency towards CPGS for centromeres in both of
these hybrids. However, whereas in H. vulgare X
5. africanum the centromeres of Hordeum chromosomes were significantly nearer the MCP than Secale
centromeres were (Fig. 5), it was the Secale centromeres that were significantly nearer the MCP than
1
.W.
Fig. 5. Polar views of positions of Hordeum (O) and Secale ( • ) centromeres on metaphase plates, and polygons of least
perimeter including all Hordeum centromeres, in nine serially sectioned reconstructed H. vulgare X S. africanum cells (data
from Bennett, 1982, and unpublished). Broken circles reveal CPGS in three cells. This figure corrects a small computer
graphics error in fig. 1 of Bennett (1982). Bar, 2um.
Genotypic control of centromere positions
301
accompanied by significant correlations within parental genomes where one case was positive and the other
negative.
The conclusion that there is no general tendency for
the centromeres of small chromosomes to be nearer the
MCP than those of large chromosomes is further
supported by results for 10 reconstructed mitotic
metaphase plates in root-tip cells of another stable
intergeneric hybrid (Aegilops squarrosa X Secale
cereale, 2n = 2x = 14) where the centromeres of the
seven markedly smaller squarrosa chromosomes were
on average farther from the MCP than were Secale
centromeres (Bennett, 1984a, and unpublished).
Moreover, support for the conclusion that CPGS is
under genotypic control comes from our recent observation that in young seeds of H. vulgare X H. marinum ssp. marinum the polarity of CPGS is tissue
specific. Thus, in 4-day-old seeds the marinum centromeres were on average significantly peripheral to
vulgare centromeres in the diploid embryo cells
(Finch & Bennett, 1983; Finch, unpublished), but
the opposite was true in triploid endosperm, as the
vulgare centromeres were significantly peripheral to
those of marinum (Finch, unpublished).
Fig. 6. Effect of flat cell shape on test for CPGS. Outer
( • ) and inner (O) genome centromere positions and cell
corners have the same horizontal coordinates in cells A and
B, but corresponding vertical coordinates are halved in B.
In both cells, the mean distance of centromeres from the
cell mean centromere position ( + ) is 1-7 times greater
(P<0-01) for outer than inner genomes. However, the
smallest circle including all inner genome centromeres
excludes all outer genome ones only in A, and so only A
shows CPGS.
Hordeum centromeres were in H. chilense X 5. africanum (Fig. 4). As centromeres of the parental genome
with the larger chromosomes can be either inside, or
outside, centromeres from another parent with smaller
chromosomes, then clearly CPGS, or a tendency
towards it, is not a packing phenomenon determined
by chromosome size per se, but is presumably under
genotypic control. This conclusion was previously
drawn for H. vulgare X 5. africanum (Finch et al.
1981) because most correlations between chromosome
volume and centromere distance from the MCP within
genomes were negative, while all those for whole cells
were positive. It is further supported by pooled data
from nine H. chilense X 5. africanum cells (Table 4)
in that the very highly significant negative correlation
between these characters for whole cells was not
302
T. Schwarzacher-Robinson et al.
Direct or indirect genetic control. The nature of
the presumed genetic control of concentric parental
genome separation is unresolved. In particular, it is
unclear whether the different positions of chromosomes belonging to different parental genomes are
subject to a direct control, or whether they are caused
indirectly by some other process.
Pohler & Clauss (1984) reported that Secale
chromosomes lagged behind Hordeum ones at premeiotic anaphase and at metaphase to telophase of
both meiotic divisions in amphidiploid H. vulgare X
S. montanum. They suggested that such differences in
the rhythm of nuclear division between the parental
genomes may offer a plausible explanation for the
spatial separation of the parental chromosomes seen by
Finch & Bennett (1981) in H. vulgare X S. africanum
and H. vulgare X S. cereale. Similarly, LindeLaursen & Jensen (1984) discussed the possibility that
observed differences in metaphase spatial distributions
between H. vulgare centromeres and others in their
hybrids of diploid H. vulgare with four different
polyploid species might be due to postulated mitotic
asynchrony between the different genomes.
If such ideas were right, the spatial separation of
parental genomes in Hordeum hybrids would probably not be determined by a direct control but arise
indirectly as an effect of whatever causes the asynchronous congression or separation of chromosomes.
Whether the suggestions made by Pohler & Clauss
(1984) and raised by Linde-Laursen & Jensen (1984)
are correct is still unknown, but a comparison of
the temporal events of the mitotic cell cycle in
H. chilense X S. africanum (where 5. africanum centromeres are central) with those in H. vulgare X
5. africanum (where S. africanum centromeres are
peripheral) would provide a telling test. If a tendency
to occupy the peripheral position were caused by
lagging at anaphase, then the centromeres from S.
africanum should lag in H. vulgare X S. africanum
while those of H. chilense should lag in H. chilense X
5. africanum. This comparison has not been made
and, while no consistent lagging of chromosomes
of either parental set was seen in H. chilense X
S. africanum roots in the present work, evidence of
allocycly in this hybrid was noted above. However, the
larger difference in total volume between H. chilense
and S. africanum observed than expected is consistent
with either (1) early condensation of H. chilense
relative to 5. africanum chromosomes or, (2) delayed
condensation of S. africanum relative to H. chilense
chromosomes. It is incompatible with delayed condensation of H. chilense relative to S. africanum chromosomes, which would decrease the difference in the
total volumes of parental genomes compared with that
expected assuming strict proportionality with their
DNA C values. Thus at the moment there is no reason
to expect that H. chilense chromosomes, which are
peripheral in H. chilense X S. africanum, tend to lag
in mitosis, but rather the reverse.
The possibility that the relative positions of
chromosomes within the nucleus are subject to direct
control(s) has been repeatedly suggested (e.g. see
Feldman & Avivi, 1973; Horn & Walden, 1978;
Comings, 1980), as has the idea that variation in the
intranuclear locations of chromosomes may affect or
relate to the extent or effectiveness of their genetic
activity (Feldman & Avivi, 1973; Bennett, 1984a).
Indeed, the question was recently raised as to whether
the intranuclear dispositions of chromosomes and
their segments may vary in a regular way within an
organism, so that they display different arrangements
characteristic of different tissues or stages and, if so,
whether such variation may play a causal role in the
control of development (Mathogef al. 1984; Bennett,
1984a,6; Heslop-Harrison & Bennett, 1984). Experiments to test these ideas are being done using animals
(e.g. Drosophila, Gruenbaum et al. 1984) and angiosperm plants (e.g. grasses, Bennett, Smith & HeslopHarrison, unpublished). Hybrid plants like those
discussed in the present work, which provide variation
in the intranuclear location of centromeres of a given
haploid parental set, so that these are strongly peripheral in one hybrid but strongly central in another,
should be valuable materials in which to study: (1) the
control and consequences of variation in higher-order
nuclear architecture between organisms; and (2)
whether parental genomes vary in their intranuclear
positions between tissues or developmental stages
within an organism.
Until the molecular basis of control of higher-order
nuclear architecture is more fully understood it will be
difficult, if not impossible, to discover if there are
genes whose prime function is to control, or modulate,
the intranuclear dispositions of basic or parental
genomes, individual chromosomes, and/or segments
of chromosomes. However, if it were shown that
variation in higher-order genome architecture is causally correlated with different developmental states,
this would strongly suggest that such genes do exist.
We are grateful to Dr J. S. Heslop-Harrison, who
conducted the computer simulations mentioned in this
paper.
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