Copyright  1998 by the Genetics Society of America
Effect of the Pairing Gene Ph1 and Premeiotic Colchicine Treatment on Intraand Interchromosome Pairing of Isochromosomes in Common Wheat
Juan M. Vega and Moshe Feldman
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
ABSTRACT
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.
P
AIRING 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),
Corresponding author: Moshe Feldman, Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel.
E-mail: lpfeld@wiccmail.weizmann.ac.il
Genetics 150: 1199–1208 (November 1998)
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 (Aragón-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 chro-
1200
J. M. Vega and M. Feldman
mosome. 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 crossing-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 5 6x 5 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, 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 crossingover) 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
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. Monoisosomicmonotelosomic 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 5
4x 5 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, 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 6 58. Spikes at
meiosis were fixed in ethanol:chloroform:acetic acid (3:2:1,
v/v/v) for 2 days at 48 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.
Ph1 Effect on Isochromosome Pairing
1201
sis. 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 z4 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 3 1025 m, 1 3 1024 m, or 2 3 1024 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
Figure 1.—Production of monoisosomic-monotelosomic
1AL lines with (A) zero, (B) two, or (C) four doses of Ph1
and (D) a line with zero dose of Syn-B1. For details see materials and methods. Monosomic, monotelosomic, ditelosomic,
and monoisosomic are abbreviated as mono, momotelo, ditelo, and monoiso, respectively.
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 meio-
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 monoisosomicmonotelosomic 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
2
0
4
Ditelosomic 3BS c
Colchicine treated
ph1b mutant
Tetrasomic 5B
4
2
2
2
2
0
2
2
2
Syn-B1
49
128
60
60
140
100
480
100
No.
cells
2.30 6 0.28
(0–18)
30.82 6 0.48
(22–38)
16.95 6 0.58
(7–25)
9.30 6 0.79
(0–26)
2.53 6 0.19
(0–9)
0.06 6 0.02
(0–2)
0.26 6 0.06
(0–2)
2.74 6 0.20
(0–8)
Univalentsa
8.49 6 0.23
(1–14)
3.92 6 0.23
(1–8)
8.13 6 0.30
(3–13)
8.93 6 0.35
(1–15)
5.84 6 0.21
(1–11)
2.42 6 0.07
(0–8)
3.35 6 0.18
(0–8)
7.27 6 0.18
(3–12)
Rod
10.34 6 0.26
(4–19)
0.67 6 0.12
(0–4)
2.17 6 0.18
(0–5)
7.22 6 0.39
(1–14)
11.44 6 0.21
(5–17)
17.55 6 0.07
(12–20)
17.08 6 0.18
(13–20)
10.36 6 0.20
(4 –16)
Ring
Bivalentsa
18.83 6 0.14
(11–20)
4.59 6 0.24
(1–9)
10.30 6 0.31
(5–15)
16.15 6 0.40
(8–21)
17.28 6 0.16
(12–20)
19.97 6 0.01
(19–20)
20.43 6 0.09
(19–21)
17.63 6 0.10
(15–19)
Total
—
—
0.01 6 0.01
(0–1)
—
0.10 6 0.04
(0–1)
0.03 6 0.02
(0–1)
0.16 6 0.04
(0–1)
—
0.08 6 0.03
(0–1)
—
0.63 6 0.10
(0–3)
0.10 6 0.04
(–1)
0.27 6 0.05
(0–2)
—
Quadrivalentsa
0.57 6 0.07
(0–3)
—
Trivalentsa
—
—
0.02 6 0.02
(0–1)
—
—
—
0.01 6 0.01
(0–1)
—
Pentavalentsa
The doses of the pairing homeologous gene Ph1 on chromosome arm 5BL and of the synaptic gene Syn-B1 on chromosome arm 3BL are indicated. Range values are in
parentheses.
a
Values are means 6SE.
b
Monoisosomic-monotelosomic 1AL. Pairing data of the isochromosome and telochromosome 1AL are given in Table 2.
c
Pairing data of the two 3BS telochromosomes are not included. These telocentrics paired in a rod bivalent configuration in 64% of the cells.
d
Pairing data of the two 5BL isochromosomes are not included.
Colchicine treated
4
4
Tetrasomic 5B
Diisosomic 5BLd
Untreated
2
0
Ph1
Wild type
Monoiso-monotelo 1ALb
Untreated
ph1b mutant
Genotype
Dose of
Pairing configurations of conventional chromosomes at first meiotic metaphase of
the indicated untreated and premeiotically colchicine-treated genotypes,
all derived from the common wheat cultivar Chinese Spring
TABLE 1
1202
J. M. Vega and M. Feldman
Ph1 Effect on Isochromosome Pairing
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 2, A and B). This type of configuration was observed only in those cases where the pairing of conventional chromosomes was also reduced (Table 2).
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 2, C
and 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 2,
E and F).
Frying-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 2,
G and 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
1203
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 ,
1204
J. M. Vega and M. Feldman
Figure 2.—Pairing configurations involving isochromosome and telochromosome 1AL at first meiotic metaphase. See text
for explanation. (A and B) Asynapsis. (B) Monoisosomic-monotelosomic 1AL (MIMT 1AL) ditelosomic 3BS meiocyte, showing
4 univalents, 9 rod bivalents, and 10 ring bivalents. Arrow points to the isochromosome 1AL univalent, and arrowhead to the
telochromosome 1AL univalent. The other two univalents are the 3BS telocentrics. (C and D) Univalent pairing. (D) MIMT 1AL
tetrasomic 5B meiocyte treated with 2 3 1024 m colchicine, showing 18 univalents, 4 rod bivalents, and 9 ring bivalents. Arrow
points to the isochromosome 1AL ring univalent, and arrowhead to the telochromosome 1AL univalent. (E and F) Rod bivalent.
(F) MIMT 1AL ditelosomic 3BS meiocyte, showing 6 univalents, 7 rod bivalents, and 11 ring bivalents. Arrow points to the rod
bivalent between the isochromosome and telochromosome 1AL. (G and H) Frying-pan bivalent. (H) MIMT 1AL ph1b mutant
meiocyte, showing 7 univalents, 5 rod bivalents, 1 frying-pan bivalent, 10 ring bivalents, and 1 trivalent. Arrow points to the
frying-pan bivalent between the isochromosome and telochromosome 1AL. (I and J) Homeologous pairing. (I) MIMT 1AL ph1b
mutant meiocyte, showing 4 univalents, 5 rod bivalents, 9 ring bivalents, 2 trivalents, and 1 quadrivalent. Arrow points to the
isochromosome 1AL ring univalent, and arrowhead to the telochromosome 1AL involved in a trivalent. ( J) MIMT 1AL ph1b
mutant meiocyte treated with 5 3 1025 m colchicine, showing 14 univalents, 11 rod bivalents, and 2 trivalents. Arrow points to
the isochromosome 1AL involved in a trivalent, and arrowhead to the telochromosome 1AL univalent.
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
Ph1 Effect on Isochromosome Pairing
1205
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 (%)
Dose of
Genotypea
Untreated
ph1b mutant
Wild type
Tetrasomic 5B
Ditelosomic 3BS
Colchicine treated
ph1b mutant
Tetrasomic 5B
Asynapsis
(%)
Univalent
pairing
(%)
Rod
bivalent
(%)
Frying-pan
bivalent
(%)
Homeologous
pairing
(%)
Ph1
Syn-B1
0
2
4
2
2
2
2
0
1
—
—
10
34
56
53
40
18
13
14
40
42
31
33
10
—
—
0
4
2
2
8
7
53
77
17
13
12
3
10c
—
5b
The doses of the pairing homeologous gene Ph1 and the synaptic gene Syn-B1 are indicated.
a
All plants are monoisosomic-monotelosomic 1AL. The number of cells analyzed is presented in Table 1.
b
In all cases the telo paired with a homeologous chromosome and the isochromosome paired intrachromosomally.
c
In all cases the isochromosome paired interchromosomally with a homeologous chromosome and, at the
same time, paired also intrachromosomally; the telo did not pair.
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
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 SynB1 revealed that these two genes control different events
TABLE 3
Arm pairing of conventional chromosomes, pairing of the three 1AL arms, and intra- and interchromosome pairing
of iso 1AL at first meiotic metaphase of untreated and premeiotically colchicine-treated monoisosomic-monotelosomic
1AL plants derived from the common wheat cultivar Chinese Spring
Ph1
Syn-B1
Arm pairing of
conventional
chromosomesb
0
2
4
2
2
2
2
0
0.77
0.94
0.91
0.74
0.82
0.77
0.78
0.63*
0.81
0.87
0.86
0.50*
0.60*
0.44
0.47
0.50
0
4
2
2
0.35
0.57
0.65*
0.63*
0.75
0.80
0.29*
0.16*
Dose of
Genotypea
Untreated
ph1b mutant
Wild type
Tetrasomic 5B
Ditelosomic 3BS
Colchicine treated
ph1b mutant
Tetrasomic 5B
Arm pairing
of 1ALb
Intrachromosome
pairing of iso 1ALb
Interchromosome
pairing of iso 1AL
with telo 1ALb
The doses of Ph1 and Syn-B1 are indicated.
a
The number of cells analyzed is presented in Table 1.
b
The ratio between the number of paired arms and total number of arms. Values are means.
*P , 0.001. The levels of significance in data of ph1b mutant and ditelosomic 3BS plants correspond to comparison with values
from wild-type plants. The levels of significance in data of treated ph1b mutant and treated tetrasomic 5B plants correspond to
comparison with values from untreated plants.
1206
J. M. Vega and M. Feldman
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 crossingover 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
TABLE 4
Arm pairing of conventional chromosomes and
intrachromosome pairing of isochromosomes at first
meiotic metaphase of untreated and premeiotically
colchicine-treated diisosomic 5BL plants of the
common wheat cultivar Chinese Spring
Diisosomic 5BLa
Untreated
Colchicine treated
a
Arm pairing
of conventional
chromosomesb
Intrachromosome
pairing of 5BL
isochromosomesb
0.73
0.13
0.37
0.39
The number of cells analyzed is presented in Table 1.
The ratio between the number of paired arms to the total
number of arms. Values are means.
b
Ph1 Effect on Isochromosome Pairing
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 (Aragón-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).
Aragón-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
1207
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 1Am of T. monococcum. In their analysis of chromosomes 1A having interstitial segments of 1Am, 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 (AragónAlcaide 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,
1208
J. M. Vega and M. Feldman
which prevents the polymerization of continuous microtubules but not of centromeric microtubules (MoléBajer 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
(Aragón-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).
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
Aragón-Alcaide, L., S. Reader, A. Beven, P. Shaw, T. Miller et
al., 1997 Association of homologous chromosomes during floral
development. Curr. Biol. 7: 905–908.
Avivi, L., and M. Feldman, 1980 Arrangement of chromosomes in
the interphase nucleus of plants. Hum. Genet. 55: 281–295.
Dover, G., and R. Riley, 1973 The effect of spindle inhibitors applied before meiosis on meiotic chromosome pairing. J. Cell Sci.
12: 143–161.
Driscoll, C. J., and N. L. Darvey, 1970 Chromosome pairing: effect
of colchicine on an isochromosome. Science 169: 290–291.
Driscoll, C. J., N. L. Darvey and H. N. Barber, 1967 Effect of
colchicine on meiosis of hexaploid wheat. Nature 216: 687–688.
Dubcovsky, J., M.-C. Luo and J. Dvorák, 1995 Differentiation between homoeologous chromosomes 1A of wheat and 1Am of Triticum monococcum and its recognition by the wheat Ph1 locus. Proc.
Natl. Acad. Sci. USA 92: 6645–6649.
Feldman, M., 1966 The effect of chromosomes 5B, 5D and 5A on
chromosomal pairing in Triticum aestivum. Proc. Natl. Acad. Sci.
USA 55: 1447–1453.
Feldman, M., 1993 Cytogenetic activity and mode of action of the
pairing homeologous (Ph1) gene of wheat. Crop Sci. 33: 894–897.
Feldman, M., and L. Avivi, 1988 Genetic control of bivalent pairing
in common wheat: the mode of Ph1 action, pp. 269–279 in Kew
Chromosome Conference III, edited by P. E. Brandham. Her Majesty’s Stationery Office, London.
Feldman, M., and T. Mello-Sampayo, 1967 Suppression of homoeologous pairing in hybrids of polyploid wheats 3 Triticum speltoides. Can. J. Genet. Cytol. 9: 307–313.
Feldman, M., F. G. H. Lupton and T. Miller, 1995 Wheats, pp.
184–192 in Evolution of Crop Plants, edited by J. Smartt and N. W.
Simmonds. Longmann Group, London.
Gill, B. S., 1987 Chromosome banding methods, standard chromosome band nomenclature, and applications in cytogenetic analysis, pp. 243–254 in Wheat and Wheat Improvement, edited by E. G.
Heyne. American Society of Agronomy, Madison, WI.
Gill, K. S., and B. S. Gill, 1991 A DNA fragment mapped within the
submicroscopic deletion of Ph1: a chromosome pairing regulator
gene in polyploid wheat. Genetics 129: 257–259.
Gill, K. S., B. S. Gill, T. R. Endo and Y. Mukai, 1993 Fine physical
mapping of Ph1, a chromosome pairing regulator gene in polyploid wheat. Genetics 134: 1231–1236.
Hawley, R. S., and T. Arbel, 1993 Yeast genetics and the fall of
the classical view of meiosis. Cell 72: 301–303.
Holm, P. B., and X. Wang, 1988 The effect of chromosome 5B on
synapsis and chiasma formation in wheat, Triticum aestivum cv.
Chinese Spring. Carlsberg Res. Commun. 53: 191–208.
John, B., 1976 Myths and mechanisms of meiosis. Chromosoma 54:
295–325.
Kato, T., and H. Yamagata, 1980 Reduction of meiotic homologous
chromosome pairing due to high temperature in common wheat.
Jpn. J. Genet. 55: 337–348.
Kato, T., and H. Yamagata, 1982 Effect of 3B chromosome deficiency on the meiotic pairing between the arms of an isochromosome in common wheat. Jpn. J. Genet. 57: 403–406.
Kato, T., and H. Yamagata, 1983 Analysis of the action of 3B chromosome on meiotic homologous chromosome pairing in common wheat, pp. 321–325 in Proceedings of the 6th International Wheat
Genetics Symposium, edited by S. Sakamoto. Faculty of Agriculture,
Kyoto, Japan.
Kempanna, C., and R. Riley, 1962 Relationships between the genetic effects of deficiencies for chromosomes III and V on meiotic
pairing in Triticum aestivum. Nature 195: 1270–1273.
Kleckner, N., 1996 Meiosis: How could it work? Proc. Natl. Acad.
Sci. USA 93: 8167–8174.
Kleckner, N., and B. M. Weiner, 1993 Potential advantages of unstable interactions for pairing of chromosomes in meiotic, somatic,
and premeiotic cells. Cold Spring Harbor Symp. Quant. Biol. 58:
553–565.
Loidl, J., 1990 The initiation of meiotic chromosome pairing: the
cytological view. Genome 33: 759–778.
Lukaszewski, A., 1997 The development and meiotic behavior of
asymmetrical isochromosomes in wheat. Genetics 145: 1155–
1160.
Luo, M.-C., J. Dubcovsky and J. Dvorák, 1996 Recognition of homeology by the wheat Ph1 locus. Genetics 144: 1195–1203.
Maguire, M. P., 1967 Evidence for homologous pairing of chromosomes prior to meiotic prophase in maize. Chromosoma 21: 221–
231.
Molé-Bajer, J., 1969 Fine structural studies of apolar mitosis. Chromosoma 26: 427–448.
Rasmussen, S. W., and P. B. Holm, 1978 Chromosome pairing and
recombination nodules in human spermatocytes. Carlsberg Res.
Commun. 43: 275–327.
Riley, R., and V. Chapman, 1967 Effect of 5BS in suppressing the
expression of altered dosage of 5BL on meiotic chromosome
pairing in Triticum aestivum. Nature 216: 60–62.
Scherthan, H., S. Weich, H. Schwegler, C. Heyting, M. Harle
et al., 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.
Sears, E. R., 1952 The behavior of isochromosomes and telocentrics
in wheat. Chromosoma 4: 551–562.
Sears, E. R., 1954 The aneuploids of common wheat. Mo. Agric.
Exp. Stn. Res. Bull. 572: 1–58.
Sears, E. R., 1976 Genetic control of chromosome pairing in wheat.
Annu. Rev. Genet. 10: 31–51.
Sears, E. R., 1977 An induced mutant with homoeologous pairing
in common wheat. Can. J. Genet. Cytol. 19: 585–593.
Smith, S. G., 1942 Polarization and progression in pairing. II. Premeiotic orientation and the initiation of pairing. Can. J. Res.
Sect. C. Bot. Sci. 20D: 221–229.
Vega, J. M., and M. Feldman, 1998 Effect of the pairing gene Ph1
on centromere misdivision in common wheat. Genetics 148:
1285–1294.
von Wettstein, D., S. W. Rasmussen and P. B. Holm, 1984 The
synaptonemal complex in genetic segregation. Annu. Rev. Genet.
18: 331–413.
Weiner, B. M., and N. Kleckner, 1994 Chromosome pairing via
multiple interstitial interactions before and during meiosis in
yeast. Cell 77: 977–991.
Yacobi, Y., T. Mello-Sampayo and M. Feldman, 1982 Genetic induction of bivalent interlocking in common wheat. Chromosoma
87: 165–175.
Communicating editor: R. S. Hawley