1761
Journal of Cell Science 112, 1761-1769 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS3883
Homologous chromosome pairing in wheat
Enrique Martínez-Pérez, Peter Shaw, Steve Reader, Luis Aragón-Alcaide*, Terry Miller and Graham Moore‡
John Innes Centre, Colney, Norwich NR4 7UH, UK
*Present address: NIH, Bethesda, Maryland 20892, USA
‡Author for correspondence (e-mail: foote@bbsrc.ac.uk)
Accepted 24 March; published on WWW 11 May 1999
SUMMARY
Bread wheat is a hexaploid (AABBDD, 2n=6x=42)
containing three related ancestral genomes, each having 7
chromosomes, giving 42 chromosomes in diploid cells.
During meiosis true homologues are correctly associated in
wild-type wheat, but a degree of association of related
chromosomes (homoeologues) occurs in a mutant (ph1b).
We show that the centromeres are associated in nonhomologous pairs in all floral tissues studied, both in wildtype wheat and the ph1b mutant. The non-homologous
centromere associations then become homologous
premeiotically in wild-type wheat in both meiocytes and the
tapetal cells, but not in the mutant. In wild-type wheat, the
homologues are colocalised along their length at this stage,
but the telomeres remain distinct. A single telomere cluster
INTRODUCTION
Chromosome pairing has been studied in detail in budding
yeast, fission yeast, maize and humans (Dawe et al., 1994; Bass
et al., 1997; Scherthan et al., 1996, 1998; Chikashige et al.,
1997; Weiner and Kleckner, 1994). The chromosomes of
hexaploid wheat (Abranches et al., 1998) and other Triticeae,
budding yeast (Jin et al., 1998) and fission yeast (Chikashige
et al., 1997) are organised in the Rabl configuration in
interphase cells, with their centromeres clustered at one pole
and telomeres spread around the other pole. However, this is
not the case for humans (Scherthan et al., 1996), maize (Dong
and Jiang, 1998) and many other species. In all studied species,
a telomere clustering or bouquet is observed during meiotic
prophase (Scherthan et al., 1996; Bass et al., 1997). In maize,
humans and budding yeast (Trelles-Sticken et al., 1999), the
bouquet occurs during the transition between leptotene and
zygotene. In maize and humans, where there is no pre-meiotic
association of homologues, this is suggested to be the first stage
of homologue pairing.
Bread wheat, Triticum aestivum (AABBDD, 2n = 6x = 42)
has seven pairs of chromosomes derived from three ancestral
diploid progenitors (i.e. it is an allopolyploid). In contrast,
maize is proposed to be either a degenerate autotetraploid or a
segmental allopolyploid, having arisen through a whole
genome duplication event (Skrabanek and Wolfe, 1998).
Although each chromosome of hexaploid wheat has the
(bouquet) is formed in the meiocytes only by the onset of
leptotene. The sub-telomeric regions of the homologues
associate as the telomere cluster forms. The homologous
associations at the telomeres and centromeres are
maintained through meiotic prophase, although, during
leptotene, the two homologues and also the sister
chromatids within each homologue are separate along the
rest of their length. As meiosis progresses, first the sister
chromatids and then the homologues associate intimately.
In wild-type wheat, first the centromere grouping, then the
bouquet disperse by the end of zygotene.
Key words: Wheat, Chromosome pairing, Centromere, Telomere,
Ph1
potential to pair with either its homologue or one of the four
equivalent chromosomes from the other two genomes
(homoeologues), chromosome pairing is in fact largely
restricted to homologues. Thus hexaploid wheat behaves as a
diploid with 21 bivalents observed at metaphase 1 (Riley and
Chapman, 1958; Sears, 1976). The major locus (Ph1)
controlling this pairing behaviour is located on chromosome
5B. A mutant (ph1b) carrying a deletion of the Ph1 locus
exhibits a degree of pairing of homoeologous chromosomes
and hence multivalent formation at metaphase 1 (Sears, 1976).
However, most chromosomes are still observed as bivalents at
metaphase 1 in the ph1b mutant and it is stable for several
generations. In wheat hybrids where the opportunity for
homologue pairing does not exist, homoeologues pair and
synapse in either the presence or absence of the Ph1 locus
(Gillies, 1987). Thus the Ph1 locus must function by actively
promoting homologous pairing, and cannot simply be
preventing homoeologous pairing.
Studies on chromosome pairing in yeast, humans or
Drosophila have been able to use single copy probes (Weiner
and Kleckner, 1994; Fung et al., 1998; Scherthan et al., 1996)
to follow the pairing of individual sites on pairs of homologous
chromosomes as against non-homologous chromosomes. This
approach is not possible for hexaploid wheat. In the simplest
case, for each pair of genes on the homologues, there are four
genes on the two homoeologous pairs. Therefore, up to six sites
will be visualized by in situ methods, with no means of
1762 E. Martínez-Pérez and others
assessing whether any observed association involves the genes
on homoeologues or homologues. In practice, the situation is
more complicated; detailed comparative mapping reveals that
many regions in wheat, including the region covering the Ph1
locus, are not only found on the four homoeologues, but are
duplicated on other ‘non-homologous’ chromosomes
(chromosome groups 1, 2 and 7 in the case of the Ph1 region;
Foote et al., 1997). Thus any associations of related genes
could potentially involve 24 different relatives located on
homologous,
homoeologous
and
non-homologous
chromosomes.
Because of the polyploid genome in wheat, it is possible to
substitute or add single chromosome pairs from other related
species (listed in Shepherd and Islam, 1988). For example,
many modern feed wheats possess a pair of rye chromosome
arms. We have taken advantage of wheat carrying either rye or
barley chromosomes to study chromosome dynamics during
meiosis by fluorescence in situ hybridization and confocal
microscopy. We have determined the behaviour of
centromeres, telomeres and three different pairs of labelled
chromosomes, allowing us to formulate a detailed scheme for
meiotic homologue pairing in wheat.
MATERIALS AND METHODS
Wheat genotypes
Anthers used in this study came from 6 different genotypes: Ph1/Ph1
wheat (Triticum aestivum, cv Chinese Spring), termed wild-type
wheat below; ph1b/ph1b homozygous mutant, termed ph1b mutant
below (Sears, 1976); addition lines 3RS and 1RL (hexaploid wheat
carrying an extra pair of 3RS or 1RL rye (Secale cereale) telocentric
chromosomes) (listed in Shepherd and Islam, 1988. 1RL: Sears P6028.2-1; 3RS: Sears P67-29.2-4); a 3RS addition line carrying a single
3RS rye telocentric chromosome; substitution line 5Hch(5A)
(hexaploid wheat with the 5A pair of chromosomes substituted by a
pair of 5Hch wild barley (Hordeum chilensis) chromosomes. All
experiments described were carried out on summer (May-August)
grown plants.
Anther sections
Anther sections were prepared as described by Aragon-Alcaide et al.
(1998). Spikes were harvested at different developmental stages and
immediately fixed for 1 hour in 4% formaldehyde in PEM (50 mM
Pipes, 5 mM EGTA, 5 mM MgSO4, pH 6.9). To facilitate penetration
of the fixative, vacuum infiltration was used. After fixation, spikes
were transferred to 0.1% formaldehyde in PEM, and could be stored
for up to 4 days. Single spikelets were detached from the spike, and
were sectioned (50-100 µm thick sections) under water using a
Vibratome Series 1000 (TAAB Laboratories Equipment Ltd,
Aldermaston, UK). Spikelet sections were placed on multi-well slides
(ICN Biomedicals Inc., Costa Mesa, CA) coated with 2% (v/v)
gamma-aminopropyl triethoxy silane (APTES, Sigma Chemical Co.,
St Louis, MO) and dried overnight at 37°C.
Root squashes
Root tips were germinated for 3-4 days, excised, and fixed in 3:1
ethanol-acetic acid for 1 hour at room temperature, and then stored at
−20°C. After fixation, roots were washed in 1× enzyme buffer (10×
stock: 0.1 M tri-sodium citrate, 0.1 citric acid, pH 4.5) 3 times for 5
minutes. Once washed, roots were digested in 1× enzyme mix (2%
cellulase (w/v), 20% pectinase (w/v), in 1× enzyme buffer) for 2 hours
at 37°C in a shaking incubator. Roots were then washed for 15 minutes
in 1× enzyme buffer, and transferred on to a slide in a drop of 45%
acetic acid. After 1 to 3 minutes in the acetic acid a coverslip was
placed on top of the root and squashed. The slide was then frozen for
a few seconds in liquid nitrogen, and the coverslip removed using a
razor blade. Root preparations were dried overnight at 37°C.
Fluorescence in situ hybridization
Spikelet sections on multi-well slides were treated with 2% (w/v)
cellulase in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4
7H2O, 1.4 mM KH2PO4, pH 7.2) at 37°C for 1 hour, and then washed
2 times for 5 minutes in 2× SSC (1× SSC: 150 mM NaCl, 15 mM
sodium citrate), before dehydration in a series of steps in 70%, 90%
and 100% ethanol. Slides were air dried for 15 minutes. The
hybridization mix consisted of 50% formamide, 10% dextran
sulphate, 2× SSC, 125 ng/µl of blocking DNA (salmon sperm or
sonicated total wheat genomic DNA), 0.125% SDS, 10% water, 1.5
ng/µl of labelled probe. Probes (Cereals Centromeric Sequence 1
(CCS1, Abbo et al., 1995; Aragon-Alcaide et al., 1996); telomeric
repeat (TTTAGGG, Cheung et al., 1994); total rye genomic DNA; and
total barley genomic DNA) were labelled as described by AragonAlcaide et al. (1996). Whenever barley or rye total genomic DNA
were used as a probe, sonicated wheat DNA was used as blocking
DNA. If only centromeric and telomeric probes were used, salmon
sperm DNA was used as blocking DNA. The hybridization mix was
denatured at 100°C for 7 minutes, cooled on ice for 5 minutes and
then immediately added to the sections. The slides were placed in a
modified thermocycler (Omnislide; Hybaid Ltd, Long Island, NY).
Denaturation was carried out at 77°C for 10 minutes, and then
hybridization overnight at 37°C. Post-hybridization washes were
carried out at 42°C with 20% formamide in 0.1× SSC for ten minutes.
Probes were labelled with digoxigenin-11-dUTP (Boehringer
Mannheim Corp., Indianapolis, IN) or biotin-16-dUTP (Boehringer
Mannheim). Probes labelled with digoxigenin were detected using
fluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxigenin
antibody (Boehringer Mannheim), and biotin-labelled probes were
detected with extravidin-Cy3 (Sigma Chemical Co.). Both antibodies
were prepared in 4× SSC, 0.1% Tween-20, 5% BSA. Antibody
incubations were carried out for 1 hour in a humid chamber at 37°C.
After 3 washes with 4× SSC, 0.1% Tween-20, slides were
counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma),
and then mounted in antifade solution (Vectashield; Vector
Laboratories Inc., Burlingame, CA). In the case of the root squashes,
in situ hybridization was carried out as described by Aragon-Alcaide
et al. (1996).
Confocal, fluorescence microscopy and imaging
processing
All the slides were checked after in situ hybridization in a Nikon
Microphot-SA fluorescence microscope (Nikon Inc., Melville, NY).
Confocal optical sections stacks were collected using a Leica TCS SP
confocal microscope (Leica Microsystems, Heidelberg GmbH,
Germany) equipped with a Krypton and an Argon laser. All the DAPI
confocal images were collected using a Bio-Rad MRC-1000 confocal
microscope (Bio-Rad Laboratories, Hercules, CA) equipped with an
UV laser. Images were then transferred to a Macintosh computer, and
composite images were produced using Photoshop (Adobe Systems
Inc., Mountain view, CA) or NIH image (public domain program for
the Macintosh available via anonymous ftp from zippy.nimh.nih.gov).
RESULTS
Staging of meiocyte development
Meiocytes are visually indistinguishable from the
neighbouring somatic cells until a tapetum forms around the
developing meiocytes. Early in premeiotic interphase, the
meiocytes contain two or more nucleoli and have a similar
Homologous chromosome pairing in wheat 1763
Fig. 1. Confocal images of fluorescence in situ
hybridization to 1RL wheat floral tissue at early
developmental stages. Anthers were labelled
with rye genomic DNA (green) to visualize the
pair of 1RL chromosomes. The images shown
are projections of 7 individual consecutive
sections taken with an interval of 1 µm.
(a) Typical interphase chromosomes; the
heterochromatin knob of the distal end is clearly
visible. The arrow indicates a cell with the pair
of rye chromosomes in the V shape. (b) Later
stage than a. Two of the three cells shown have the rye chromosomes associated along their length, but at the distal end the two heterochromatic
knobs are still visible as two discrete signals. Bars, 5 µm.
volume to the surrounding somatic cells. Determination of the
timing of pre-meiotic S phase is problematic (Bennett et al.,
1973, 1979), but the identification of sister chromatids can
provide an unequivocal marker for completion of S phase.
Leptotene is classically defined as the stage at which lateral
element formation occurs between the sister chromatids. By
leptotene there is only a single nucleolus, which is located
adjacent to the nuclear membrane and the nucleus is
significantly larger than somatic cell nuclei. Squashing of the
meiocytes at this stage reveals that the chromatin is starting to
become thread-like. By zygotene, the nucleolus is flattened
against the nuclear membrane and the tapetal cells are
binucleate (Bennett et al., 1973, 1979). By the end of zygotene,
the central element of the synaptonemal complex has formed
and synapsis is complete.
Centromere and telomere dynamics
In all somatic wheat cells so far examined, the centromeres are
grouped at the nuclear periphery at one pole of the nucleus and
the telomeres are spread around the opposite side of the
nucleus, in a typical Rabl configuration (Abranches et al.,
1998). In the early stages of meiocyte development, as shown
in Fig. 1, this centromere/telomere organization is maintained
in both wild-type and ph1b mutant wheat (Fig. 2a,b). Later, the
telomeres begin to cluster to form a single telomere bouquet,
at the opposite pole to the centromere cluster. In wild-type
wheat, the centromeres remain grouped at the nuclear
membrane during telomere clustering (Fig. 2c), whereas in the
ph1b mutant, the centromeres disperse from the membrane
during telomere clustering (Fig. 2d). At the stage when the
bouquet is fully formed, the centromeres remain grouped on
the membrane in the wild-type wheat, but have completely
dispersed in the ph1b mutant (Fig. 2e,f). From the staging
defined by Bennett et al. (1973, 1979) telomere clustering
would occur in wild-type wheat during late pre-meiotic
interphase and in the ph1b mutant during early meiotic
prophase. Later during meiotic prophase, both telomeres and
centromeres disperse (Fig. 2g-j).
Using 3-D data stacks we counted the numbers of telomeres
and centromeres, both in meiocytes at different stages and in
surrounding tapetal and other somatic cells in the flowers
(Table 1). Although hexaploid wheat has 42 centromeres, all
the cells examined from floral tissue, whether somatic cells or
meiocytes, or from wild-type or ph1b mutant, had close to 21
centromere sites. The number of centromere sites remained
unchanged through to meiotic prophase (Table 1). Seedling
roots displayed the diploid number of 42 centromere sites (data
not shown). At present we do not know at which precise stage
of development the centromeres reduce to 21. However, the
presence of only 21 centromeric sites in floral tissue implies
the centromeric regions are associated in pairs.
In contrast to the centromeres, during premeiotic interphase,
telomeres remain spread around the opposite pole of the
Premeiotic chromosome alignment
Fig. 1 shows different premeiotic stages of meiocyte development
from a wheat genotype which carries an additional pair of rye
telocentric (1RL) chromosomes. The presence of a sub-telomeric
heterochromatin knob on this rye chromosome allows easy
identification of the distal telomere. In the earliest stages, the two
homologous chromosomes are separated, as they are in all other
somatic cells we have examined (Fig. 1a,b). Next, the
centromeres associate, giving a V-shaped configuration (arrow in
Fig. 1a). Finally, the two chromosomes colocalise along their
entire length (Fig. 1b). However, the two telomeric
heterochromatin knobs remain distinct, implying that the
chromosomes are not intimately associated. Throughout these
stages, the chromosomes remain in a typical interphase
configuration (Abranches et al., 1998). These results confirm and
extend our previous observations on a wheat genotype containing
a pair of homologous wild barley 5Hch chromosomes (AragonAlcaide et al., 1997b). Premeiotic pairing in wheat has also been
confirmed by the studies by Schwarzacher (1997) and Michailova
et al. (1998). In the Ph1 mutant, this pre-meiotic association is at
a much lower level (Aragon-Alcaide et al., 1997b).
Table 1. Average number of centromeres and telomeres in different cell types from wheat flowers
Somatic cells
Non tapetal cells
Wild-type wheat
ph1b mutant
Meiocytes
Tapetal cells
Pre-telomere cluster
Late zygotene
Cent.
Tel.
Cent.
Tel.
Cent.
Tel.
Cent.
Tel.
20.7 (2.3)
22.5 (2.1)
>60*
>60*
19.9 (1.5)
21.9 (2.1)
>60*
>60*
18.9 (1.4)
19.7 (1.6)
>60*
>60*
21.1 (2.1)
20.3 (2.0)
45.1 (3.2)
40.7 (3.8)
3-D focal section stacks from 15 individual cells were analysed for each cell type or stage. Standard deviation is given in brackets.
*In all cases indicated, the number of telomere sites observed in 100% of the cells was greater than 60.
1764 E. Martínez-Pérez and others
nucleus to the centromeres, but unassociated with each other
(of the 84 telomeres present, more than 60 could be counted in
every nucleus examined, Table 1). Formation of the bouquet in
wild-type wheat is completed in most meiocytes at the two
Fig. 2. Fluorescence in situ hybridization
to centromeres (green) and telomeres (red)
of wheat floral tissue at different stages.
Chromatin was counterstained with DAPI.
Confocal image stacks were recorded with
a section spacing of 1 µm. In the panels
shown here 5-15 sections have been
projected to give a clearer view of the
centromere and telomere distributions, but
it is not possible to show the entire depth
of the nuclei of interest without
obscuration from overlying and
underlying cells. The counting of
telomeres and centromeres was carried out
on the original 3-D focal section stacks. In
each panel a region of the anther section
including the developing meiocytes and
some of the surrounding tapetum is
shown. A single meiocyte from the
section is inset, and one or more single
DAPI sections from the same meiocyte
are shown in order to demonstrate the
nuclear chromatin organization. The
nucleoli, visible as dark holes in the DAPI
image are indicated by arrowheads. Bar, 5
µm, for all panels. (a) Wild-type wheat at
an early pre-meiotic stage. Meiocytes are
barely distinguishable. The nuclei display
a clear Rabl configuration with
centromeres clustered at one nuclear pole,
and telomeres at the opposite pole. Three
nucleoli are visible in the inset cell, the
number of centromeres is haploid and the
number of telomeres is diploid (not all
visible on this projection). (b) Equivalent
stage in the ph1b mutant. Two nucleoli
present, at different depths, Rabl
configuration with a haploid number of
centromeres an a diploid number of
telomeres. (c) Wild-type wheat at a
slightly later stage than a. Rabl
configuration maintained, telomeres
beginning to cluster. Two nucleoli, haploid
number of centromeres. (d) ph1b mutant.
Telomeres are beginning to cluster,
centromeres (haploid number) have
declustered. One central nucleolus is
present. (e) Wild-type wheat at a later
stage than c, showing a tight telomere
bouquet at the opposite pole to the
centromeres, which remain clustered at
the nuclear periphery (haploid number).
Two nucleoli, indicating pre-meiotic
interphase. (f) ph1b mutant. Fully formed
telomere bouquet. Centromeres (haploid
number) dispersed. Nucleolus against
nuclear periphery, indicating late premeiotic interphase/early leptotene.
(g) Meiocyte from wild-type wheat at
later stage than e showing completely
dispersed telomeres and centromeres
(both in a haploid number). (h) DAPI image of the meiocyte shown in g. A single nucleolus is flattened against the nuclear periphery (arrowhead).
(i) ph1b mutant meiocyte at the same stage as g. Centromeres and telomeres are dispersed and in haploid number. (j) DAPI image of i.
Homologous chromosome pairing in wheat 1765
Fig. 3. Confocal sections from 3RS
anthers labelled with rye genomic DNA
(green), and telomeres (red). The
sections are shown in consecutive order
and the last panel of each series is the
projection of the whole stack. Because
of the overlapping of the red and the
green channel when the signals are very
strong, the heterochromatin knobs
present in the telomere cluster appear as
yellow signals. (a) Two meiocytes at
different stages of the telomere cluster.
On panel 7, the arrow indicates two
DNA threads visible. In panel 8
(projection) the two heterochromatin
knobs on the left hand meiocyte are
clearly separated (arrowheads), the
telomere cluster is still forming, and the
two homologous chromosomes are
separated along most of their length. In
the right hand side meiocyte the
telomere cluster is very tight, the two
knobs are only seen as one structure, and
the two homologous chromosomes are
more condensed than the left meiocyte,
and colocalize along their length. In b
and c the consecutive sections only
shows the green channel (rye
chromosome), and the projection shows
the green and the red (telomeres)
channels. (b) Single meiocyte displaying
a telomere cluster. The two homologues
appear as a circular structure, linked at
their centromeres and telomeres. At
some points (indicated by arrows in
panels 3 and 5) the two sister chromatids
can be observed. On panel 6 the two
heterochromatin knobs can still be
identified individually (arrowheads). On
the top right hand corner the telomere
cluster, with a single heterochromatin
knob, from another meiocyte can be
seen. (c) Single meiocyte at a later stage
of the telomere cluster than in b. The
two knobs are visible as a single site,
and in the distal regions the two
homologues appear as a single thick
thread. The two sister chromatids of
both homologues can be seen in the
proximal region (arrows in panels 5 and
9). Section spacing 1 µm. Bars, 5 µm.
nucleoli stage (31 out of 50 nuclei examined) when the
centromeres are still located in 21 pairs at the other pole. In
the ph1b mutant it is completed in meiocytes at the single
nucleolus stage (41 nuclei out of 50 examined) when their
centromeres are dispersed.
Behaviour of homologous chromosomes during
meiotic prophase
To relate the association of homologues with telomere
clustering, we carried out double labelling on the wheat
genotype carrying a pair of 3RS chromosomes (Fig. 3). Of 20
nuclei examined containing a tight telomere cluster, all
possessed a single rye heterochromatin knob, indicating
association of the homologues in the distal regions at the onset
of meiotic prophase. Fig. 3a shows two meiocytes in different
stages of telomere clustering. In the left hand meiocyte,
clustering has just begun. The two telomeric heterochromatin
knobs are apart, although within the developing cluster. In the
right hand meiocyte, the cluster is fully formed and the two
knobs have fused together. In the right hand meiocyte in Fig.
3a, the two homologues are colocalised for most of their length,
but in some regions there is clearly more than one strand visible
1766 E. Martínez-Pérez and others
Fig. 4. 3RS wheat addition line carrying a single chromosome instead
of a pair of homologues. (a) Root tissue squash labelled with rye
genomic DNA, verifying the presence of a single chromosome.
(b,c,d) Confocal images (projections) of meiocytes labelled with rye
genomic DNA (green), and a telomere probe (red). In all three panels
the telomere cluster is present and different degrees of sister chromatid
separation can be observed. In d the two sister chromatids are fully
separated except at the telomeres and centromeres. Bars, 5 µm.
(arrow in panel 7). In Fig. 3b, the two telomeric knobs are very
close together (arrowheads in panel 6). The two homologues
then run apart along different sides of the nucleus, and then
must join at their centromeres since there are no breaks in the
labelled structure, and we have already shown that the
centromeres at this stage are located at the opposite pole of the
nucleus in pairs (Fig. 2). In some places along the
chromosomes, two strands are visible, indicating the presence
of sister chromatids after DNA replication (arrows in panels 3
and 5). Fig. 3c shows another example, but with the homologues
more closely associated. In the region near the telomere cluster,
only one thick strand is visible, but further along four separate
strands can be clearly seen (arrows in panels 5 and 9).
To demonstrate that the individual strands must correspond
to sister chromatids, we also studied a wheat genotype carrying
a single telocentric rye chromosome (3RS), rather than a
homologous pair. Fig. 4a shows somatic root tip cells from this
line, confirming the presence of only a single rye chromosome.
Fig. 4 b-d shows different examples of the single rye
chromosome at the telomere cluster stage. In all cases, two
strands are visible for at least part of the length of the
chromosome, and in Fig. 4d the two chromatids are separated
along their whole length, and only joined at the centromere and
telomere.
Finally, Fig. 5 shows a wheat genotype containing a pair of
wild barley 5Hch metacentric chromosomes at the stage when
the telomeric bouquet is beginning to decluster, and the
centromeres have already dispersed from the membrane
(arrows in panel 10). The homologues are intimately associated
along virtually their entire length. The telomeres from the two
arms are located in different parts of the telomere cluster.
DISCUSSION
We have shown that centromeres associate in non-homologous
pairs in wheat in all floral tissues, and that in wild-type wheat,
but not in ph1b, these associations become homologous premeiotically in tapetum and meiocyte precursors. Telomeres are
not associated prior to meiosis, but are brought together into a
single bouquet at the onset of leptotene, resulting in the
association of homologous telomeric and sub-telomeric
Fig. 5. Confocal sections from a wheat genotype carrying a pair of 5Hch wild barley homologous chromosomes. Panels 1 to 9 show consecutive
sections, taken with a 1 µm interval, of the labelled barley chromosome (green). Panel 10 is the projection of the whole stack with telomeres
and centromeres both labelled in red. The two barley homologues are intimately associated along nearly their whole length, and only the
proximal regions appears more diffuse. The telomere cluster is visible as bright red in panel 10 and is beginning to decluster. The distal end of
the two arms of the barley homologues can be seen at different sides of the telomere cluster. The centromeres are visible as darker red sites on
panel 10 (arrows), and have already dispersed from the nuclear membrane. Bars, 5 µm.
Homologous chromosome pairing in wheat 1767
Fig. 6. Diagram of chromosome pairing events before and during meiosis. The first row shows summary images of chromosome behaviour, in
the appropriate wheat addition/substitution lines. The third row shows summary images of telomeres (red) and centromeres (green). The middle
row is a diagram summarizing the events at each stage.The first 3 columns, a to c, correspond to developmental stages prior to leptotene.
(a) When meiocytes and tapetal cells are morphologically indistinguishable, homologous chromosomes can be clearly seen separated and with
a typical interphase structure (1RL line shown). The cells display a Rabl configuration with centromeres grouped at one pole and telomeres
spread at the opposite pole. Telomeres are present in a diploid number, centromeres are present in a haploid number, indicating they are
associated in pairs, but because chromosomes at this point are separated the centromeres associations must be mostly non-homologous. (b) A
typical V-shaped conformation of chromosomes is shown (1RL line shown). Centromeres and telomeres are still in a Rabl configuration. The
centromeres are present in haploid number, but the associations have now changed to homologous as can be seen by the V shape of the
chromosomes. (c) A later cell than in b, the two homologues colocalize along nearly their whole length. Two heterochromatin knobs are visible,
implying that the chromosomes are not intimately associated (1RL line shown). Centromeres and telomeres still in Rabl configuration, with
centromeres in a haploid number and telomeres in a diploid number. (d) Late S phase/onset of meiotic prophase. The chromosomes are
replicated, as can be seen in the single 3RS chromosome showing with two sister chromatids. The telomeres are clustering, and centromeres are
still in a haploid number grouped on the membrane. (e) The pair of 3RS chromosomes shown are starting to associate intimately from the distal
end where the two homologues are intimately associated. In the proximal regions the two homologues, and sometimes sister chromatids are
visible. The telomeres are tightly clustered, and in the opposite pole, the centromeres, haploid in number, remain grouped on the membrane.
(f) The pair of wild barley 5Hch chromosomes is visible as a single unit, indicating intimate association of homologues, and only the proximal
region appears more diffuse. Centromeres have already dispersed from the membrane (still haploid in number), and telomeres are beginning to
decluster. (g) The homologues are completely associated and are seen as a single thread. Both centromeres and telomeres are completely
dispersed and present in a haploid number.
regions. Thus both centromeres and telomeres are clustered at
opposite nuclear poles, in an extension of the interphase Rabl
configuration. This configuration and the centromere and
telomere associations are maintained through to the completion
of homologue association at the end of zygotene, but at the
onset of leptotene, both sister chromatids and homologues are
visibly separated along the rest of their length. Our results are
based on the analysis of all wheat centromeres and telomeres
and on lines containing either rye telocentric or barley
metacentric chromosomes. All these probes show a consistent
behaviour, so we are confident that the rye and barley
chromosomes behave in a similar way to the endogenous wheat
chromosomes.
Some of the data presented contradict results reported by
previous studies using spread and squashed preparations. For
instance, Michailova et al. (1998) did not observe pre-meiotic
association of homologues in tapetal cells. However, the results
here confirm our previous observations that homologues in
both pre-meiocytes and tapetal cells associate via initial
centromere contacts. We can attribute the contradictions to a
difference in the techniques used. It has been shown both by
Bennett et al. (1979) and by Aragon-Alcaide et al. (1998) that
it is essential to examine structurally well-preserved material,
both to be confident of the 3-D preservation of delicate nuclear
structure, and to be confident of the cell types which are being
analysed. For example, tapetal cells and meiocytes can be
misclassified, and polarised centromere or telomere clusters
can easily be displaced or brought together artefactually by the
process of squashing. We overcame these problems by using
anther sections, which preserves 3-D structure and allow easy
classification of cell types based on their position in the anther.
We show a detailed scheme for premeiotic and meiotic
1768 E. Martínez-Pérez and others
events in wild-type wheat in Fig. 6. The fact that the diploid
number (42) of centromere sites is observed in root tissue, but
only the haploid number (21) in the floral tissue, implies that
the centromeres are mostly in pairs during floral development.
Since this occurs before homologues are associated in floral
development, the initial pairing must be non-homologous. This
stage is represented in Fig. 6a. Sears (1972) demonstrated that
non-homologous telocentric chromosomes can fuse at their
centromeres in wheat, implying that non-homologous
centromeres can pair in the presence of the Ph1 locus.
Although most associations seem to involve 2 centromeres,
occasionally one can count fewer than 21 sites, with some sites
being brighter than the others. This implies that associations of
more than two centromeres can occur. We do not as yet know
at what stage this pairing occurs, or whether it is between
unrelated or homoeologous centromeres. Later, these
centromere associations become homologous in both the
meiocytes and tapetal cells, implying a sorting mechanism, and
an intermediate V-shaped chromosome arrangement is often
observed (Fig. 6b). The homologous chromosomes become
colocalised along their length, but, unlike the centromeres, the
telomeres and heterochromatin knobs remain distinct (Fig. 6c),
since the diploid number of telomere sites (more than 60) are
seen. Premeiotic association of homologous chromosomes and
pairing of centromeres has also been recently supported by the
observation that premeiotic treatment with colchicine disrupts
chromosome pairing in wild-type wheat (Vega and Feldman,
1998). A haploid number of discrete centromere sites have also
been observed in the diploid Lilium during premeiotic
interphase (Suzuki et al., 1997), suggesting a similar
centromere pre-association mechanism.
Then, in the meiocytes only, the telomeres cluster to form a
bouquet by the onset of meiotic prophase. Homologous
telomeres are brought together as the bouquet forms. By the
time the telomeres cluster, the chromosomes have replicated,
and both sister chromatids and homologues are often visibly
separated along their length, although they remain associated
at centromeres and telomeres (Fig. 6d). Since we have used a
telocentric chromosome to monitor the separation, it might be
suggested that the two telomeres have both become located
within the telomere bouquet, resulting in a ‘loop-back’
configuration. This is unlikely for several reasons. First, we
have never observed the centromere of the telocentric
chromosome at the telomere cluster, which would be implied
by this explanation. Second, the chromosome remains linear
with the heterochromatic knob end in the telomere cluster and
the other end in the centromere cluster region, and there is no
sign of a looped conformation. Third, the telocentric
chromosome stretches across the nucleus, as do all the other
chromosomes, and so, if looped back, would have to stretch to
twice its normal length.
The sister chromatids then associate from the distal regions,
followed by the intimate association of the homologues (Fig.
6e). The most obvious interpretation of these observations is
that sister chromatids associate as the lateral elements are
inserted, and homologues associate as central elements are
formed. A conclusion from our work is that the central
elements can be formed before lateral element formation is
complete, and that these two processes, which are usually
regarded as separate stages of meiotic prophase in other
species, may be a continuum in wild-type wheat. Dawe et al.
(1994) have shown previously that during the leptotene/
zygotene transition stage, sister chromatids partially separate
at a time of intimate homologue association in maize. This
suggests this phenomenon may be widespread.
Gillies (1987) noted the difficulty of obtaining synaptonemal
complex spreads from wild-type wheat compared to the ph1b
mutant and concluded that the spreading was causing breakage
of the lateral elements. However an alternative explanation,
which is suggested by our results, is that the lateral element
formation is not complete before central element formation
occurs from the telomere regions. This is also consistent with
the observation that meiotic prophase is shortened in wild-type
wheat compared to its diploid relatives (and the ph1b mutant)
(Bennett et al., 1974).
When homologous chromosome pairing is virtually
complete, first the centromeres and then the telomeres disperse
(Fig. 6f). By the time all the telomeres have dispersed, they are
seen at a haploid number of sites, and homologues are
completely paired (Fig. 6g). Thus, according to classical
definitions, the telomere cluster begins prior to leptotene, and
lasts until the end of zygotene in this species. By the staging
criteria of Bennett et al. (1973, 1979), the single telomere
bouquet would be initially formed during premeiotic
interphase. The occurrence of sister chromatid separation with
the formation of the telomere bouquet places its initial
formation during the pre-meiotic S phase/meiotic prophase
transition and before the stage which has been classically
defined as leptotene. But the labelled chromosomes at this
stage clearly differ from a typical interphase configuration, and
therefore we would include it as part of meiotic prophase.
In budding yeast Weiner and Kleckner (1994) and Loidl et
al. (1994) reported up to 50% premeiotic association of
homologous chromosomes. However, the chromosomes
dissociate during S phase and then reassociate intimately
during meiotic prophase (reviewed by Kleckner, 1996; Roeder,
1997). In wild-type wheat, the premeiotic association of
homologues is much higher (90%) than budding yeast while in
the ph1b mutant it is reduced to 25%. In vegetative cells of
budding yeast, centromeres are clustered at the spindle pole
body with the telomeres separated around the opposite pole of
the nucleus (Jin et al., 1998). In both wild-type wheat and the
ph1b mutant, centromeres are grouped at one pole of the
nucleus and telomeres are spread around the opposite pole. In
wild-type wheat, the association of centromeres in pairs either
in pre-meiotic or meiotic cells is maintained after squashing.
However, diffuse centromere structures, in which individual
centromeres are not well enough differentiated to count, are
observable after squashing nuclei from the ph1b mutant
(Aragon-Alcaide et al., 1997a). This implies that the Ph1 locus
affects the centromere structure.
In both budding and fission yeast, the centromeres decluster
from the spindle pole body as the telomeres cluster to the
spindle pole body (Chikashige et al., 1997; Trelles-Sticken et
al., 1999). This implies a major reorganization of the
chromosomes and loss of the Rabl configuration. In the ph1b
mutant, the telomeres cluster when the centromeres are no
longer grouped. Thus the telomere cluster occurs at a later
stage than in wild-type wheat, and is accompanied by the loss
of the Rabl configuration in the meiocytes. This is similar to
the situations described in maize (Bass et al., 1997) and
humans (Scherthan et al., 1996, 1998).
Homologous chromosome pairing in wheat 1769
To date meiosis has been described in detail in two plants,
wheat and maize (Dawe et al., 1994; Bass et al., 1997), and
significant differences in the mechanism of homologue pairing
have been discovered. Both species are polyploid, but maize
has recently been proposed to be either a degenerate
autotetraploid or a segmental allopolyploid (Skrabanek and
Wolfe, 1998), while wheat is clearly an allopolyploid. The
requirement for stable maintenance of these two different types
of polyploids could account for the differences in meiotic
mechanism. Our studies have shown that the presence of the
Ph1 locus results in a premeiotic association of homologues
via centromeres during floral development and an earlier
association of sub-telomeric regions via telomeric clustering at
the onset of meiotic prophase. Other studies have shown that
the presence of the locus eliminates recombination between
homoeologous chromosomes or segments (Gillies et al., 1987;
Luo et al., 1996). As yet it is unclear whether these two effects
of the Ph1 locus are functionally related, or whether the Ph1
locus comprises two or more genes which have differing effects
on premeiotic and meiotic events. The recent identification of
new deletions of the Ph1 locus should resolve this issue (Miller
et al., 1998; Snape et al., 1998).
We thank Tracie Foote and Alison Beven for technical assistance.
This work was funded by the Biotechnology and Biological Sciences
Research Council of the UK, and the John Innes Foundation (E. M.P.).
REFERENCES
Abbo, S., Dunford R. P., Foote, T. N., Reader, S. M., Flavell, R. B. and
Moore, G. (1995). Organisation of retroelement and stem-loop repeat
families in the genomes and nuclei of cereals. Chromosome Res. 3, 5-15.
Abranches, R., Beven, A. F., Aragon-Alcaide, L. and Shaw, P. J. (1998).
Transcription sites are not correlated with chromosome domains in wheat
nuclei. J. Cell Biol. 143, 5-12.
Aragon-Alcaide, L., Miller, T., Schwarzacher, T., Reader, S. and Moore,
G. (1996). A cereal centromeric sequence. Chromosoma 105, 261-268.
Aragon-Alcaide, L., Reader, S., Miller, T. and Moore, G. (1997a).
Centromeric behaviour in wheat with high and low homoeologous
chromosomal pairing. Chromosoma 106, 327-333.
Aragon-Alcaide, L., Reader, S., Beven, A., Shaw, P., Miller, T. and Moore,
G. (1997b). Association of homologous chromosomes during floral
development. Curr. Biol. 7, 905-908.
Aragon-Alcaide, L., Beven, A., Moore, G. and Shaw, P. (1998). The use of
vibratome sections of cereal spikelets to study anther development and
meiosis. The Plant J. 14, 503-508
Bass, H. W., Marshall, W. F., Sedat, J. W., Agard, D. A. and Cande, W. Z.
(1997). Telomeres cluster de novo before the initiation of synapsis: A threedimensional spatial analysis of telomere positions before and during meiotic
prophase. J. Cell Biol. 137, 5-18.
Bennett, M. D., Smith, J. B. and Bayliss, M. W. (1973). Cell development
in the anther, the ovule, and the young seed of Triticum aestivum L. var.
Chinese Spring. Phil. Trans. R. Soc. Lond. Ser. B. 266, 39-81.
Bennett, M. D., Dover, G. A. and Riley, R. (1974). Meiotic duration in wheat
Genotypes with and without homoeologous meiotic chromosomes. Proc. R.
Soc. Lond. Ser. B. 187, 191-207.
Bennett, M. D., Smith, J. B. Simpson, S. and Wells, B. (1979). Intranuclear
fibrillar material in cereals pollen mother cells. Chromosoma 71, 289-332.
Cheung, W. Y., Money T. A., Abbo, S., Devos, K. M., Gale, M. D. and
Moore, G. (1994). A family of related sequences associated with
(TTTAGGG)n repeats are located in the interstitial regions of wheat
chromosomes. Mol. Gen. Genet. 245, 349-354.
Chikashige, Y., Ding, D. Q., Imai, Y., Yamamoto, M., Haraguchi, T. and
Hiraoka, Y. (1997). Meiotic nuclear reorganization: switching the position
of centromeres and telomeres in the fission yeast Schizosaccharomyces
pombe. EMBO J. 16, 193-202.
Dawe, K. R., Sedat, J. W., Agard, D. A. and Cande, W. Z. (1994). Meiotic
Chromosome pairing in maize is associated with a novel chromatin
organisation. Cell 76, 901-912.
Dong, F. and Jiang, J. (1998). Non-Rabl patterns of centromere and telomere
Distribution in interphase nuclei of plant cells. Chrom. Res. 6, 551-558.
Foote, T., Roberts, M., Kurata, N., Sasaki, T. and Moore, G. (1997).
Detailed comparative mapping of cereal chromosome regions corresponding
to the Ph1 locus in wheat. Genetics 147, 801-807.
Fung, J. C., W. F. Marshall, W. F., Dernburg, A., Agard, D. A. and Sedat,
J. W. (1998). Homologous chromosome pairing in Drosophila
melanogaster proceeds through multiple independent initiations. J. Cell
Biol. 141, 5-20.
Gillies, C. B. (1987). The effect of the Ph gene alleles on synaptonemal
complex formation in Triticum aestivum x T kotshyi hybrids. Theor. Appl.
Genet. 74, 430-438.
Jin, Q. W., Trelles-Sticken, E., Scherthan, H. and Loidl. J. (1998). Yeast
nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase. J. Cell Biol. 141, 21-29.
Kleckner, N. (1996). Meiosis-how does it work?. Proc. Nat. Acad. Sci. USA
93, 8167-8174.
Loidl, J., Klein, F. and Scherthan, H. (1994). Homologous pairing is reduced
but not abolished in asynaptic mutants in yeast. J. Cell Biol. 125, 1191-1200.
Luo, M. C., Dubcousky, J. and Dvorak, J. (1996). Recognition of
homoeology by wheat Ph1 locus. Genetics 144, 1195-1203.
Michailova, E. I., Naranjo, T., Shepherd, K., Wennekes-van Eden, J.,
Heyting, C., de Jong, J. H. (1998). The effect of the wheat Ph1 locus on
chromatin organization and meiotic chromosome pairing analysed by
genomic painting. Chromosoma 107, 339-350.
Miller T., Reader, S., Shaw, P. and Moore, G. (1998). Towards an
understanding of the biological action of the Ph1 locus in wheat. Proc. 9th
Int. Wheat Genetics Symp. 1, 17-19.
Riley, R. and Chapman, V. (1958). Genetic control of the cytologically
diploid behaviour of hexaploid wheat. Nature 182, 713-715.
Roeder, G. S. (1997). Meiotic chromosomes: it takes two to tango. Genes Dev.
11, 2600-2621.
Scherthan, H., Weich, S., Schwegler, H., Heyting, C., Harle, M. and
Cremer. T. (1996). Centromere and telomere movements during early
meiotic prophase of mouse and man are associated with the onset of
chromosome pairing. J. Cell Biol. 134, 1109-1125.
Scherthan, H., Eils, R., Trelles-Sticken, E., Dietzel, S., Cremer, T., Walt,
H. and Jauch, A. (1998). Aspects of three-dimensional chromosome
reorganisation during the onset of human male meiotic prophase. J. Cell Sci.
111, 2337 2351.
Schwarzacher, T. (1997). Three stages of meiotic homologous chromosome
pairing in wheat: cognition, alignment and synapsis. Sex. Plant Reprod. 10,
324-331.
Sears, E. R. (1972). Chromosome engineering in wheat. Stadler Symposia 4,
23-38.
Sears, E. R. (1976). Genetic control of chromosome pairing in wheat. Annu.
Rev. Genet. 10, 31-51.
Shepherd, K. W. and Islam. A. K. M. R. (1988). Fourth compendium of
wheat-alien chromosomes lines. Proc. Seventh Int. Wheat Genetics Symp.
1, 1373-1395.
Skrabanek, L. and Wolfe. K. H. (1998). Eukaryote genome duplicationwhere’s the evidence? Curr. Opin. Genet. Dev. 8, 694-700.
Snape S. W, Miller, T. E., Roberts, M., Reader, S. M., Fish, L. J., Foote,
T. N. and Moore, G. (1998). A new hexaploid wheat mutant exhibiting
high homoeologous chromosome pairing. Annual Wheat Newsletter 44,
254-255.
Suzuki, T., Ide, N. and Tanaka, I. (1997). Immunocytochemical visualization
of the centromeres during male and female meiosis in Lilium longiflorum.
Chromosoma 106, 435-445.
Trelles-Sticken, E., Loidl, J. and Scherthan, H. (1999). Bouquet formation
in budding yeast: Initiation of recombination is not required for meiotic
telomere clustering. J. Cell Sci. 112, 651-658.
Vega, J. M. and Feldman, M. (1998). Effect of the pairing gene Ph1 and
premeiotic colchicine treatment on intra and interchromosome pairing of
isochromosomes in common wheat. Genetics 150, 1199-1208.
Weiner, B. and Klecker, N. (1994). Chromosome pairing via multiple
interactions before and during meiosis in yeast. Cell 77, 977-991.