Effect of the Pairing Gene Ph1 and Premeiotic Colchicine Treatment on Intra- and Interchromosome Pairing of Isochromosomes in Common Wheat

Corresponding author: Moshe Feldman, Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel., lpfeld{at}wiccmail.weizmann.ac.il (E-mail).

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The analysis of the pattern of isochromosome pairing allows one to distinguish factors affecting presynaptic alignment of homologous chromosomes from those affecting synapsis and crossing-over. Because the two homologous arms in an isochromosome are invariably associated by a common centromere, the suppression of pairing between these arms (intrachromosome pairing) would indicate that synaptic or postsynaptic events were impaired. In contrast, the suppression of pairing between an isochromosome and its homologous chromosome (interchromosome pairing), without affecting intrachromosome pairing, would suggest that homologous presynaptic alignment was impaired. We used such an isochromosome system to determine which of the processes associated with chromosome pairing was affected by the Ph1 gene of common wheat—the main gene that restricts pairing to homologues. Ph1 reduced the frequency of interchromosome pairing without affecting intrachromosome pairing. In contrast, intrachromosome pairing was strongly reduced in the absence of the synaptic gene Syn-B1. Premeiotic colchicine treatment, which drastically decreased pairing of conventional chromosomes, reduced interchromosome but not intrachromosome pairing. The results support the hypothesis that premeiotic alignment is a necessary stage for the regularity of meiotic pairing and that Ph1 relaxes this alignment. We suggest that Ph1 acts on premeiotic alignment of homologues and homeologues as a means of ensuring diploid-like meiotic behavior in polyploid wheat.

PAIRING of homologous chromosomes at first meiotic metaphase results from three successive processes: alignment, synapsis, and crossover formation (LOIDL 1990 ; HAWLEY and ARBEL 1993 ; KLECKNER and WEINER 1993 ; KLECKNER 1996 ). Alignment or association refers to any tendency of homologues to lie closer to each other than nonhomologues previous to synapsis. It may occur in the absence of any detectable physical contact, with some contact at specific chromosomal regions, or with contact over most or all of the homologues length. Synapsis refers to the intimate contact between homologues within the frame of the synaptonemal complex at zygotene, thereby facilitating the processes involved in crossing-over at pachytene.

Despite the consensus that presynaptic alignment of homologues ensures the regularity of pairing, there is little agreement on the timing of the first alignment of homologous chromosomes (reviewed by LOIDL 1990 ). In contrast to those who assume that homologues are already associated at the last premeiotic interphase (SMITH 1942 ; FELDMAN 1966 ; MAGUIRE 1967 ), others hold that homologues do not associate before the beginning of zygotene (JOHN 1976 ; RASMUSSEN and HOLM 1978 ). Although there have been indications in a number of organisms that premeiotic alignment is a characteristic feature of meiosis (AVIVI and FELDMAN 1980 ), it was difficult to demonstrate this phenomenon conclusively because individual chromosomes could not be clearly distinguished. In a few species this problem has recently been circumvented by fluorescence in situ hybridization with DNA probes that detect a specific pair of homologous chromosomes or chromosome segments. In the budding yeast Saccharomyces cerevisiae the homologues were found to be associated via multiple interstitial interactions during the last premeiotic interphase (WEINER and KLECKNER 1994 ). Genomic in situ hybridization in a wheat line carrying a pair of homologues originating from barley showed that the hybridization signals of the two barley homologues fused into a single fluorescent signal during the last premeiotic interphase, indicating their complete association (ARAGON-ALCAIDE et al. 1997 ). In contrast, homologues were not found to be associated until early meiotic prophase in mouse and humans (SCHERTHAN et al. 1996 ). However, in this study the distance between the hybridization signals of homologues was not compared with that between nonhomologues, and, therefore, the results from premeiotic stages were inconclusive.

Another approach to studying the timing of homologous alignment is based on a system that distinguishes between factors affecting alignment and those affecting synapsis and crossing-over. The pairing behavior of an isochromosome serves that purpose. An isochromosome, consisting of two homologous arms, can undergo either intrachromosome pairing between the two arms to form a ring univalent at first meiotic metaphase or interchromosome pairing with a homologous chromosome. Factors that disrupt homologous alignment would reduce the frequency of interchromosome pairing without affecting intrachromosome pairing because the homologous arms of an isochromosome are connected by the common centromere and their relative position remains undisrupted. On the other hand, factors that prevent synapsis or crossing-over would affect both types of pairing.

In common wheat, Triticum aestivum L., SEARS 1952 and DRISCOLL and DARVEY 1970 observed an almost complete intrachromosome pairing at a frequency similar to that of pairing between homologous arms of conventional chromosomes. In this species, application of the antimicrotubule agent colchicine during the last premeiotic interphase resulted in pairing failure of conventional homologues at the first meiotic metaphase (DRISCOLL et al. 1967 ; DOVER and RILEY 1973 ), but did not affect pairing between the two arms of an isochromosome (DRISCOLL and DARVEY 1970 ). Hence, these authors concluded that colchicine inhibits premeiotic association of homologues rather than their synapsis and crossover. Similarly, high-temperature treatment during the last premeiotic interphase, which considerably reduced pairing of conventional homologous chromosomes, did not interfere with synapsis and chiasma formation between the two arms of an isochromosome (KATO and YAMAGATA 1980 ). In contrast to the effect of colchicine and high temperature, a deficiency for wheat chromosome 3B reduces homologous pairing not only between the arms of conventional chromosomes (SEARS 1954 ) but also between those of an isochromosome (KATO and YAMAGATA 1982 ). This suggests that the pairing gene located on 3B controls either synaptic or postsynaptic events.

A mechanism for the recognition of homologues previous to synapsis might be especially relevant in allopolyploid plants where it could help to prevent cross-ing-over between identical DNA sequences residing in homeologues, namely, partially homologous chromosomes from related genomes. This possibility was studied in common wheat, an allohexaploid (2n = 6x = 42; genomes AABBDD) that originated from hybridization events involving three closely related diploid species (reviewed by FELDMAN et al. 1995 ). The 21 pairs of homologous chromosomes (7 pairs of each genome) of common wheat are classified into seven homeologous groups, each containing 1 pair of chromosomes from the A, B, and D genomes (SEARS 1954 ). Homeologous group 1, for example, contains the pairs 1A, 1B, and 1D. In spite of their genetic similarity, homeologues do not pair at meiosis of common wheat. The suppression of pairing between homeologues, while homologues are allowed to pair regularly, is due mainly to the action of the pairing homeologous gene (Ph1; reviewed by SEARS 1976 ). Several hypotheses have been proposed to explain the mode of action of Ph1, all of which fall into two main categories: (i) those assuming that this gene exerts its effect at premeiotic stages, affecting the alignment of homologous and homeologous chromosomes (FELDMAN 1966 , FELDMAN 1993 ; FELDMAN and AVIVI 1988 ), and (ii) those assuming that Ph1 operates exclusively following the commencement of synapsis, affecting processes involved in synapsis and recombination (HOLM and WANG 1988 ; DUBCOVSKY et al. 1995 ; LUO et al. 1996 ).

To determine which of the processes involved in chromosome pairing (alignment or synapsis and crossing-over) is affected by Ph1, we studied the effect of this gene on the pairing of an isochromosome and a telochromosome for the same chromosome arm. The frequency of intrachromosome pairing of the isochromosome and that of interchromosome pairing between the isochromosome and the telochromosome were analyzed in plants with different doses of Ph1. The effect of Ph1 on intra- and interchromosome pairing was compared with the effect of premeiotic colchicine treatment, which inhibits premeiotic alignment, and with the effect of the deficiency for the long arm of chromosome 3B, associated with synaptic or postsynaptic events.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Aneuploid lines developed by the late E. R. Sears from the standard common wheat cultivar Chinese Spring were used in this study. To investigate the effect of Ph1 on intra- and interchromosome pairing we selected the long arm of chromosome 1A (1AL) as a representative of the wheat chromosome arms because of its medium length (GILL 1987 ) and lack of major genes affecting chromosome pairing. Monoisosomic-monotelosomic 1AL (MIMT 1AL) lines, having zero, two, or four doses of Ph1, were produced as illustrated in Figure 1. For the production of MIMT 1AL plants with zero doses of Ph1, we used the mutant line ph1b/ph1b (Figure 1A; SEARS 1977 ), which is deficient for the Ph1 gene because of an interstitial deletion in the critical chromosome region (GILL and GILL 1991 ; GILL et al. 1993 ). Homozygous ph1b/ph1b plants were identified by test-crossing with Aegilops variabilis (2n = 4x = 28) as described (VEGA and FELDMAN 1998 ). MIMT 1AL plants with two doses of Ph1 were disomic for chromosome 5B (Figure 1B), which carries Ph1 on its long arm, 5BL. The tetrasomic 5B line was used for the production of MIMT 1AL plants with four doses of Ph1 (Figure 1C).

The effect of Ph1 on intra- and interchromosome pairing was compared with the effect of a gene(s) located on the long arm of chromosome 3B, 3BL, whose activity is responsible for normal synapsis and chiasma-formation (SEARS 1954 ; KEMPANNA and RILEY 1962 ; KATO and YAMAGATA 1982 , KATO and YAMAGATA 1983 ). We designated this synaptic gene Syn-B1. The ditelosomic 3BS line, deficient for 3BL, was used for the production of MIMT 1AL plants lacking Syn-B1 (Figure 1D).

Intraisochromosome pairing was also analyzed in the diisosomic 5BL line, having two 5BL isochromosomes and lacking the short arm of 5B, 5BS.

Plants were grown in a greenhouse at 20 ± 5°. Spikes at meiosis were fixed in ethanol:chloroform:acetic acid (3:2:1, v/v/v) for 2 days at 4° and then transferred into ethanol: acetic acid (3:1, v/v) and refrigerated until analyzed. Anther squashes were made in 1% acetocarmine. Chromosome pairing was analyzed on semipermanent slides sealed with a gelatine-acetic acid medium. The data were analyzed using a contingency chi-square test.

Colchicine treatment:
To verify the developmental stage of the anthers at the time of colchicine application we determined the ploidy of the tapetum cells and the meiocytes at first meiotic metaphase (DOVER and RILEY 1973 ). Briefly, treatments before the last mitosis of the meiocytes resulted in doubling of the chromosome number, and nearly all the chromosomes paired as bivalents during the subsequent meiosis. Application of colchicine between the last mitosis of the meiocytes and the penultimate mitosis in tapetal cells, which takes place at about the middle of premeiotic interphase, induced asynapsis in the meiocytes and increased the tapetal ploidy level to 8N. In our experimental conditions, this stage took place ~4 days before first meiotic metaphase. Finally, treatments between the penultimate and the last division in tapetal cells, which is synchronous with leptotene, resulted in normal pairing in the meiocytes and increased the tapetal ploidy level to 4N.

When the tip of the spike was at the height of the third leaf node, colchicine (I.C.N.) was injected with a hypodermic syringe through the leaf sheaths into the space surrounding the developing spike. In each application, about 0.50 ml of 5 x 10^-5 M, 1 x 10^-4 M, or 2 x 10^-4 M colchicine solution was injected. The treated spikes were dissected out and fixed 4 days later. Because there were no significant differences between the three colchicine concentrations in their effect on chromosome pairing, the data of the treatments were pooled.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Pairing of conventional chromosomes in untreated and colchicine-treated plants carrying different doses of Ph1 and Syn-B1:
Conventional chromosomes paired as expected at first meiotic metaphase in monoisosomic-monotelosomic 1AL plants carrying different doses of Ph1 and Syn-B1 (Table 1). Briefly, in wild-type plants carrying two doses of both Ph1 and Syn-B1, homologous chromosomes paired regularly in bivalents; two univalents were observed in only 3% of the meiocytes. Mutants lacking Ph1, namely homozygous for ph1b, showed a mean of 0.85 multivalents per cell, accompanied by partial reduction in pairing (the mean number of univalents per cell was 2.53 and that of rod bivalents, 5.84). Tetrasomic 5B plants, carrying four doses of Ph1, showed regular pairing except for the four 5B chromosomes, which formed a quadrivalent in 16% of the cells and a trivalent and univalent in 8% of the cells. Ditelosomics for 3BS, lacking Syn-B1, showed partial reduction in pairing (the mean number of univalents was 2.74 and that of rod bivalents, 7.27). The reduction in pairing was similar to that observed in the absence of Ph1, but, in contrast, multivalents were never observed.

In plants lacking Ph1, premeiotic colchicine treatment increased the mean number of univalents from 2.53 to 16.95 and of rod bivalents from 5.84 to 8.13. There was a 40% reduction in the number of homologous chromosomes involved in bivalents and a 16% reduction in the number of chromosomes, presumably homeologous, involved in multivalents. Premeiotic colchicine treatment also resulted in a high reduction in pairing in plants carrying four Ph1 doses. The mean number of univalents increased from 0.26 to 9.30 and of rod bivalents from 3.35 to 8.93. There was a 21% reduction in the number of homologous chromosomes involved in bivalents.

The pairing data of the conventional chromosomes in untreated diisosomic 5BL plants carrying two 5BL isochromosomes and, therefore, four doses of Ph1, are presented in Table 1. The mean number of univalents per cell was 2.30 and that of rod bivalents was 8.49. This contrasts with the other genotype carrying four doses of Ph1, tetrasomic 5B, which did not show a reduction in pairing. The reason for this difference is that diisosomic 5BL plants are deficient for the short arm of chromosome 5B, 5BS, which carries pairing promoting genes (FELDMAN 1966 ; FELDMAN and MELLO-SAMPAYO 1967 ; RILEY and CHAPMAN 1967 ). One quadrivalent was observed in one of the diisosomic 5BL meiocytes, most probably the result of homeologous pairing. Following premeiotic colchicine treatment diisosomic 5BL plants showed almost complete absence of pairing (Table 1); the mean number of univalents per cell was increased from 2.30 to 30.82, and there was a 76% reduction in the number of homologous chromosomes involved in bivalents.

Pairing of the isochromosome (iso) and telochromosome (telo) 1AL in untreated and colchicine-treated plants carrying different doses of Ph1 and Syn-B1:
The following five types of pairing configurations involving iso and telo 1AL were observed at first meiotic metaphase (Figure 2). The frequencies of these configurations in the different MIMT 1AL lines are presented in Table 2.

• Asynapsis: The iso and the telo appeared as univalents, with no pairing between the two arms of the iso (Figure 2A and Figure B). This type of configuration was observed only in those cases where the pairing of conventional chromosomes was also reduced (Table 2).

  
  

  View this table: In this window In a new window   Table 2. Pairing configuration at first meiotic metaphase of iso- and telochromosomes 1AL in untreated and premeiotically colchicine-treated plants derived from the common wheat cultivar Chinese Spring (%)

• Univalent pairing: The iso and the telo appeared as univalents, with intrachromosome pairing between the two arms of the iso to form a ring univalent (Figure 2C and Figure D).

• Rod bivalent: Interchromosome pairing between the iso and the telo where the telo paired with one arm of the iso leaving the second arm unpaired (Figure 2E and Figure F).

• cFrying-pan bivalent: Interchromosome pairing between the iso and the telo where, in addition to the terminal pairing of one of the iso arms with the telo, the two arms of the iso paired interstitially with each other to produce a "frying-pan" shaped bivalent (Figure 2G and Figure H). Frying-pan bivalents with terminal pairing of the iso arms and interstitial pairing of the telo with one of the iso arms were never observed. Considering that synapsis initiates at or near the telomeres (VON WETTSTEIN et al. 1984 ), and that subsequent synapsis in intercalary chromosome regions may depend on the successful distal pairing (LUKASZEWSKI 1997 ), the pairing that gave rise to frying-pan bivalents must have started between the distal region of one of the iso arms and the distal region of the telo and later shifted to proximal pairing between the two arms of the iso.

• Homeologous pairing: In some cells of the ph1b mutant, iso or telo 1AL paired with other chromosomes, most likely their homeologues 1B and 1D. In untreated plants, iso 1AL was not involved in homeologous pairing, but telo 1AL paired with chromosomes different from iso 1AL in 5% of the meiocytes (Table 2); in those cases, the iso paired intrachromosomally (Figure 2I). In colchicine-treated spikes, however, telo 1AL was not involved in homeologous pairing but iso 1AL paired with chromosomes different from telo 1AL in 10% of the meiocytes (Table 2); in those cells, the iso also paired intrachromosomally while the telo remained unpaired (Figure 2J).

Effect of Ph1, Syn-B1, and premeiotic colchicine treatment on intra- and interchromosome pairing of isochromosomes:
The following parameters (Table 3) were calculated based on the pairing data of conventional chromosomes (Table 1) and of iso and telo 1AL (Table 2): (1) mean arm pairing of conventional chromosomes, i.e., the ratio between the number of paired arms to the total number of arms; (2) mean arm pairing of the three 1AL arms; (3) mean intrachromosome pairing of iso 1AL, calculated from the sum of the frequencies of univalent pairing, frying-pan bivalents, and homeologous pairing in Table 2; and (4) mean interchromosome pairing of iso 1AL and telo 1AL, calculated from the sum of the frequencies of rod bivalents and frying-pan bivalents in Table 2.

In the absence of Ph1, there was a reduction in the frequency of pairing of conventional chromosomes from 0.94, in the wild type, to 0.77. In contrast, the frequency of pairing of the three 1AL arms was slightly higher in plants lacking Ph1, and the frequency of intrachromosome pairing of iso 1AL was somewhat lower, though not significantly, than that of wild-type plants. The frequency of interchromosome pairing of iso 1AL with telo 1AL was significantly higher than in plants with two doses of Ph1 (P < 0.001) or in any other genotype.

In tetrasomic 5B, four Ph1 doses did not significantly modify the frequency of intrachromosome and interchromosome pairing compared to two Ph1 doses.

In the absence of Syn-B1, the frequency of pairing of conventional chromosomes was reduced to 0.74, and the frequency of pairing of the three 1AL arms was reduced significantly compared to its frequency in plants carrying the gene (P < 0.001). The intrachromosome pairing of iso 1AL decreased from 0.87 (wild type) to 0.50 (P < 0.001). This reduction of intrachromosome pairing in the absence of Syn-B1 had two components: first, 10% of the cells showed asynapsis of the iso and telo (Table 2), and second, the ratio of rod bivalents to frying-pan bivalents, involving iso and telo 1AL, was drastically increased when compared with the unchanged value in zero, two, and four doses of Ph1 (Table 2). Plants lacking Syn-B1 also showed a decrease in the frequency of interchromosome pairing of iso 1AL with telo 1AL when compared with plants lacking Ph1 (P < 0.05), but there was no significant change when they were compared with wild-type plants (P > 0.20).

Following premeiotic colchicine treatment, the mean arm pairing of conventional chromosomes was drastically reduced to 0.35 in ph1b mutant plants and to 0.57 in tetrasomic 5B plants. The mean arm pairing of 1AL was partially reduced to 0.65 in ph1b mutant plants (P < 0.001) and to 0.63 in tetrasomic 5B plants (P < 0.001). While the frequency of intrachromosome pairing of iso 1AL was not significantly affected by colchicine treatments (Table 3), that of interchromosome pairing of iso with telo 1AL was decreased to 0.29 in treated ph1b mutants (P < 0.001) and to 0.16 in tetrasomic 5B plants (P < 0.001).

In diisosomic 5BL plants, premeiotic colchicine treatment drastically reduced the frequency of pairing of conventional chromosomes from 0.73 to 0.13 (Table 4). However, the frequency of intrachromosome pairing of the two isochromosomes 5BL was unchanged, being 0.37 in untreated plants and 0.39 in colchicine-treated plants.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effect on the pattern of isochromosome pairing:
The analysis of intra- and interchromosome pairing of iso 1AL in plants carrying different doses of Ph1 and Syn-B1 revealed that these two genes control different events of meiotic pairing. Two and four doses of Ph1 reduced the frequency of interchromosome pairing without affecting intrachromosome pairing. This shows that Ph1 does not modify synaptic or postsynaptic events, but rather suppresses presynaptic homologous alignment, as suggested by FELDMAN and AVIVI 1988 . Because in an isochromosome the two homologous arms are connected by a common centromere, their alignment cannot be disrupted, and therefore the frequency of intrachromosome pairing remains unchanged even when zero or extra doses of Ph1 induce reduction in pairing of conventional chromosomes. In diisosomic 5BL plants, four doses of Ph1 reduced pairing of conventional chromosomes to 73%, but the frequency of intrachromosome pairing of the two 5BL isochromosomes was higher than the expected value for such pairing reduction (data not shown). In triisosomic 5BL plants, six doses of Ph1 reduced pairing of conventional chromosomes to 43% (FELDMAN 1966 ), yet, despite this conspicuous reduction in pairing, the frequency of intrachromosome pairing of the three 5BL isochromosomes was higher than the expected value (FELDMAN and AVIVI 1988 ). Hence, when homologous arms are connected by a common centromere, their pairing is not reduced by an extra dose of Ph1, demonstrating that this gene is a suppressor of presynaptic alignment and not of the processes of synapsis or chiasma formation.

In contrast to Ph1, in the absence of Syn-B1 the intrachromosome pairing of iso 1AL was drastically reduced while the interchromosome pairing between iso and telo 1AL was unaffected. Actually, the reduction in pairing was higher between the arms of the isochromosome (42%) than between conventional chromosomes (21%). A similar phenomenon was found by KATO and YAMAGATA 1982 . While it is not clear why the frequency of interchromosome pairing is not reduced in the absence of Syn-B1, the pronounced decrease in intrachromosome pairing indicates that Syn-B1 does not affect premeiotic alignment but rather synaptic or postsynaptic events. This is in accord with the conclusion of KATO and YAMAGATA 1982 .

Although premeiotic colchicine treatment of ph1b mutant and tetrasomic 5B plants greatly suppressed interchromosome pairing between iso and telo 1AL, the intrachromosome pairing between the arms of iso 1AL was not significantly reduced by the treatment. Likewise, colchicine did not reduce the frequency of intrachromosome pairing of the two 5BL isochromosomes in diisosomic 5BL plants, even when the treatment suppressed pairing of conventional chromosomes almost completely (82% reduction). These observations in plants with zero and four doses of Ph1 are in agreement with those of DRISCOLL and DARVEY 1970 in monoisosomic 5DL plants carrying two doses of Ph1. They showed that premeiotic treatment with colchicine did not change the frequency of intrachromosome pairing of iso 5DL, although it reduced the pairing of conventional chromosomes by 54%. Taken together, these results indicate that premeiotic colchicine treatments, like Ph1, do not alter the processes of synapsis or crossing-over but rather the premeiotic alignment of homologous chromosomes.

Previous studies have shown that the effect of extra doses of Ph1 on chromosome pairing is similar to that induced by premeiotic treatments with colchicine (reviewed by FELDMAN and AVIVI 1988 ). In triisosomic 5BL plants, six doses of Ph1 reduce homologous pairing to about one half of the normal level, and at the same time they induce homeologous pairing and interlocking of bivalents (FELDMAN 1966 ; YACOBI et al. 1982 ). Premeiotic colchicine treatment of plants having the normal two doses of Ph1 also induces homeologous pairing and interlocking of bivalents (DRISCOLL et al. 1967 ; FELDMAN and AVIVI 1988 ). In the present work, Ph1 and premeiotic colchicine treatment induced a reduction in the frequency of pairing between iso and telo 1AL without affecting the frequency of pairing between the arms of the iso. All the above observations indicate that a premeiotic event that is essential for the regularity of meiotic pairing is affected by both colchicine treatment and the Ph1 gene. Because the distinctive feature of an isochromosome is the connection of the two homologous arms via the centromere, this suggests that the premeiotic event necessary for the regularity of meiotic pairing is the alignment of homologous chromosomes.

The mode of action of Ph1:
The similar outcome of premeiotic colchicine treatment and Ph1 on the pattern of isochromosome pairing supports the hypothesis accounting for the effect of different doses of Ph1 on the premeiotic alignment of homologues and homeologues and the subsequent pattern of pairing at first meiotic metaphase (FELDMAN 1966 ). In plants with a zero dose of Ph1, homologues as well as homeologues would be closely associated at premeiotic stages—though the latter to a lesser extent. This results in some homeologous pairing at first meiotic metaphase superimposed on the homologous pairing, in interlocking of homeologous bivalents, and in asynapsis of those homologues whose pairing initiation or completion was interrupted by the homeologues. With the normal two doses of Ph1, the premeiotic association of homeologues would be completely suppressed, resulting in regular and exclusive pairing of homologues at first meiotic metaphase. The reduction in interchromosome pairing between iso and telo 1AL suggests that the premeiotic association of homologous chromosomes is also somewhat suppressed in the presence of two doses of Ph1, but that they stay close enough to each other to ensure regular pairing at meiotic prophase. In six doses of Ph1, premeiotic chromosome association would be further suppressed, leading to increased distance between homologues. This results in asynapsis of homologues that are relatively far from one another, in pairing of homeologues that happen to lie close to each other, and in interlocking of bivalents as a result of pairing between somewhat separated partners. According to this model, premeiotic colchicine treatment would disrupt the premeiotic association of homologues resulting in a similar pattern of pairing to the one observed in the presence of six doses of Ph1.

Direct evidence supporting this model is lacking because of the difficulty in identifying homologues and homeologues at premeiotic stages. However, using fluorescence in situ hybridization with DNA probes to homologous chromosome segments in budding yeast, WEINER and KLECKNER 1994 presented the first conclusive evidence for premeiotic homologous association. Before the initiation of meiotic S phase, the array of distances between any given pair of homologous segments fell far below the array of distances between nonhomologous segments. Recently, premeiotic association was also found for a pair of homologous barley chromosomes added to common wheat, which were visualized by genomic in situ hybridization (ARAGON-ALCAIDE et al. 1997 ). Three stages were identified at premeiotic interphase: the barley homologues were first observed separated, then in contact at the centromere, and, finally, in contact along their entire length. Premeiotic association was also observed for a pair of homologous rye telocentrics added to common wheat (E. I. MIKHAILOVA, T. NARANJO, K. SHEPHERD, J. WENNEKES, C. HEYTING and J. H. DE JONG, unpublished results). These findings in yeast and wheat demonstrate that homologous chromosomes recognize each other and associate before meiosis. Such interaction would lead to exclusive synapsis of homologues at first meiotic prophase (KLECKNER and WEINER 1993 ).

ARAGON-ALCAIDE et al. 1997 observed that in the absence of Ph1 the barley homologues were not in contact along their length. These authors proposed that the absence of Ph1 disrupts premeiotic homologues association. However, the fact that the barley homologues pair almost regularly at first meiotic metaphase of plants deficient for Ph1 argues against this assumption. It is possible that the anthers the authors analyzed in the mutant were slightly younger than those in the wild type. Because in the analyzed anthers of the Ph1 mutant the barley homologues were observed in contact at the centromere in 25% of the meiocytes, it is possible that late anthers would show the homologues in contact along their entire length. Moreover, from the fact that in our study the interchromosomal pairing of iso and telo 1AL was higher in the absence of the gene than in its presence (Table 3), it was concluded that homologues are more closely aligned at premeiotic stages in plants deficient for Ph1 than in plants carrying the gene.

A different approach to the analysis of Ph1 action was taken by DUBCOVSKY et al. 1995 and LUO et al. 1996 who studied recombination between wheat chromosome 1A and its closely related homeologous chromosome 1A^m of T. monococcum. In their analysis of chromosomes 1A having interstitial segments of 1A^m, no recombination was detected between those segments and a normal chromosome 1A in the presence of Ph1, whereas the levels of recombination were close to normal in the juxtaposed homologous segments. These authors concluded that Ph1 prevents homeologous pairing in polyploid wheat by processing homology along the entire length of the chromosomes. This is consistent with the earlier assumption that Ph1 regulates homology recognition at the level of individual DNA heteroduplexes (HOLM and WANG 1988 ). However, on the basis of this hypothesis one would expect a high incidence of homeologous pairing in the absence of Ph1. Yet, in the present study, the frequency of homeologous pairing was only 0.05 in the ph1b mutant plants analyzed, and at least one-third of the meiocytes showed exclusive homologous pairing. Moreover, their assumption does not explain the pairing of homeologous chromosomes in plants carrying six doses of Ph1, nor the similarity between the effects of an extra dose of Ph1 and premeiotic colchicine treatment on the pairing of conventional chromosomes as well as on the pairing of isochromosomes.

Colchicine was found to disrupt meiotic pairing when applied at the first half of the premeiotic interphase but pairing was normal when colchicine was applied at the second half of this interphase (DOVER and RILEY 1973 ). The sensitive stage to colchicine coincides with the G1 phase, when association of homologues was observed in yeast (WEINER and KLECKNER 1994 ) and, most probably, with the stage when the two barley homologues are still separated or associated at their centromeres at premeiotic interphase in wheat (ARAGON-ALCAIDE et al. 1997 ). It was suggested (FELDMAN and AVIVI 1988 ) that microtubules are involved in the process of intimate homologous association, and that the disruption of microtubules by colchicine would inhibit further association between homologues resulting in asynapsis at first meiotic metaphase. Chloral hydrate, which prevents the polymerization of continuous microtubules but not of centromeric microtubules (MOLE-BAJER 1969 ), did not disturb meiotic pairing in wheat when applied at stages from the last mitosis to meiotic prophase (DOVER and RILEY 1973 ). This indicates that the microtubules involved with the chromosome movements related to pairing are those that interact with the centromere. This might explain why the contact between the barley homologues in wheat commences at the centromere and not at other chromosome regions (ARAGON-ALCAIDE et al. 1997 ). The parallelism between the effects of Ph1 and colchicine points to a similar molecular target of Ph1 action. In agreement with this view, we have previously shown that Ph1 affects centromere-microtubules interaction at meiotic anaphases and discussed how the effect of Ph1 on the stability of this interaction might affect the arrangement of chromosomes in somatic and premeiotic cells (VEGA and FELDMAN 1998 ).

	ACKNOWLEDGMENTS

The authors are grateful to Mr. Yigal Avivi for editing the manuscript. This research was supported by a doctoral fellowship from the Spanish Ministry of Education and Science to J.M.V. and by the Leo and Julia Forchheimer Foundation to M.F.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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