Journal of Cell Science 112, 1761-1769 (1999) 1761 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: [email protected])

Accepted 24 March; published on WWW 11 May 1999

SUMMARY Bread wheat is a hexaploid (AABBDD, 2n=6x=42) (bouquet) is formed in the meiocytes only by the onset of containing three related ancestral genomes, each having 7 leptotene. The sub-telomeric regions of the homologues chromosomes, giving 42 chromosomes in diploid cells. associate as the telomere cluster forms. The homologous During meiosis true homologues are correctly associated in associations at the telomeres and centromeres are wild-type wheat, but a degree of association of related maintained through meiotic prophase, although, during chromosomes (homoeologues) occurs in a mutant (ph1b). leptotene, the two homologues and also the sister We show that the centromeres are associated in non- chromatids within each homologue are separate along the homologous pairs in all floral tissues studied, both in wild- rest of their length. As meiosis progresses, first the sister type wheat and the ph1b mutant. The non-homologous chromatids and then the homologues associate intimately. centromere associations then become homologous In wild-type wheat, first the centromere grouping, then the premeiotically in wild-type wheat in both meiocytes and the bouquet disperse by the end of zygotene. tapetal cells, but not in the mutant. In wild-type wheat, the homologues are colocalised along their length at this stage, Key words: Wheat, Chromosome pairing, Centromere, Telomere, but the telomeres remain distinct. A single telomere cluster Ph1

INTRODUCTION potential to pair with either its homologue or one of the four equivalent chromosomes from the other two genomes Chromosome pairing has been studied in detail in budding (homoeologues), chromosome pairing is in fact largely yeast, fission yeast, maize and humans (Dawe et al., 1994; Bass restricted to homologues. Thus hexaploid wheat behaves as a et al., 1997; Scherthan et al., 1996, 1998; Chikashige et al., diploid with 21 bivalents observed at metaphase 1 (Riley and 1997; Weiner and Kleckner, 1994). The chromosomes of Chapman, 1958; Sears, 1976). The major locus (Ph1) hexaploid wheat (Abranches et al., 1998) and other Triticeae, controlling this pairing behaviour is located on chromosome budding yeast (Jin et al., 1998) and fission yeast (Chikashige 5B. A mutant (ph1b) carrying a deletion of the Ph1 locus et al., 1997) are organised in the Rabl configuration in exhibits a degree of pairing of homoeologous chromosomes interphase cells, with their centromeres clustered at one pole and hence multivalent formation at metaphase 1 (Sears, 1976). and telomeres spread around the other pole. However, this is However, most chromosomes are still observed as bivalents at not the case for humans (Scherthan et al., 1996), maize (Dong metaphase 1 in the ph1b mutant and it is stable for several and Jiang, 1998) and many other species. In all studied species, generations. In wheat hybrids where the opportunity for a telomere clustering or bouquet is observed during meiotic homologue pairing does not exist, homoeologues pair and prophase (Scherthan et al., 1996; Bass et al., 1997). In maize, synapse in either the presence or absence of the Ph1 locus humans and budding yeast (Trelles-Sticken et al., 1999), the (Gillies, 1987). Thus the Ph1 locus must function by actively bouquet occurs during the transition between leptotene and promoting homologous pairing, and cannot simply be zygotene. In maize and humans, where there is no pre-meiotic preventing homoeologous pairing. association of homologues, this is suggested to be the first stage Studies on chromosome pairing in yeast, humans or of homologue pairing. Drosophila have been able to use single copy probes (Weiner Bread wheat, Triticum aestivum (AABBDD, 2n = 6x = 42) and Kleckner, 1994; Fung et al., 1998; Scherthan et al., 1996) has seven pairs of chromosomes derived from three ancestral to follow the pairing of individual sites on pairs of homologous diploid progenitors (i.e. it is an allopolyploid). In contrast, chromosomes as against non-homologous chromosomes. This maize is proposed to be either a degenerate autotetraploid or a approach is not possible for hexaploid wheat. In the simplest segmental allopolyploid, having arisen through a whole case, for each pair of genes on the homologues, there are four genome duplication event (Skrabanek and Wolfe, 1998). genes on the two homoeologous pairs. Therefore, up to six sites Although each chromosome of hexaploid wheat has the 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 acetic acid. After 1 to 3 minutes in the acetic acid a coverslip was on homoeologues or homologues. In practice, the situation is placed on top of the root and squashed. The slide was then frozen for more complicated; detailed comparative mapping reveals that a few seconds in liquid nitrogen, and the coverslip removed using a many regions in wheat, including the region covering the Ph1 razor blade. Root preparations were dried overnight at 37¡C. locus, are not only found on the four homoeologues, but are Fluorescence in situ hybridization duplicated on other ‘non-homologous’ chromosomes Spikelet sections on multi-well slides were treated with 2% (w/v) (chromosome groups 1, 2 and 7 in the case of the Ph1 region; cellulase in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 Foote et al., 1997). Thus any associations of related genes 7H2O, 1.4 mM KH2PO4, pH 7.2) at 37¡C for 1 hour, and then washed could potentially involve 24 different relatives located on 2 times for 5 minutes in 2× SSC (1× SSC: 150 mM NaCl, 15 mM homologous, homoeologous and non-homologous sodium citrate), before dehydration in a series of steps in 70%, 90% chromosomes. and 100% ethanol. Slides were air dried for 15 minutes. The Because of the polyploid genome in wheat, it is possible to hybridization mix consisted of 50% formamide, 10% dextran substitute or add single chromosome pairs from other related sulphate, 2× SSC, 125 ng/µl of blocking DNA (salmon sperm or species (listed in Shepherd and Islam, 1988). For example, sonicated total wheat genomic DNA), 0.125% SDS, 10% water, 1.5 µ many modern feed wheats possess a pair of rye chromosome ng/ l of labelled probe. Probes (Cereals Centromeric Sequence 1 arms. We have taken advantage of wheat carrying either rye or (CCS1, Abbo et al., 1995; Aragon-Alcaide et al., 1996); telomeric repeat (TTTAGGG, Cheung et al., 1994); total rye genomic DNA; and barley chromosomes to study chromosome dynamics during total barley genomic DNA) were labelled as described by Aragon- meiosis by fluorescence in situ hybridization and confocal Alcaide et al. (1996). Whenever barley or rye total genomic DNA microscopy. We have determined the behaviour of were used as a probe, sonicated wheat DNA was used as blocking centromeres, telomeres and three different pairs of labelled DNA. If only centromeric and telomeric probes were used, salmon chromosomes, allowing us to formulate a detailed scheme for sperm DNA was used as blocking DNA. The hybridization mix was meiotic homologue pairing in wheat. 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 MATERIALS AND METHODS hybridization overnight at 37¡C. Post-hybridization washes were carried out at 42¡C with 20% formamide in 0.1× SSC for ten minutes. Wheat genotypes Probes were labelled with digoxigenin-11-dUTP (Boehringer Anthers used in this study came from 6 different genotypes: Ph1/Ph1 Mannheim Corp., Indianapolis, IN) or biotin-16-dUTP (Boehringer wheat (Triticum aestivum, cv Chinese Spring), termed wild-type Mannheim). Probes labelled with digoxigenin were detected using wheat below; ph1b/ph1b homozygous mutant, termed ph1b mutant fluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxigenin below (Sears, 1976); addition lines 3RS and 1RL (hexaploid wheat antibody (Boehringer Mannheim), and biotin-labelled probes were carrying an extra pair of 3RS or 1RL rye (Secale cereale) telocentric detected with extravidin-Cy3 (Sigma Chemical Co.). Both antibodies chromosomes) (listed in Shepherd and Islam, 1988. 1RL: Sears P60- were prepared in 4× SSC, 0.1% Tween-20, 5% BSA. Antibody 28.2-1; 3RS: Sears P67-29.2-4); a 3RS addition line carrying a single incubations were carried out for 1 hour in a humid chamber at 37¡C. 3RS rye telocentric chromosome; substitution line 5Hch(5A) After 3 washes with 4× SSC, 0.1% Tween-20, slides were (hexaploid wheat with the 5A pair of chromosomes substituted by a counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma), pair of 5Hch wild barley (Hordeum chilensis) chromosomes. All and then mounted in antifade solution (Vectashield; Vector experiments described were carried out on summer (May-August) Laboratories Inc., Burlingame, CA). In the case of the root squashes, grown plants. in situ hybridization was carried out as described by Aragon-Alcaide et al. (1996). Anther sections Anther sections were prepared as described by Aragon-Alcaide et al. Confocal, fluorescence microscopy and imaging (1998). Spikes were harvested at different developmental stages and processing immediately fixed for 1 hour in 4% formaldehyde in PEM (50 mM All the slides were checked after in situ hybridization in a Nikon Pipes, 5 mM EGTA, 5 mM MgSO4, pH 6.9). To facilitate penetration Microphot-SA fluorescence microscope (Nikon Inc., Melville, NY). of the fixative, vacuum infiltration was used. After fixation, spikes Confocal optical sections stacks were collected using a Leica TCS SP were transferred to 0.1% formaldehyde in PEM, and could be stored confocal microscope (Leica Microsystems, Heidelberg GmbH, for up to 4 days. Single spikelets were detached from the spike, and Germany) equipped with a Krypton and an Argon laser. All the DAPI were sectioned (50-100 µm thick sections) under water using a confocal images were collected using a Bio-Rad MRC-1000 confocal Vibratome Series 1000 (TAAB Laboratories Equipment Ltd, microscope (Bio-Rad Laboratories, Hercules, CA) equipped with an Aldermaston, UK). Spikelet sections were placed on multi-well slides UV laser. Images were then transferred to a Macintosh computer, and (ICN Biomedicals Inc., Costa Mesa, CA) coated with 2% (v/v) composite images were produced using Photoshop (Adobe Systems gamma-aminopropyl triethoxy silane (APTES, Sigma Chemical Co., Inc., Mountain view, CA) or NIH image (public domain program for St Louis, MO) and dried overnight at 37¡C. the Macintosh available via anonymous ftp from zippy.nimh.nih.gov). Root squashes Root tips were germinated for 3-4 days, excised, and fixed in 3:1 RESULTS 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 Staging of meiocyte development 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% Meiocytes are visually indistinguishable from the cellulase (w/v), 20% pectinase (w/v), in 1× enzyme buffer) for 2 hours neighbouring somatic cells until a tapetum forms around the at 37¡C in a shaking incubator. Roots were then washed for 15 minutes developing meiocytes. Early in premeiotic interphase, the in 1× enzyme buffer, and transferred on to a slide in a drop of 45% 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 Centromere and telomere dynamics timing of pre-meiotic S phase is problematic (Bennett et al., In all somatic wheat cells so far examined, the centromeres are 1973, 1979), but the identification of sister chromatids can grouped at the nuclear periphery at one pole of the nucleus and provide an unequivocal marker for completion of S phase. the telomeres are spread around the opposite side of the Leptotene is classically defined as the stage at which lateral nucleus, in a typical Rabl configuration (Abranches et al., element formation occurs between the sister chromatids. By 1998). In the early stages of meiocyte development, as shown leptotene there is only a single nucleolus, which is located in Fig. 1, this centromere/telomere organization is maintained adjacent to the nuclear membrane and the nucleus is in both wild-type and ph1b mutant wheat (Fig. 2a,b). Later, the significantly larger than somatic cell nuclei. Squashing of the telomeres begin to cluster to form a single telomere bouquet, meiocytes at this stage reveals that the chromatin is starting to at the opposite pole to the centromere cluster. In wild-type become thread-like. By zygotene, the nucleolus is flattened wheat, the centromeres remain grouped at the nuclear against the nuclear membrane and the tapetal cells are membrane during telomere clustering (Fig. 2c), whereas in the binucleate (Bennett et al., 1973, 1979). By the end of zygotene, ph1b mutant, the centromeres disperse from the membrane the central element of the synaptonemal complex has formed during telomere clustering (Fig. 2d). At the stage when the and synapsis is complete. bouquet is fully formed, the centromeres remain grouped on the membrane in the wild-type wheat, but have completely Premeiotic chromosome alignment dispersed in the ph1b mutant (Fig. 2e,f). From the staging Fig. 1 shows different premeiotic stages of meiocyte development defined by Bennett et al. (1973, 1979) telomere clustering from a wheat genotype which carries an additional pair of rye would occur in wild-type wheat during late pre-meiotic telocentric (1RL) chromosomes. The presence of a sub-telomeric interphase and in the ph1b mutant during early meiotic heterochromatin knob on this rye chromosome allows easy prophase. Later during meiotic prophase, both telomeres and identification of the distal telomere. In the earliest stages, the two centromeres disperse (Fig. 2g-j). homologous chromosomes are separated, as they are in all other Using 3-D data stacks we counted the numbers of telomeres somatic cells we have examined (Fig. 1a,b). Next, the and centromeres, both in meiocytes at different stages and in centromeres associate, giving a V-shaped configuration (arrow in surrounding tapetal and other somatic cells in the flowers Fig. 1a). Finally, the two chromosomes colocalise along their (Table 1). Although hexaploid wheat has 42 centromeres, all entire length (Fig. 1b). However, the two telomeric the cells examined from floral tissue, whether somatic cells or heterochromatin knobs remain distinct, implying that the meiocytes, or from wild-type or ph1b mutant, had close to 21 chromosomes are not intimately associated. Throughout these centromere sites. The number of centromere sites remained stages, the chromosomes remain in a typical interphase unchanged through to meiotic prophase (Table 1). Seedling configuration (Abranches et al., 1998). These results confirm and roots displayed the diploid number of 42 centromere sites (data extend our previous observations on a wheat genotype containing not shown). At present we do not know at which precise stage a pair of homologous wild barley 5Hch chromosomes (Aragon- of development the centromeres reduce to 21. However, the Alcaide et al., 1997b). Premeiotic pairing in wheat has also been presence of only 21 centromeric sites in floral tissue implies confirmed by the studies by Schwarzacher (1997) and Michailova the centromeric regions are associated in pairs. et al. (1998). In the Ph1 mutant, this pre-meiotic association is at In contrast to the centromeres, during premeiotic interphase, a much lower level (Aragon-Alcaide et al., 1997b). telomeres remain spread around the opposite pole of the

Table 1. Average number of centromeres and telomeres in different cell types from wheat flowers Somatic cells Meiocytes Non tapetal cells Tapetal cells Pre-telomere cluster Late zygotene Cent. Tel. Cent. Tel. Cent. Tel. Cent. Tel. Wild-type wheat 20.7 (2.3) >60* 19.9 (1.5) >60* 18.9 (1.4) >60* 21.1 (2.1) 45.1 (3.2) ph1b mutant 22.5 (2.1) >60* 21.9 (2.1) >60* 19.7 (1.6) >60* 20.3 (2.0) 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 every nucleus examined, Table 1). Formation of the bouquet in (of the 84 telomeres present, more than 60 could be counted 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 pre- meiotic 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 nuclei examined containing a tight telomere cluster, all centromeres are still located in 21 pairs at the other pole. In possessed a single rye heterochromatin knob, indicating the ph1b mutant it is completed in meiocytes at the single association of the homologues in the distal regions at the onset nucleolus stage (41 nuclei out of 50 examined) when their of meiotic prophase. Fig. 3a shows two meiocytes in different centromeres are dispersed. stages of telomere clustering. In the left hand meiocyte, clustering has just begun. The two telomeric heterochromatin Behaviour of homologous chromosomes during knobs are apart, although within the developing cluster. In the meiotic prophase right hand meiocyte, the cluster is fully formed and the two To relate the association of homologues with telomere knobs have fused together. In the right hand meiocyte in Fig. clustering, we carried out double labelling on the wheat 3a, the two homologues are colocalised for most of their length, genotype carrying a pair of 3RS chromosomes (Fig. 3). Of 20 but in some regions there is clearly more than one strand visible 1766 E. Martínez-Pérez and others

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 Fig. 4. 3RS wheat addition line carrying a single chromosome instead centromeres have already dispersed from the membrane of a pair of homologues. (a) Root tissue squash labelled with rye (arrows in panel 10). The homologues are intimately associated genomic DNA, verifying the presence of a single chromosome. along virtually their entire length. The telomeres from the two (b,c,d) Confocal images (projections) of meiocytes labelled with rye arms are located in different parts of the telomere cluster. 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. DISCUSSION We have shown that centromeres associate in non-homologous (arrow in panel 7). In Fig. 3b, the two telomeric knobs are very pairs in wheat in all floral tissues, and that in wild-type wheat, close together (arrowheads in panel 6). The two homologues but not in ph1b, these associations become homologous pre- then run apart along different sides of the nucleus, and then meiotically in tapetum and meiocyte precursors. Telomeres are must join at their centromeres since there are no breaks in the not associated prior to meiosis, but are brought together into a labelled structure, and we have already shown that the single bouquet at the onset of leptotene, resulting in the centromeres at this stage are located at the opposite pole of 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 association of homologues in tapetal cells. However, the results opposite nuclear poles, in an extension of the interphase Rabl here confirm our previous observations that homologues in configuration. This configuration and the centromere and both pre-meiocytes and tapetal cells associate via initial telomere associations are maintained through to the completion centromere contacts. We can attribute the contradictions to a of homologue association at the end of zygotene, but at the difference in the techniques used. It has been shown both by onset of leptotene, both sister chromatids and homologues are Bennett et al. (1979) and by Aragon-Alcaide et al. (1998) that visibly separated along the rest of their length. Our results are it is essential to examine structurally well-preserved material, based on the analysis of all wheat centromeres and telomeres both to be confident of the 3-D preservation of delicate nuclear and on lines containing either rye telocentric or barley structure, and to be confident of the cell types which are being metacentric chromosomes. All these probes show a consistent analysed. For example, tapetal cells and meiocytes can be behaviour, so we are confident that the rye and barley misclassified, and polarised centromere or telomere clusters chromosomes behave in a similar way to the endogenous wheat can easily be displaced or brought together artefactually by the chromosomes. process of squashing. We overcame these problems by using Some of the data presented contradict results reported by anther sections, which preserves 3-D structure and allow easy previous studies using spread and squashed preparations. For classification of cell types based on their position in the anther. instance, Michailova et al. (1998) did not observe pre-meiotic 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 (1994) have shown previously that during the leptotene/ number (42) of centromere sites is observed in root tissue, but zygotene transition stage, sister chromatids partially separate only the haploid number (21) in the floral tissue, implies that at a time of intimate homologue association in maize. This the centromeres are mostly in pairs during floral development. suggests this phenomenon may be widespread. Since this occurs before homologues are associated in floral Gillies (1987) noted the difficulty of obtaining synaptonemal development, the initial pairing must be non-homologous. This complex spreads from wild-type wheat compared to the ph1b stage is represented in Fig. 6a. Sears (1972) demonstrated that mutant and concluded that the spreading was causing breakage non-homologous telocentric chromosomes can fuse at their of the lateral elements. However an alternative explanation, centromeres in wheat, implying that non-homologous which is suggested by our results, is that the lateral element centromeres can pair in the presence of the Ph1 locus. formation is not complete before central element formation Although most associations seem to involve 2 centromeres, occurs from the telomere regions. This is also consistent with occasionally one can count fewer than 21 sites, with some sites the observation that meiotic prophase is shortened in wild-type being brighter than the others. This implies that associations of wheat compared to its diploid relatives (and the ph1b mutant) more than two centromeres can occur. We do not as yet know (Bennett et al., 1974). at what stage this pairing occurs, or whether it is between When homologous chromosome pairing is virtually unrelated or homoeologous centromeres. Later, these complete, first the centromeres and then the telomeres disperse centromere associations become homologous in both the (Fig. 6f). By the time all the telomeres have dispersed, they are meiocytes and tapetal cells, implying a sorting mechanism, and seen at a haploid number of sites, and homologues are an intermediate V-shaped chromosome arrangement is often completely paired (Fig. 6g). Thus, according to classical observed (Fig. 6b). The homologous chromosomes become definitions, the telomere cluster begins prior to leptotene, and colocalised along their length, but, unlike the centromeres, the lasts until the end of zygotene in this species. By the staging telomeres and heterochromatin knobs remain distinct (Fig. 6c), criteria of Bennett et al. (1973, 1979), the single telomere since the diploid number of telomere sites (more than 60) are bouquet would be initially formed during premeiotic seen. Premeiotic association of homologous chromosomes and interphase. The occurrence of sister chromatid separation with pairing of centromeres has also been recently supported by the the formation of the telomere bouquet places its initial observation that premeiotic treatment with colchicine disrupts formation during the pre-meiotic S phase/meiotic prophase chromosome pairing in wild-type wheat (Vega and Feldman, transition and before the stage which has been classically 1998). A haploid number of discrete centromere sites have also defined as leptotene. But the labelled chromosomes at this been observed in the diploid Lilium during premeiotic stage clearly differ from a typical interphase configuration, and interphase (Suzuki et al., 1997), suggesting a similar therefore we would include it as part of meiotic prophase. centromere pre-association mechanism. In budding yeast Weiner and Kleckner (1994) and Loidl et Then, in the meiocytes only, the telomeres cluster to form a al. (1994) reported up to 50% premeiotic association of bouquet by the onset of meiotic prophase. Homologous homologous chromosomes. However, the chromosomes telomeres are brought together as the bouquet forms. By the dissociate during S phase and then reassociate intimately time the telomeres cluster, the chromosomes have replicated, during meiotic prophase (reviewed by Kleckner, 1996; Roeder, and both sister chromatids and homologues are often visibly 1997). In wild-type wheat, the premeiotic association of separated along their length, although they remain associated homologues is much higher (90%) than budding yeast while in at centromeres and telomeres (Fig. 6d). Since we have used a the ph1b mutant it is reduced to 25%. In vegetative cells of telocentric chromosome to monitor the separation, it might be budding yeast, centromeres are clustered at the spindle pole suggested that the two telomeres have both become located body with the telomeres separated around the opposite pole of within the telomere bouquet, resulting in a ‘loop-back’ the nucleus (Jin et al., 1998). In both wild-type wheat and the configuration. This is unlikely for several reasons. First, we ph1b mutant, centromeres are grouped at one pole of the have never observed the centromere of the telocentric nucleus and telomeres are spread around the opposite pole. In chromosome at the telomere cluster, which would be implied wild-type wheat, the association of centromeres in pairs either by this explanation. Second, the chromosome remains linear in pre-meiotic or meiotic cells is maintained after squashing. with the heterochromatic knob end in the telomere cluster and However, diffuse centromere structures, in which individual the other end in the centromere cluster region, and there is no centromeres are not well enough differentiated to count, are sign of a looped conformation. Third, the telocentric observable after squashing nuclei from the ph1b mutant chromosome stretches across the nucleus, as do all the other (Aragon-Alcaide et al., 1997a). This implies that the Ph1 locus chromosomes, and so, if looped back, would have to stretch to affects the centromere structure. twice its normal length. In both budding and fission yeast, the centromeres decluster The sister chromatids then associate from the distal regions, from the spindle pole body as the telomeres cluster to the followed by the intimate association of the homologues (Fig. spindle pole body (Chikashige et al., 1997; Trelles-Sticken et 6e). The most obvious interpretation of these observations is al., 1999). This implies a major reorganization of the that sister chromatids associate as the lateral elements are chromosomes and loss of the Rabl configuration. In the ph1b inserted, and homologues associate as central elements are mutant, the telomeres cluster when the centromeres are no formed. A conclusion from our work is that the central longer grouped. Thus the telomere cluster occurs at a later elements can be formed before lateral element formation is stage than in wild-type wheat, and is accompanied by the loss complete, and that these two processes, which are usually of the Rabl configuration in the meiocytes. This is similar to regarded as separate stages of meiotic prophase in other the situations described in maize (Bass et al., 1997) and species, may be a continuum in wild-type wheat. Dawe et al. humans (Scherthan et al., 1996, 1998). Homologous chromosome pairing in wheat 1769

To date meiosis has been described in detail in two plants, of centromeres and telomeres in the fission yeast Schizosaccharomyces wheat and maize (Dawe et al., 1994; Bass et al., 1997), and pombe. EMBO J. 16, 193-202. significant differences in the mechanism of homologue pairing 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 have been discovered. Both species are polyploid, but maize organisation. Cell 76, 901-912. has recently been proposed to be either a degenerate Dong, F. and Jiang, J. (1998). Non-Rabl patterns of centromere and telomere autotetraploid or a segmental allopolyploid (Skrabanek and Distribution in interphase nuclei of plant cells. Chrom. Res. 6, 551-558. Wolfe, 1998), while wheat is clearly an allopolyploid. The Foote, T., Roberts, M., Kurata, N., Sasaki, T. and Moore, G. (1997). Detailed comparative mapping of cereal chromosome regions corresponding requirement for stable maintenance of these two different types to the Ph1 locus in wheat. Genetics 147, 801-807. of polyploids could account for the differences in meiotic Fung, J. C., W. F. Marshall, W. F., Dernburg, A., Agard, D. A. and Sedat, mechanism. Our studies have shown that the presence of the J. W. (1998). Homologous chromosome pairing in Drosophila Ph1 locus results in a premeiotic association of homologues melanogaster proceeds through multiple independent initiations. J. Cell via centromeres during floral development and an earlier Biol. 141, 5-20. Gillies, C. B. (1987). The effect of the Ph gene alleles on synaptonemal association of sub-telomeric regions via telomeric clustering at complex formation in Triticum aestivum x T kotshyi hybrids. Theor. Appl. the onset of meiotic prophase. Other studies have shown that Genet. 74, 430-438. the presence of the locus eliminates recombination between Jin, Q. W., Trelles-Sticken, E., Scherthan, H. and Loidl. J. (1998). Yeast homoeologous chromosomes or segments (Gillies et al., 1987; nuclei display prominent centromere clustering that is reduced in non- dividing cells and in meiotic prophase. J. Cell Biol. 141, 21-29. Luo et al., 1996). As yet it is unclear whether these two effects Kleckner, N. (1996). Meiosis-how does it work?. Proc. Nat. Acad. Sci. USA of the Ph1 locus are functionally related, or whether the Ph1 93, 8167-8174. locus comprises two or more genes which have differing effects Loidl, J., Klein, F. and Scherthan, H. (1994). Homologous pairing is reduced on premeiotic and meiotic events. The recent identification of but not abolished in asynaptic mutants in yeast. J. Cell Biol. 125, 1191-1200. new deletions of the Ph1 locus should resolve this issue (Miller Luo, M. C., Dubcousky, J. and Dvorak, J. (1996). 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