Condensation of rye chromatin in somatic interphase nuclei of Ph1 and ph1b wheat.

Abstract

The Ph1 locus in hexaploid wheat (Triticum aestivum L.) enforces diploid-like behavior in the first metaphase of meiosis. To test the hypothesis that this chromosome pairing control is exercised by affecting the degree of chromatin condensation, the dispersion of rye chromatin in interphase nuclei in somatic tissues of wheat-rye chromosome translocations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL and 5RS.5BL was compared in Ph1 and ph1b isogenic backgrounds. No significant differences in rye chromatin condensation that could be attributed to the Ph1 locus were detected. Regardless of the Ph1 status, each rye chromosome arm tested conformed to the general Rabl's orientation and occupied portions of the nuclei proportional to their length. Earlier observations that indicated the involvement of Ph1 locus in rye chromatin condensation in wheat could have been due either to specific loci on the studied 5RL rye arm that control the chromosome condensation process or to damage to the genetic system controlling chromatin condensation in the existing ph1b stocks of wheat. That damage might have been caused by homoeologous recombination and uneven disjunction of chromosomes from multivalents.

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Original Article
Cytogenet Genome Res 119:263–267 (2007)
DOI: 10.1159/000112072
Condensation of rye chromatin in somatic
interphase nuclei of Ph1 and ph1b wheat
D. Kopecky
a D.C. Allen
b M. Duchoslav
c J. Dolezel
a A.J. Lukaszewski
b
a Institute of Experimental Botany, Olomouc (Czech Republic)
b Department of Botany and Plant Sciences, University of California, Riverside, CA (USA)
c Department of Botany, Faculty of Sciences, Palacky University, Olomouc (Czech Republic)
Wheat ( Triticum aestivum L., 2n = 6x = 42) is an allo-
polyploid with the nuclear genome composed of three re-
lated genomes A, B and D derived from its three diploid
ancestors. A chromosome in each genome has its genetic
equivalent in the two other genomes to the extent that most
nulli-tetrasomic combinations, where a pair of chromo-
somes from one genome is replaced by an additional pair of
chromosomes from one of the other two genomes, are fully
functional and fertile (Sears, 1966). These genetically re-
lated chromosomes from different genomes are called ho-
moeologues. With the Ph system disabled, homoeologues in
wheat are capable of frequent meiotic pairing. Of the two
loci, Ph1 has a far stronger effect (Sears, 1984). In its absence,
but in the presence of Ph2 , homoeologous recombination
takes place, not only among the three genomes of wheat but
also among any of those and other chromosomes intro-
duced into wheat from related species. This feature facili-
tates chromosome engineering and introgressing alien
chromatin into wheat (Sears, 1981).
Even though the effects of the Ph1 locus have been
known for 50 years now (Riley and Chapman, 1958; Sears
Abstract. The Ph1 locus in hexaploid wheat ( Triticum
aestivum L.) enforces diploid-like behavior in the first meta-
phase of meiosis. To test the hypothesis that this chromo-
some pairing control is exercised by affecting the degree of
chromatin condensation, the dispersion of rye chromatin in
interphase nuclei in somatic tissues of wheat-rye chromo-
some translocations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL
and 5RS.5BL was compared in Ph1 and ph1b isogenic back-
grounds. No significant differences in rye chromatin con-
densation that could be attributed to the Ph1 locus were de-
tected. Regardless of the Ph1 status, each rye chromosome
Request reprints from Adam J. Lukaszewski
Department of Botany and Plant Sciences, University of California
Riverside, CA 92521 (USA)
telephone: +1 951 827 3946; fax: +1 951 827 4437
e-mail: adam.lukaszewski@ucr.edu
© 2008 S. Karger AG, Basel
1424–8581/07/1194–0263$23.50/0
Accessible online at:
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arm tested conformed to the general Rabl’s orientation and
occupied portions of the nuclei proportional to their length.
Earlier observations that indicated the involvement of Ph1
locus in rye chromatin condensation in wheat could have
been due either to specific loci on the studied 5RL rye arm
that control the chromosome condensation process or to
damage to the genetic system controlling chromatin con-
densation in the existing ph1b stocks of wheat. That damage
might have been caused by homoeologous recombination
and uneven disjunction of chromosomes from multiva-
lents.
Copyright © 2008 S. Karger AG, Basel
Efficient production of balanced gametes depends on reg-
ular bivalent formation in the first metaphase (MI) of meio-
sis. Any deviation from bivalent pairing introduces some de-
gree of instability, hence lowers the efficiency of the entire
process. While bivalent formation is natural in a normal dip-
loid organism, polyploidy may lead to the formation of mul-
tivalents and hence, unequal disjunction in the first ana-
phase (AI). Perhaps for this reason, many polyploid species
evolved genetic systems that enforce bivalent pairing in MI
(for review, see Jenczewski and Alix, 2004). The most studied
of these is the Ph (pairing homoeologous) system of polyploid
wheat. It consists of at least two loci, Ph1 and Ph2 , located on
chromosomes 5BL and 3DS, respectively (Riley and Chap-
man, 1958; Sears and Okamoto, 1958; Sears, 1977).
Accepted in revised form for publication by B. Friebe, 17 August 2007.

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Cytogenet Genome Res 119:263–267 (2007)264
and Okamoto, 1958), its mode of action still remains un-
known. The proposed hypotheses have ranged from Ph1
controlling spatial disposition of chromosomes in all tis-
sues of a plant (Feldman and Avivi, 1988) via the control of
centromeres (Martínez-Perez et al., 2001, 2003) to the con-
trol of stringency of crossing over (Dubcovsky et al., 1995).
Recently it has been postulated that the Ph1 locus controls
chromosome pairing by affecting the level of chromosome
condensation (Mikhailova et al., 1998; Maestra et al., 2002).
In its absence, alien chromatin appeared more diffuse in
somatic interphase nuclei and in early meiosis, and so, pre-
sumably, it was more open to surveys by molecular ma-
chinery attempting to identify and juxtapose DNA seg-
ments amenable to crossing over. This effect was clearly
visible both in the meiocytes and in somatic tissues tested.
Because condensation differences are postulated as a
mechanism in the recognition of homology (Prieto et al.,
2004) and invoked in the putative identification of the
DNA sequence of the Ph1 locus (Griffiths et al., 2006) we
have initiated a survey of a collection of alien introgres-
sions into some crop species to test if such easily detectable
differences in the levels of the interphase chromatin con-
densation could indeed be responsible for large differences
in the ability of homoeologous chromosomes to pair, in-
cluding large differences in pairing affinity of the same
chromosomes in different genetic backgrounds. In the first
step, observations of Mikhailova et al. (1998) and Maestra
et al. (2002) were verified using several wheat-rye centric
translocations in the Ph1 + and Ph1 – backgrounds in wheat.
Here we report that no measurable differences in chroma-
tin condensation attributable to the status of the Ph1 locus
could be detected.
Materials and methods
The plant material consisted of lines of hexaploid spring wheat
( T. aestivum L.) cv. ‘Pavon 76’, homozygous for centric wheat-rye trans-
locations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL and 5RS.5BL. These
translocations were either produced in ‘Pavon 76’ or were transferred
to it by at least seven backcrosses. In the course of engineering wheat-
rye centric translocations (Lukaszewski, 2000; Lukaszewski et al.,
2004) or in anticipation of such attempts, these translocations were
combined with the ph1b mutation line of ‘Pavon 76’. This line was pro-
duced by seven backcrosses of the original ph1b mutation from cv. ‘Chi-
nese Spring’ (Sears, 1977) to ‘Pavon 76’, followed by selfing and selec-
tion of homozygotes. The exception to this protocol was the 3RS.3DL
ph1b line for which a double homozygote was not available and was
selected specifically for this study. One double homozygote was identi-
fied among the progeny of a plant 19‘ + 3RS.3DL + 3D + 5B ph1b ‘, where
‘ denotes disomy/homozygosity.
Seeds of the appropriate stocks were germinated on wet filter paper
in Petri dishes, root tips were collected to ice water for 26–30 h and
fixed in a mixture of absolute alcohol:glacial acetic acid (3:
for seven days and then stored at –18 ° C. Cytological preparations and
in situ hybridization with labeled DNA were made according to Mas-
soudi-Nejad et al. (2002). In all experiments, genomic in situ hybridiza-
tion (GISH) was done with a probe prepared from total genomic DNA
of rye. The probe was labeled with digoxigenin by DIG-nick translation
and detected with anti-DIG fluorescein (FITC) using standard kits
from Roche Applied Science (USA) following the manufacturer’s in-
structions. The hybridization mix contained unlabeled genomic DNA
1) at 37 ° C
of wheat sheared to ca. 200–500 bp fragments at 1:
blocking DNA). Following hybridization, preparations were counter-
stained with 1.5% propidium iodide (PI) in VectaShield antifade (Bio-
Rad, UK), mounted and observed under a microscope.
To minimize experimental error, all observations were made on
slides each with two preparations of the same translocation on it: one
in the Ph1 and one in ph1b background. Squashes were made side by
side using 18 ! 18 mm cover slips; GISH was performed on both prep-
arations with the same hybridization mixture under single 22 ! 40
mm cover slips.
Samples were collected and measurements were done in two labo-
ratories in Riverside (CA, USA) and Olomouc (Czech Republic). For the
measurements of chromatin dispersion, images of individual nuclei or
groups of nuclei were captured using Meridian InSight Point Confocal
attachment mounted on a Zeiss Axioscope 20 microscope (Riverside)
or by SensiCam B/W CCD camera attached to Olympus AX70 micro-
scope (Olomouc). The resulting images were analyzed for the total area
of each nucleus, as visualized by the red color of PI, and the area of
green fluorescence (FITC), occupied by rye chromatin within each nu-
cleus. The percentage of the total nucleus area occupied by rye chro-
matin was taken as a measure of its dispersion. Samples of nuclei ana-
lyzed per plant ranged from 25 to 52. The proportions thus obtained in
individual lines were compared for each translocation separately in the
Ph1 and ph1b backgrounds using Nested ANOVA with plants nested
within Ph1 / ph1b (NCSS 2001 software, www.ncss.com).
150 ratio (probe:
Results and discussion
Visual screening of all lines failed to detect any obvious
differences either in the arrangement or dispersion of rye
chromatin in wheat interphase nuclei, between different
translocations and between the Ph1 and ph1b backgrounds
( Fig. 1 ). In all cases, labeled rye chromosome arms con-
formed to the general Rabl’s orientation and tended to run
parallel across the nucleus. Some deviations from this pre-
dominant arrangement were evident but they were relative-
ly infrequent and no translocation-specific or Ph1 -specific
pattern was obvious. The same general pattern of chromo-
some arrangement and chromatin dispersion was observed
in the nuclei of somatic tissues of anthers in the Ph1 and
ph1b lines of the 1RS.1BL and 2BS.2RL translocations ana-
lyzed for meiotic pairing (data not shown). The conclusion
from these cursory observations was that there was no gross
difference in the arrangement or the state of dispersion
among different translocations analyzed, nor between the
Ph1 and ph1b backgrounds, in somatic tissues of root tips or
anthers. This is in striking contrast to the observations of
Mikhailova et al. (1998) where the disposition of a rye chro-
mosome arm in the ph1b background appeared haphazard
and disorganized, in all tissues analyzed.
Despite some variation in signal intensities between dif-
ferent runs of GISH, the measurements of the dispersion of
rye chromatin in wheat nuclei produced fairly uniform re-
sults ( Table 1 , Fig. 2 ). The average proportion of the nuclear
area occupied by rye chromatin for individual transloca-
tions ranged from ca. 4% to almost 8.5% of the total area of
a nucleus. Given that each nucleus contained two labeled
chromosome arms among the total of 42 chromosomes
present, and that the domains occupied by the labeled rye
arms overlapped with the counterstained wheat arms, the
area proportions were as expected, considering size differ-

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Cytogenet Genome Res 119:263–267 (2007) 265
ences among rye chromosomes. In the rye genome, chro-
mosome 2R is the longest and has the highest DNA content
(Lukaszewski et al., 1982); it has an arm ratio of about 1.2.
The shortest arm in the rye genome is 5RS; in this study, it
occupied the lowest proportion of the nuclear area. The nu-
clei sampled varied in size, either due to differences in the
mitotic cycle or degree of squashing, and the proportions of
the area occupied by rye chromatin changed with changes
in the total nucleus area ( Fig. 2 ).
With one exception, no significant differences were
found in the proportions of nuclei occupied by rye chroma-
tin in any of the pairwise comparisons of the Ph1 and ph1b
for individual translocations ( Table 1 , Fig. 1 ). The exception
was the 5RS translocation where in the ph1b line rye chro-
matin occupied significantly larger proportions of the nu-
clei. This is not surprising, as the 5RS.5BL translocations in
the Ph1 and ph1b backgrounds are not identical. Given that
Ph1 is located on 5BL and tightly linked to the centromere
(Sears, 1984), the two lines had to be produced separately:
transfer of 5RS.5BL from the Ph1 to the ph1b background or
vice versa would have required screening large samples to
identify rare recombinants between the centromere and the
Ph1 locus. The translocation breakpoint in the 5RS.5BL Ph1
line is in the centromere; in the ph1b background it is on the
Chromosome
translocation
Number of
nuclei evaluated
Proportion of rye chromatin
in total nucleus area, %
Probability
(ANOVA)
Ph1 ph1bPh1 ph1b
1RS.1BL
2RS.2BL
2BS.2RL
3RS.3BL
5RS.5BL
Total
51 50
5.04 8 0.21
6.27 8 0.14
7.07 8 0.28
7.83 8 0.43
4.06 8 0.10
6.05 8 0.18
5.41 8 0.22
6.19 8 0.14
6.93 8 0.30
8.44 8 0.50
5.07 8 0.14
6.41 8 0.18
0.222
0.860
0.902
0.361
0.001
0.292
177
82
25
150
485
176
82
25
150
483
Table 1. Proportion of the total nucleus
area occupied by rye chromatin in several
Ph1 and ph1b wheat-rye translocation lines
of cv. ‘Pavon 76’ (mean 8 standard error)
Fig. 1. Centric wheat-rye translocation in mitotic metaphase and in
interphase nuclei in Ph1 and ph1b wheat as visualized by in situ hybrid-
ization with total rye genomic DNA labeled with FITC and counter-
stained with propidium iodide.
Fig. 2. The relationship between the total nucleus area and the area
occupied by rye chromatin in the Ph1 and ph1b lines of the ‘Pavon 76’
2BS.2RL translocation line.
2
1
0
20
40
60
80
100
120
140
160
180
200
050010001500
Nucleus Area (μm²)
Area of Rye Chromatin (μm²)
Ph1
ph1b
Linear (Ph1)
Linear (ph1b)

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Cytogenet Genome Res 119:263–267 (2007)266
long arm, 10–12% of the relative arm’s length away from the
centromere. Consequently, the rye segment in the latter is
longer and so rye chromatin occupies a larger proportion of
the nuclei.
In the final test, all Ph1 lines were compared to all ph1b
lines and no statistically significant difference was ob-
served. The only conclusion that can be drawn from these
results is that among the lines analyzed in this study, the Ph1
locus did not affect rye chromatin condensation in somatic
tissues of wheat. This is in clear contrast to the observations
of Mikhailova et al. (1998) where the differences in the ap-
pearance of the 5RL rye arm between Ph1 and ph1b lines
were so obvious, both in the somatic tissue and in meio-
cytes, that no attempts to quantify the effect were made.
Based on those differences and using the same stocks, Mae-
stra et al. (2002) felt justified to state that ‘Our results…are
strongly in favor of the putative effect of the Ph1 locus on
chromatin condensation and organization’. This putative
effect of Ph1 on chromatin condensation was invoked by
Griffiths et al. (2006) in an attempt to identify the coding
DNA sequence of Ph1 in the sequenced segment of chromo-
some 5B known to contain the locus.
The difference between the studies of Mikhailova et al.
(1998) and Maestra et al. (2002) on the one hand and this
study on the other may lay in the nature of the stocks used.
The 5RL ditelosomic in the ph1b background that was used
by Mikhailova et al. (1998) and Maestra et al. (2002) was not
analyzed in this study. Assuming that the difference was not
due the environment in which the respective studies were
conducted, two explanations seem plausible: either the 5RL
arm of rye affects chromatin condensation in wheat in some
interaction with the Ph1 locus, or the ph1b lines used in the
two experiments were different.
It is known that rye chromosome 5R interferes with the
diploidizing system in wheat and that this effect appears to
be dosage-dependent (Riley et al., 1973; Lelley, 1976). Per-
haps the effect observed by Mikhailova et al. (1998) and
Maestra et al. (2002) in the disomic 5RL addition to the ph1b
line of wheat was not a consequence of the absence of the Ph1
locus but rather a full expression of the loci on 5RL, uninter-
rupted by Ph1 . No stocks suitable for a critical comparison
are currently available. It must be noted, however, that Riley
et al. (1973) associated Ph1 suppression by 5R with the short
and not the long arm which contradicts numerous observa-
tions of the last author (Lukaszewski, unpublished data).
It has been recently illustrated (Sanchez-Moran et al.,
2001) that the ph1b lines of wheat suffer major genome rear-
rangements. Initially, this must be a consequence of ho-
moeologous pairing that takes places in the absence of Ph1 .
This is, after all, why Ph1 is considered so critical for the
stability and fertility of wheat. As the genome damage due
to homoeologous exchanges (and resulting unequal dis-
junction of chromosomes in AI) accumulate other effects of
other chromosomes and chromosome regions may also
start playing a role in the chromosome behavior and further
contribute to the general genome instability. Several wheat
chromosomes are known to affect meiotic behavior, such as
the asynaptic effect of nullisomy 3B (Sears, 1954). Because
homoeologous recombination events are unpredictable, it is
likely that the ph1b lines currently available in many labo-
ratories around the world, over 30 years after the first isola-
tion of the ph1b mutant (Sears, 1977), differ substantially in
their chromosome constitution and hence their genetics.
While they are suitable for the induction of homoeologous
pairing in chromosome engineering efforts, they may be
quite unsuitable for any studies of the mode of action of the
Ph1 locus itself. At best, they represent the combined effects
of the absence of Ph1 and all consequences of its absence that
have accumulated since the line was created. In this sense,
many effects now attributed to the Ph1 locus may in fact be
due to the presence of various genome rearrangements that
have accumulated since the Ph1 locus had been removed.
No reports have been published that would suggest or
imply that efforts have been undertaken to clean up the ex-
isting ph1b lines or to maintain them with the compensat-
ing presence of Ph1 so that no further damage to the genome
occurs. The ph1b lines used in this study were newly devel-
oped by repeated backcrosses to euploid cv. ‘Pavon 76’. This
approach guaranteed that in every backcross generation a
functional dominant Ph1 allele was present thus preventing
homoeologous recombination. At the same time, the seven
backcrosses used to develop the ph1b line of cv. ‘Pavon 76’
plus a cross and a backcross to each of the translocations
lines must have removed most, if not all, chromosome aber-
rations that might have been present in the starting Chinese
Spring ph1b line obtained from Dr. E. R. Sears. None of the
lines of the present study had more than two generations in
the ph1b status making chromosome rearrangements less
likely than in a line maintained by selfing for 30 years.
This study does not shed any new light on the mode of
action of the Ph1 locus in wheat. However, it provides evi-
dence that the locus does not affect plant-wide chromatin
condensation to any appreciable effect. Disorganized early
meiosis in the 5RL ph1b line of Mikhailova et al. (1998) and
Maestra et al. (2002) was likely a consequence of altered
chromatin organization in the entire plant including the
germ line, and not the other way around. Meiotic behavior
was not the object of this study; however, MI pairing of the
two translocations studied in detail, 1RS.1BL and 2BS.2RL,
was perfectly normal for rye introgressions in wheat and
resulted in wheat-rye recombined chromosomes among
progeny (Lukaszewski, 2000; Lukaszewski et al., 2004).
In maize, chromosomes undergo a structural conforma-
tion change as they enter meiosis (Dawe et al., 1994) and this
change may be related to the homology search. A similar
change is postulated to also take place in wheat (Mikhailova
et al., 1998). Perhaps this stage may be affected by the Ph1
locus. However, as this stage is short and transient there ap-
pears no reliable method of comparing the Ph1+ and Ph1–
lines in sufficiently large and homogenous samples to deter-
mine if Ph1 affects this stage. Still, given all accumulated
evidence, primarily from the observations of the synaptone-
mal complex formation (Holm, 1988) and crossing over in
introgressed alien segments (Dubcovsky et al., 1995; Luo et
al., 1996), the argument seems difficult to dismiss that the
Ph1 locus in wheat affects the stringency of recombination.

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Cytogenet Genome Res 119:263–267 (2007) 267
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