Inter- and Intragenomic Chromosome Pairing
in Haploids of Durum Wheat
P. P. Jauhar, A. B. Almouslem, T. S. Peterson, and L. R. Joppa
To assess inter- and intragenomic chromosome pairing in durum wheat (Triticum
turgidum L.), chromosome pairing and chiasma frequency were studied in durum
haploids (2n 5 2x 5 14; AB genomes) with the Ph1 allele, haploids with the ph1c
allele, and substitution haploids with chromosome 5B replaced by 5D. The Ph1haploids extracted from seven durum cultivars had little pairing; on the average
only 3.1% of the chromosome complement was paired with 0.23 chiasma per pollen
mother cell (PMC). Variation in haploid chromosome pairing frequency was observed among the seven genotypes. Chromosomes of the A and B genomes in the
ph1c-haploids showed increased pairing, with 38.6% of the complement paired and
3.0 chiasmata per cell. The potential of intergenomic pairing was more fully realized
in the substitution haploids, which had 51.3% of the complement paired with a
chiasma frequency of 4.1 per cell. Fluorescent GISH (genomic in situ hybridization)
analysis of PMCs revealed that most of the pairing was intergenomic, that is, between the chromosomes of the A and B genomes. Up to six intergenomic bivalents
were observed. Ring bivalents were common; a few showed interlocking. A low
frequency of intragenomic pairing within the A genome and within the B genome
was observed; the GISH analysis confirmed that this was not caused by intergenomic translocations. Bivalents within the A genome likely involved chromosomes
4A and 7A, and were more frequent than those within the B genome. Chromosome
pairing and chiasma frequencies in the 5D(5B) substitution haploids were similar
to those in amphihaploids obtained by hybridization between the putative progenitors of durum wheat. It is obvious that the homoeologous pairing control mechanism present in 5B exercises almost total pairing control that exists in durum wheat
and that the effect of other pairing control genes, if any, is insignificant. Moreover,
the A and B genomes have undergone little structural modification since the evolution of durum wheat several thousand years ago. Durum haploids, therefore, offer
excellent tools for studying genomic relationships in durum wheat.
From the USDA Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58105-5677
(Jauhar, Peterson, and Joppa) and the Department of
Botany, Faculty of Sciences, University of Aleppo, Aleppo, Syria (Almouslem). This research was supported in
part by a Fulbright Scholarship granted to A.B.A. by
the Council for International Exchange of Scholars,
Washington, D.C. Dr. Almouslem worked in Dr. Jauhar’s
lab in Fargo. Mention of a trademark or proprietary
product does not constitute a guarantee or warranty
of the product by the USDA or imply approval to the
exclusion of other products that also may be suitable.
Address correspondence to Prem P. Jauhar at the address above or e-mail: [email protected].
q 1999 The American Genetic Association 90:437–445
Durum wheat (Triticum turgidum L. ssp. durum Desf. Husr.) is an economically important cereal used for human consumption
worldwide. It is an allotetraploid (2n 5 4x
5 28; AABB genomes) whose genomes
were derived from two diploid wild species. The donor of the A genome is T. urartu Tum. ( Nishikawa 1983). Although
some controversy surrounds the donor of
B genome ( Kimber and Feldman 1987),
the available evidence shows that the B
genome was derived from Aegilops speltoides Tausch ( Daud and Gustafson 1996;
Dvořák 1998; Dvořák and Zhang 1990; Jaaska 1980; Sarkar and Stebbins 1956; Wang
et al. 1997). The two progenitors hybridized in the wild, about 10,000 years ago
( Harlan 1992), resulting in emmer wheat
(T. turgidum var. dicoccoides Körn). Either
unreduced gametes of the female and
male parents functioned or spontaneous
chromosome doubling took place in the
hybrid to give rise to tetraploid wheat.
Concurrently with this phenomenon occurred a spontaneous mutation leading to
the origin of the homoeologous chromosome pairing suppresser gene, Ph1, which
conferred meiotic regularity and hence reproductive stability to emmer wheat. Durum wheat of today is a domesticated
form of emmer ( Figure 1). Ph1, located on
the long arm of chromosome 5B, about 1.0
cM from the centromere (Sears 1984), exercises a precise control on meiotic chromosome pairing. Using a combination of
cytogenetic and molecular techniques, Gill
et al. (1993) reported the physical location
of Ph1 to a submicroscopic chromosome
region—the Ph1-gene region about 3 Mb in
size. With Ph1 present, only homologous
437
chromosome 5D. The Cappelli mutant has
a small deletion in the long arm of chromosome 5B and lacks the dominant allele
Ph1 (Giorgi 1978).
Diploid Hybrid Between Ae. speltoides
and T. urartu
Diploid hybrids between the two putative
progenitors, T. urartu and Ae. speltoides,
were synthesized by the embryo rescue
technique (Jauhar and Peterson 1996), using Ae. speltoides as the female parent.
Chromosome pairing in one of these hybrids was studied.
Chromosomal Studies by Conventional
Staining
The haploid status of young plantlets was
confirmed by counting somatic chromosomes from their root tips according to
the techniques described earlier (Almouslem et al. 1998; Jauhar 1993). For meiotic
analyses, spikes at appropriate stages
were fixed and anthers squashed according to Jauhar (1991). Three or more haploids from each cultivar were studied ( Table 1). Fifty pollen mother cells (PMCs)
per haploid were scored for various meiotic configurations and chiasma frequency.
Figure 1. Steps in the evolution of durum wheat.
partners pair, resulting in diploid-like pairing and disomic inheritance in durum
wheat. However, Ph1 restricts intergenomic or homoeologous pairing.
The chromosome complement of durum
wheat consists of seven homoeologous
groups of two chromosomes each (the
corresponding chromosomes in the A and
B genomes). Although the homoeologous
relationships between the A and B genomes have been known for over four decades (Sears 1954), the degree of pairing
between chromosomes of the two genomes has not been assessed. Because
the B genome donor (Ae. speltoides) contains active genes that promote homoeologous pairing, it is difficult to ascertain
chromosome pairing relationships between the A and B genomes. The only viable option, therefore, is to go backward
on the evolutionary scale ( Figure 1) and
extract polyhaploids (2n 5 2x 5 14; AB)
of durum wheat with one dose each of the
A and B genomes and then study chromosome pairing between them. Haploids
of polyploids are referred to as polyhaploids. In this article, the terms polyhaploid,
euhaploid, and haploid have been used
interchangeably because they all refer to
durum haploids with half the chromosome
number (2n 5 2x 5 14; AB). However, in
the presence of even a single dose of Ph1
438 The Journal of Heredity 1999:90(4)
in the haploids, the potential of pairing is
not realized because pairing among the
homoeologous chromosomes is suppressed. Therefore we synthesized durum
euhaploids with and without Ph1 (Almouslem et al. 1998) and studied chromosome
pairing and chiasma frequency. Specificity
of chromosome pairing was studied using
fluorescent genomic in situ hybridization
(GISH) on meiotic chromosomes. Details
of chromosome pairing in the synthetic
haploids are reported, and intergenomic
and intragenomic relationships are discussed in this article. This is the first report of chromosome pairing in synthetic
haploids of durum wheat.
Materials and Methods
Haploids with and without Ph1
We extracted haploids with and without
Ph1 by pollinating appropriate durum genotypes with maize, Zea mays L. (Almouslem et al. 1998). Haploids with Ph1 were
generated from seven durum cultivars
[Durox, Langdon ( LDN), Lloyd, Medora,
Monroe, Renville, and Vic]. Haploids without Ph1 were derived from the disomic
mutant ph1c ph1c of Cappelli (Giorgi
1978), and from a D-genome disomic substitution line LDN 5D(5B) in which chromosome 5B carrying Ph1 was replaced by
Chromosomal Studies by Fluorescent
GISH
Fluorescent GISH provides an excellent
tool for genomic painting and was used to
distinguish meiotic chromosomes of the A
and B genomes and to study the specificity of pairing in PMCs. GISH was carried
out by probing the A genome with Triticum
urartu DNA while blocking the B genome
with Ae. speltoides DNA. In the case of the
5D(5B) substitution line, the same scheme
was followed. With the B genome blocked,
any A-genome sequences in chromosome
5D fluoresced partially, that is, they
showed a banding or speckling effect (partial hybridization).
The A-genome probe was prepared using total genomic DNA extracted from
young, actively growing T. urartu leaves using the CTAB method ( Doyle and Doyle
1990). The DNA was sheared by passing
through a 25-gauge needle 400–500 times.
Fragment size was determined by electrophoresis on a 2% agarose gel with markers
of appropriate molecular weight. Shearing
was considered adequate when most of
the DNA bands were 600 to 300 bp in
length. The sheared DNA was labeled with
biotin-14-dATP using a BioNick Labeling
System (Gibco-BRL) following the protocol
supplied with the kit. Activity of the probe
Table 1. Chromosome pairing and chiasma frequency in durum wheat haploids (2n 5 2x 5 14; AB) with Ph1
I
Per
cell
Per
II
Percentage of
complement
paired
0.22
(0–2)
0.31
(0–2)
0.34
(0–1)
0.24
(0–2)
0.17
(0–1)
0.16
(0–2)
0.11
(0–1)
13.56
(10–14)
13.37
(10–14)
13.32
(10–14)
13.52
(10–14)
13.67
(12–14)
13.68
(12–14)
13.77
(12–14)
0.23
(0–2)
0.32
(0–2)
0.35
(0–2)
0.24
(0–2)
0.17
(0–1)
0.16
(0–1)
0.12
(0–2)
1.0
3.18
1.0
4.52
1.0
4.89
1.0
3.43
1.0
2.38
1.03
2.29
1.0
1.68
0.22
13.56
0.23
1.0
3.14
Mean and range of chromosome configurations at metaphase I
No. of
PMCs
scored
IV
Cultivar
No. of
haploids
studied
Durox
4
200
—
—
Langdon
3
150
—
—
Lloyd
4
200
—
Medora
4
200
—
0.005
(0–1)
—
Monroe
3
150
—
Renville
5
250
Vic
4
200
Overall mean
III
Fry pan
II
Ring
Chain
Total
0.005
(0–1)
0.007
(0–1)
—
—
—
0.005
(0–1)
0.007
(0–1)
0.005
(0–1)
—
—
—
—
—
—
—
—
—
—
—
0.005
(0–1)
0.005
(0–1)
0.004
(0–1)
—
0
0.001
0.002
0.003
was determined by Dot-Blot (Gustafson et
al. 1990) assay and only samples of probe
which showed good activity were combined and used. The final concentration of
the probe was verified using a spectrophotometer. The size of the probe fragments
was determined by electrophoresis on a
2% agarose gel, the preferred fragment
size being between 200 and 300 bp. Blocking DNA for the B genome was prepared
by autoclaving Ae. speltoides total genomic DNA for 15 min at 1218C. The autoclaved DNA was then ethanol precipitated
and resuspended in TE ( Tris-EDTA) buffer.
The concentration of the blocking DNA
was determined using a spectrophotometer, and fragment size was determined by
electrophoresis on 2% agarose gel.
Meiotic chromosome slides were prepared from spikes fixed in ethanol : chloroform : glacial acetic acid (6:3:1 by volume) fixative. Anthers were squashed in
45% acetic acid and viewed under phase
contrast. Well-spread chromosome preparations were stored for up to 1 week in a
2808C freezer.
—
—
—
0.001
Chiasma frequency
Rod
Total
0.22
(0–2)
0.31
(0–2)
0.34
(0–1)
0.24
(0–2)
0.17
(0–1)
0.16
(0–2)
0.11
(0–1)
0.22
Coverslips were removed and the GISH
protocol of N. Faridi (personal communication, 1997) was followed. The hybridization mixture used was as follows: 50 ml
formamide, 20 ml 50% dextran sulfate, 10
ml 203 SSC, 400 ng biotin-14-dATP-labeled
probe DNA, and 2000 ng blocking DNA. TE
buffer (pH 8.0) was used to bring the final
volume of the mixture to 100 ml. Twentyfive microliters of the hybridization mixture was applied to each slide, covered
with a plastic coverslip, hybridized for 10
min at 808C, and left overnight in an incubator at 378C. Fluorescein isothiocyanate
( FITC)-conjugated avidin DCS (10 mg/ml)
was used to detect the biotinylated probe,
while propidium iodide (PI) (20 mg/ml)
was used as a counterstain. A Zeiss Axioscope with a 50-W UV light source, 09 wide
bypass UV filter set for FITC detection,
and a 15 UV filter set for PI detection were
used to view the prepared slides. A Zeiss
MC-80 camera with Kodak Ektachrome
Elite 100 ASA slide film ( Eastman Kodak
Company, Rochester, New York) was used
to capture color images.
Results
We studied 27 synthetic haploids with Ph1
from seven durum cultivars ( Table 1), 2
haploids with the ph1c allele ( Table 2),
and 7 substitution haploids in which chromosome 5B was replaced by chromosome
5D ( Table 3). All the haploids had 2n 5 14
somatic chromosomes ( Figure 2A). Fluorescent GISH analysis of PMCs confirmed
that seven chromosomes each were derived from the A and B genomes ( Figure
4A,B). Intergenomic pairing between the
chromosomes of the two genomes and
intragenomic pairing within the two genomes were studied.
Haploids with Ph1
As expected, very little pairing occurred in
the presence of Ph1 ( Figure 2B,D; Table 1).
In the 27 Ph1-haploids, only 1.68–4.89% of
the chromosome complement paired with
a chiasma frequency of 0.12–0.35 per cell.
On average, 3.14% of the chromosome
complement paired with 0.23 chiasma per
cell. Thus, in most PMCs the 14 chromo-
Table 2. Chromosome pairing and chiasma frequency in durum wheat haploids (2n 5 2x 5 14; AB) without Ph1 derived from Cappelli mutant ph1c ph1c
Mean and range of chromosome configurations at metaphase I
Chiasma frequency
IV
III
Haploids
No. of
PMCs
scored
Chain
Fry pan
Chain
Total
Ring
Rod
Total
HP-124
50
—
—
HP-125
50
—
0.02
(0–1)
0.10
(0–1)
0.16
(0–1)
0.10
(0–1)
0.18
(0–1)
0.14
(0–1)
0.30
(0–2)
2.08
(0–5)
2.46
(0–5)
2.22
(0–5)
2.76
(0–5)
Overall mean
—
—
0.01
0.13
0.14
0.22
2.27
2.49
II
Per
cell
Per
II
Percentage of
complement
paired
9.26
(4–14)
7.94
(3–14)
2.56
(0–6)
3.44
(0–7)
1.02
33.86
1.11
43.29
8.60
3.00
1.07
38.57
I
Jauhar et al • Chromosome Pairing in Durum Wheat Haploids 439
Table 3. Chromosome pairing and chiasma frequency in substitution haploids (2n 5 2x 5 14; AB) without Ph1 derived from the Langdon 5D(5B) substitution
line
Mean and range of chromosome configurations at metaphase I
Chiasma frequency
IV
III
Haploids
No. of
PMCs
scored
Chain
Fry pan
Chain
Total
Ring
Rod
Total
HP-2
50
—
—
HP-7
50
—
—
HP-24
50
—
HP-42
50
—
0.02
(0–1)
—
HP-44
50
HP-63
50
0.02
(0–1)
—
HP-64
50
—
0.04
(0–1)
0.02
(0–1)
0.02
(0–1)
0.20
(0–1)
0.06
(0–1)
0.18
(0–1)
0.12
(0–1)
0.14
(0–1)
0.26
(0–1)
0.20
(0–2)
0.20
(0–1)
0.06
(0–1)
0.20
(0–1)
0.12
(0–1)
0.18
(0–2)
0.28
(0–1)
0.22
(0–2)
0.30
(0–2)
0.22
(0–2)
0.44
(0–2)
0.26
(0–2)
0.62
(0–3)
0.62
(0–3)
0.32
(0–2)
2.94
(1–5)
2.62
(0–5)
2.94
(1–6)
3.32
(1–6)
3.18
(1–6)
2.70
(1–5)
2.76
(0–5)
3.24
(1–6)
2.84
(0–5)
3.38
(1–6)
3.58
(1–6)
3.80
(1–6)
3.32
(1–6)
3.08
(0–5)
0.014
0.166
0.18
0.40
2.92
3.32
Overall mean
0.003
somes remained unpaired ( Figure 2B) and
sometimes showed 7:7 distribution to the
two poles ( Figure 2C). Any pairing between the chromosomes of the A and B
genomes was limited to the formation of
rod bivalents ( Figure 2D); ring bivalents
were rarely observed. Thus, the average
chiasma frequency was only 1.0 per bivalent ( Table 1).
Haploids without Ph1
The disomic mutant ph1c ph1c of the durum cultivar Cappelli and the appropriate
disomic substitution line of Langdon,
namely LDN 5D(5B), were used to extract
haploids without Ph1. The two genotypes
differed in their response to produce haploids. Whereas the substitution line, when
crossed with maize, produced 10 (1.05 %)
haploid plants from 956 florets pollinated,
the Cappelli mutant yielded only 2 (0.36
%) haploid plants (Almouslem et al. 1998).
Jauhar et al. (1991) also found that the mutant ph1b ph1b of the hexaploid wheat cultivar Chinese Spring gave a low yield of
haploids.
Because of the size differences between
the chromosomes of the A and B genomes
(Gill 1987), intergenomic pairing was characterized by the formation of heteromorphic configurations. Thus, heteromorphic
bivalents ( Figure 3A,C,D) and asymmetrical trivalents ( Figure 3H) were observed.
ph1c Haploids
Unlike the Ph1-haploids ( Table 1), the
ph1c-haploids showed substantial intergenomic pairing ( Table 2). In a total of 100
PMCs from two haploids, 38.57% of the
chromosomes were paired and this is al-
440 The Journal of Heredity 1999:90(4)
II
Per
cell
Per
II
Percentage of
complement
paired
6.92
(2–12)
8.14
(0–14)
6.70
(2–12)
6.48
(2–10)
5.78
(2–10)
6.52
(2–11)
7.18
(1–12)
3.94
(1–7)
3.18
(0–6)
4.20
(1–7)
4.08
(2–7)
4.88
(2–7)
4.52
(2–8)
3.86
(1–8)
1.09
50.57
1.08
41.86
1.13
52.14
1.07
53.71
1.16
58.71
1.19
53.43
1.10
48.71
6.82
4.10
1.12
51.33
I
most a 12-fold increase over that in the
Ph1-haploids; chiasma frequency increased by almost 13-fold. Meiotic associations of 5 II 1 4 I and 1 III 1 4 II 1 3 I
occurred, respectively, in 3% and 2% of the
PMCs. Pairing in these haploids was mostly limited to rod-bivalent formation as evidenced by chiasma frequency of 1.07 per
bivalent. Although ring bivalents and trivalents were observed, their frequency
was low ( Table 2).
Substitution Haploids
In the substitution haploids, which lacked
chromosome 5B and hence did not have
the Ph1 gene, homoeologous pairing was
greatly elevated. Almost 51% of the chromosomes paired, which is about 16-fold of
that observed in the Ph1-haploids; chiasma frequency increased almost 18-fold
( Table 3). Both chromosome pairing and
chiasma frequency were higher than that
in the ph1c-haploids ( Table 2). The fre-
Figure 2. Somatic and meiotic chromosomes of durum haploids with Ph1. (A) 14 somatic chromosomes. Note
the satellited chromosomes 1B and 6B; 1B has a smaller satellite. (B) 14 univalents at meiotic metaphase I. Note
the complete lack of pairing in the presence of Ph1. (C) Anaphase I with 7:7 distribution. (D) Metaphase I with 1
II 1 12 I.
scribed above. The hybrid had very good
pairing, with 50.43% of the chromosome
complement paired, with 4.02 chiasmata
per cell. The data on this hybrid are given
in Table 4 along with data on similar hybrids studied by other workers. The
A-genome donors listed in Table 4 are
chromosomally equivalent.
Figure 3. Chromosome pairing at meiotic metaphase I (MI) in durum haploids without Ph1. Note some conspicuously heteromorphic bivalents (arrows). (A) 4 rod II 1 6 I. (B) 2 ring II 1 2 rod II 1 6 I. (C) 1 III 1 2 rod II 1 7
I; note an asymmetrical trivalent and heteromorphic bivalents. (D) 1 III 1 1 ring II 1 3 rod II 1 3 I. (E) 2 ring II 1
4 rod II 1 2 I; note the interlocked ring bivalents. (F) 1 III 1 5 rod II 1 1 I; note the high pairing. (G) 1 III 1 1 ring
II 1 4 rod II 1 1 I; note the almost complete pairing. (H) 2 III 1 8 I; note the asymmetrical trivalents.
quency of ring bivalents was 0.40 per cell
( Table 3) compared to 0.001 per cell in the
Ph1-haploids ( Table 1) and 0.22 in the
ph1c-haploids ( Table 2). Of the 350 PMCs
scored, 59 had 5 or more bivalents per cell
( Figure 3E,F,G); 15 cells had 6 II 1 2 I ( Figure 3E). The meiotic configuration with
the highest amount of pairing recorded
was 1 III 1 5 II 1 1 I ( Figure 3F,G) in 3 of
the 350 PMCs. Thus, only one chromo-
some remained unpaired in these cells.
However, 7 II were not observed in any of
the cells. Interlocking of two ring bivalents
was observed in three cells ( Figure 3E).
Diploid Hybrid Between Ae. speltoides
and T. urartu
This synthetic hybrid (2n 5 2x 5 14; AB)
is essentially an equivalent of a substitution haploid without the Ph1 gene de-
GISH Analysis of Inter- and
Intragenomic Chromosome Pairing
Haploids with Ph1. The specificity of chromosome pairing was studied by observing
meiosis in the haploid complement by
counterstaining with propidium iodide
(PI) and also probing with the A-genome
probe using the GISH protocol. Although
some workers (e.g., Kimber and Feldman
1987) do not believe Ae. speltoides to be
the donor of the B genome, genomic DNA
from Ae. speltoides effectively blocked the
B genome in our study. As revealed by
GISH, the Ph1 haploids had seven chromosomes derived from the A genome and
seven from the B genome ( Figure 4A,B).
As shown in Table 1, pairing was very low
and limited to intergenomic rod-bivalent
formation ( Figure 4C,D). Intragenomic
pairing was not observed in haploids containing the Ph1 gene.
Haploids without Ph1. Substitution haploids (without Ph1), derived from Langdon
5D(5B) substitution line, showed extensive pairing ( Table 3), the specificity of
which was studied by GISH. Intergenomic
bivalent formation, both rods and rings
( Figure 5), accounted for most of the pairing. GISH analysis showed that most of the
heteromorphic bivalents were formed by
intergenomic pairing ( Figure 4C,D). Intergenomic trivalents formed by two chromosomes of the A genome and one of the
B genome or vice versa were observed. Intergenomic quadrivalents formed by two
chromosomes each of the A and B genomes were also recorded ( Figure 5E,F).
Intragenomic bivalents formed by chromosomes within the A genome or within
the B genome were observed. Figure 5B,
for example, shows an intragenomic bivalent formed by two A-genome chromosomes. It is interesting that such intragenomic bivalents were more frequently
formed within the A genome than within
the B genome.
Discussion
Pairing between the chromosomes of the
A and B genomes of durum wheat has not
been studied except in a few rarely occurring spontaneous haploids having the Ph1
Jauhar et al • Chromosome Pairing in Durum Wheat Haploids 441
Figure 4. GISH analysis of chromosome pairing at meiotic metaphase I (MI) in durum haploids with Ph1. (A) MI cell with 14 univalents of the A and B genomes uniformly
counterstained with propidium iodide (PI). (B) The same cell [as in (A)] probed with biotinylated A-genome DNA; the preparation was blocked with the genomic DNA of Ae.
speltoids ( B genome) and the probe was detected with FITC. The seven A-genome chromosomes are brightly lit in green color. (C) MI cell with 1 rod II 1 12 I counterstained
with PI. (D) The same cell [shown in (C)] was probed as in ( B) to visualize the chromosomes of the A and B genomes. Note the rod bivalent formed by chromosomes of the
A and B genomes, and six univalents of each of the genomes clearly visualized.
Table 4. Chromosome pairing and chiasma frequency in diploid hybrids between the putative donors of the A and B genomes
Hybrid
Ae. speltoides
3 T. urartu
Ae. speltoides ligustica I
3 T. monococcum
Ae. speltoides ligustica II
3 T. monococcum
Ae. speltoides ligustica III
3 T. monococcum
Ae. speltoides
3 T. monococcum
Chromosome
no. 2n
No. of
PMCs
scored
IV
AB
14
50
AB
14
50
AB
14
150
0.02
(0–1)
0.02
(0–4)
—
AB
14
50
AB
14
75
Overall mean
a
Mean and range of chromosome configurations at M I
Genomic
constitution
NS 5 not scored.
442 The Journal of Heredity 1999:90(4)
III
II
I
Ring
Rod
Total
0.04
(0–1)
—
0.14
(0–2)
0.18
(0–2)
0.15
(0–2)
0.16
(0–1)
—
0.40
(0–3)
2.44
(0–5)
0.44
(0–2)
0.38
(0–3)
NS a
2.88
(0–6)
3.84
(0–6)
2.34
(0–6)
3.28
(0–6)
NS a
3.28
(0–6)
6.28
(0–6)
2.78
(0–6)
3.66
(0–6)
3.37
(0–7)
6.94
(2–14)
0.82
(0–4)
8.42
(2–14)
6.04
(1–12)
7.25
(0–14)
0.011
0.125
0.76
2.84
3.55
6.66
Chiasma
frequency
Per
cell
Per
II
Percent
complement
paired
4.02
1.12
50.43
This study
9.14
1.39
94.14
Sears 1941
3.53
1.16
43.00
Sears 1941
4.48
1.10
56.86
Sears 1941
NS a
NS a
48.19
Kimber and Riley 1963
4.70
1.21
53.70
Reference
Figure 5. GISH analysis of chromosome pairing at meiotic metaphase I (MI) in durum haploids without Ph1 derived from LDN 5D(5B) substitution line. (A) MI with 1 ring
II 1 4 rod II 1 4 I counterstained with PI. (B) The same cell [shown in (A)] probed with the A-genome probe as in Figure 4B. The chromosomes of the A genome are lit up
in green color. Note the four intergenomic bivalents (formed by chromosomes of the A and B genomes) and an intragenomic bivalent within the A genome (arrow). (C) MI
with 2 ring II 1 1 rod II 1 8 I counterstained with PI. (D) The same cell [shown in (C)] probed with the A-genome probe (as described for Figure 4B). Note the three
intergenomic bivalents. (E) MI with 1 IV 1 2 rod II 1 6 I counterstained with PI. (F) The same cell [shown in ( E)] probed with the A-genome probe. Note the intergenomic
ABBA quadrivalent formed by chromosomes of the A and B genomes (arrow).
gene ( Kimber et al. 1978; Lacadena and
Ramos 1968; Romero and Sendino 1982;
see also Jauhar 1991). Synthetic durum
haploids (2n 5 2x 5 14; AB), with or without the homoeologous pairing suppresser,
Ph1, provide an opportunity for studying
intergenomic chromosome pairing rela-
tionships. Chromosome pairing in the two
sets of haploids without Ph1 ( Tables 2 and
3) was compared with that in haploids
with Ph1 ( Table 1). GISH helped to study
the specificity of chromosome pairing and
to discriminate between intergenomic and
intragenomic associations.
The Ph1-haploids showed very little
pairing, which was limited mostly to intergenomic rod bivalent formation. In 1,350
PMCs in the 27 haploids studied, only one
ring bivalent was observed ( Table 1). The
amount of pairing and chiasma frequency
in the durum Ph1-haploids was much less
Jauhar et al • Chromosome Pairing in Durum Wheat Haploids 443
than was observed in the common wheat
Ph1-haploids (Jauhar et al. 1991; Kimber
and Riley 1963). Only 3.14% of the chromosomes were paired in durum haploids,
while in bread wheat haploids pairing was
8.61% (Jauhar et al. 1991). This could be
due to the fact that the haploids of hexaploid wheat have in each homoeologous
set three chromosomes, which have a
greater potential of pairing, compared to
only two chromosomes per homoeologous set in durum wheat. Alternatively, the
genetic control of pairing may be more
stringent in durum with seven homoeologous sets of two chromosomes each than
in common wheat with seven homoeologous sets of three chromosomes each.
However, haploids (2n 5 3x 5 21) of hexaploid oat (Avena byzantina C. Koch)
showed only 0.17 rod bivalents per cell
and a ‘‘ring-shaped bivalent was never observed’’ in the 714 PMCs analyzed ( Nishiyama and Tabata 1964; see also Jauhar
1977).
There was some variation in the amount
of pairing and chiasma frequency among
the Ph1-haploids of the seven cultivars;
chiasma frequency per cell varied from
0.12 to 0.35 ( Table 1). Similar apparently
genotypic variation has been observed
among the spontaneous haploids of durum (see Jauhar 1991) and among the synthetic haploids of hexaploid wheat (Jauhar et al. 1991; Kimber and Riley 1963).
In the ph1c-haploids, 38.57% of the chromosome complement paired, with a chiasma frequency of 3.00, which resulted in
0.22 ring bivalent per cell ( Table 2). Obviously a much higher pairing occurred in
haploids with the recessive allele ph1c
than in those with Ph1. However, chromosome pairing and chiasma frequency
were less in the ph1c-haploids than in the
ph1b-haploids of spring wheat (Jauhar et
al. 1991), even though the latter carried on
chromosome 3D another suppresser of homoeologous chromosome pairing (MelloSampayo 1971; Sears 1976, 1984; Upadhya
and Swaminathan 1967).
Chromosome pairing and chiasma frequency were greater in the LDN 5D(5B)
substitution haploids that lacked the entire chromosome 5B ( Table 3) than in the
ph1c-haploids ( Table 2), although in both
cases Ph1 was absent. The ph1c-haploids
had less pairing despite the presence of
chromosome 5B, which has a pairing promoter in its short arm (Sears 1976). This
is in contrast to the situation in hexaploid
wheat in which the amount of chromosome pairing and chiasma frequency were
higher in the ph1b-haploids (Jauhar et al.
444 The Journal of Heredity 1999:90(4)
1991) than in the nulli-5B haploids ( Kimber and Riley 1963). The replacement of
5B with 5D in the substitution haploids of
durum wheat is analogous to the nulli-5B
condition in hexaploid wheat. ( Nullisomy
for chromosome 5B or any other chromosome is not tolerated in durum wheat.
Hence nulli-5B plants have not been reported. Therefore, to study the effect of
the absence of 5B in durum it has to be
replaced by its homoeologue 5D. It appears that 5D does not alter the pairing
pattern in the substitution haploids.) It is
interesting that the amount of homoeologous pairing (51.3% of complement) in
the seven LDN 5D(5B) substitution haploids was substantially lower than that
(60.7% of the complement; Kimber and Riley 1963) in nulli-5B haploids of spring
wheat, although in both cases chromosome 5B was missing.
An interesting phenomenon observed
was the interlocking of ring bivalents (Figure 3E). This phenomenon, which is generally attributed to trapping of a bivalent between two homologues of another during
synapsis at zygotene, is usually very rare.
However, it was increased in common wheat
by genetic means in which the dose of the
long arm of chromosome 5B (i.e., 5BL), and
hence of the Ph1 gene, was increased stepwise to higher doses of 3, 4, and 6 ( Yacobi
et al. 1982). A linear relationship between
Ph1 dose and the frequency of interlocking
was observed. These authors found that
wheat plants with a reduced dose (one or
zero) of Ph1 also showed an increased frequency of interlocking, but to a lesser extent
than those with high Ph1 dosage. Our durum haploids were without Ph1 because
chromosome 5B was replaced by 5D. In
these haploids the frequency of interlocking
was not only low but also involved no more
than two ring bivalents (Figure 3E). Clearly
this interlocking was restricted to homoeologous ring bivalents. It has been reported
that the short arm of chromosome 5B (i.e.,
5BS) carries a gene(s) that counteracts the
effect of Ph1 on interlocking in common
wheat ( Yacobi et al. 1982). In our durum
haploids, chromosome 5B was missing altogether. Thus, the interlock-inducing effect
of Ph1 (located in the long arm) and the
interlock-reducing effect of genes located in
the short arm were missing. Yet, durum
wheat haploids showed bivalent interlocking—a phenomenon known to be genetically
controlled in common wheat.
Jauhar et al. (1991) estimated the degree of pairing affinity and thereby the degree of relatedness among the A, B, and D
genomes of bread wheat by studying the
specificity of chromosome pairing in the
Ph1- and ph1b-haploids (2n 5 3x 5 21;
ABD). They observed that in the presence
of the allele ph1b, the chromosomes of the
A and D genome paired preferentially compared to those of the A-B and B-D genomes. The question then remained how
the chromosomes of the A and B genomes
would pair in the absence of the D genome. Clearly, chromosomes of the A and
B genomes pair extremely well in the durum haploids without Ph1 ( Figure 3A–H;
Table 3), with 51.3% of the chromosome
complement showing pairing; most of this
pairing is due to intergenomic pairing between the chromosomes of the A and B
genomes ( Figures 4 and 5). Unlike the
spring wheat haploids, conditions for preferential pairing do not exist in the durum
haploids and hence chromosomes of the
A genome have only the option of pairing
with chromosomes of the B genome.
Using C-banding ( Naranjo 1990; Naranjo
et al. 1987) and N-banding (Gill and Chen
1987), specificity of chromosome pairing
was studied, which helped in meiotic genome analysis in wheat. GISH is another
powerful tool for chromosome mapping
and for elucidating genomic relationships
(Anamthawat-Jónsson and Heslop-Harrison 1993; Heslop-Harrison and Schwarzacher 1996; Mukai 1996; Schwazacher et
al. 1989). We used GISH to discriminate between intergenomic and intragenomic
chromosome pairing in durum haploids
( Figures 4 and 5). Most pairing was intergenomic in nature, that is, between the
chromosomes of the A and B genomes,
and up to six intergenomic bivalents were
observed. Multivalent formation involved
both inter- and intragenomic pairing ( Figure 5F). Some multivalents probably arose
from translocations involving chromosomes 4A, 5A, and 7B. Although AAB and
ABB trivalents were observed, most associations were still intergenomic. Using Giemsa banding, Naranjo (1990) and Naranjo
et al. (1987) found a 4AL-5AL-7BS cyclic
translocation specific to T. turgidum and T.
aestivum. It is difficult to conclude that the
AAB trivalents involved chromosomes 4A,
5A, and 7B.
Intragenomic rod bivalents were also
observed ( Figure 5B), as shown by Naranjo (1990). The GISH analysis showed
that they were not caused by intergenomic translocations. The autosyndetic
bivalents may be attributed to residual homology within the A or B genome caused
perhaps by ancient intragenomic translocations. They most likely involved
chromosomes 5A-7A and 5B-7B (in ph1c-
haploids). However, intragenomic rod bivalents were more commonly formed
within the A genome than within the B genome.
The synthetic durum haploids without
Ph1 are almost the equivalent of synthetic
diploid hybrids between the putative donors of the A and B genomes. It would,
therefore, be interesting to compare chromosome pairing in the two cases. In five
diploid hybrids between Ae. speltoides and
T. monococcum, 53.7% of the complement
paired ( Table 4), which is very similar to
chromosome pairing (51.33% of complement) observed in the seven substitution
haploids lacking chromosome 5B ( Table
3). It would appear, therefore, that the A
and B genomes have undergone little
structural modification since their merger
to produce durum wheat. Mochizuki and
Okamoto (1961) and Kimber and Riley
(1963) also showed that the chromosomes
of the three diploid progenitors have undergone little change during the evolution
of hexaploid common wheat. Hence studying chromosome relationships among the
A, B, and D genomes in the synthetic haploids of hexaploid wheat is a sound approach (Jauhar et al. 1991). It is further
obvious that the homoeologous pairing
control mechanism present in chromosome 5B is responsible for almost the entire control on pairing that exists in durum
wheat and that other pairing control genes
on other chromosomes, if any, have minor
effects, too low to be noticeable in the
presence of the major controller. It is nevertheless possible that it is more than the
Ph1 locus on chromosome 5B that gives
the regulatory effect because pairing frequency is higher in the absence of 5B than
in the durum haploids with ph1c mutation.
Because of genetically enforced chromosome pairing in tetraploid durum
wheat and hexaploid bread wheat, only
strictly homologous chromosomes pair,
resulting in diploid-like pairing and disomic inheritance. Consequently, the constituent genomes in polyploid wheats
have maintained their meiotic integrity
and hence have undergone little structural
modification since the evolution of durum
and hexaploid wheats. Therefore, synthetic haploids of these wheats, with and without Ph1, provide excellent tools for study-
ing intergenomic chromosome relationships.
Kimber G and Feldman M 1987. Wild wheat: an introduction. Special report 353. Columbia: University of
Missouri.
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Received October 14, 1998
Accepted February 18, 1999
Corresponding Editor: Reid G. Palmer
Jauhar et al • Chromosome Pairing in Durum Wheat Haploids 445