21
Development 1990 Supplement. 21-28
Printed in Great Bntmn © The Company of Biologists Limited 1990
Gene expression and parental dominance in hybrid plants
J. S. HESLOP-HARRISON
Karyobtology Croup, Jl Centre for Plant Science Research, Colney Lane, Norwich, NR4 7UJ, UK
Summary
Genomic imprinting, where the genes from one parent
have different expression properties to those of the other
parent, occurs in plants. It has potentially significant
consequences because of the importance of hybrids in
plant evolution and plant breeding, and provides a
mechanism that can hide genetic variation for many
generations. The study of nuclear organization shows
that chromosome and genome position relates to
imprinting in F| hybrids, with peripheral genomes
tending to be expressed preferentially. In some inbred,
polyploid hybrids, such as Triticale (a wheat x rye
hybrid), treatment with the demethylation agent azacytidine releases hidden variation, which was perhaps lost
because of imprinting phenomena.
Occurrence and importance
after several generations of inbreeding, which would
lead to non-uniformity in crop stands.
Although the mechanism of imprinting is not certain,
it is possible that imprinting might be manipulated. For
example, particular sets of genes could be incorporated
in a crop, which could them be activated in a
subsequent generation. Such a property would be
useful both for the induction of new characteristics at
particular times (e.g. the period when cereal grains are
filling) or years (e.g. drought). Alternatively, genomic
imprinting could be used to ensure the expression of
certain desirable groups of genes in all the progeny of a
cross, without the possibility of non-expression because
of dominance relationships or supression.
Genetic phenomena that do not follow Mendelian
segregation ratios have been noted - and occasionally
published - in hybrid plants over the last 50 years. They
have been explained by phrases including 'block
transferance of characters,' 'genetic affinity,' 'suppression,' 'selectivity' of expression and 'cryptic structural differentiation.' Other unusual segregation ratios
have been explained using 'skewed backcross ratios,'
'polygenes,' 'expression modifiers/modification,' 'linkage' and 'homoeostasis.' In some cases, elimination of
chromosome sets, leaving the chromosomes of only one
parent, may be involved (e.g. Davies, 1958, 1974;
Lange, 1971). However, some of the non-Mendelian
events can be explained by genomic imprinting - where
genes from one parent have different expression
properties to those of genes from the other parent,
solely as a consequence of their parental origin. In the
present work, 'parents' will be used to describe both the
direct parents of an individual plant, and, for inbred
and hybrid plants, the original lines used to make the
cross that gave rise to the stock. Even within inbred
lines, parental differences may give imprinting, but, in
plants, the evidence for this remains limited and largely
unpublished.
In crop plants
When breeding crop plants, phenomena leading to
genomic imprinting can prevent the production of new
plant hybrids that combine desirable characteristics of
the parents. Another consequence of imprinting can be
the unexpected and undesirable release of variation
Key words: gene expression, parental dominance, hybrid
plants, Triticale, demethylation, azacytidine.
In wide hybrids
In the plant kingdom, sexual interspecific and even
intergeneric hybrids can be made relatively easily (see,
e.g. Stephens, 1949; Finch and Bennett, 1980). In
evolutionary terms, such wide hybridization may lead
to genetic introgression in plants, where genes are
transferred between different evolutionary lines. The
recombination of characters between alien species is
also important for enabling the introduction of new
genetic variation into the gene pools of crops which may
have been restricted by many centuries of inbreeding or
intensive selection for homozygosity. Many such
hybrids do not resemble intermediates between the
parental species, but exhibit a form of genomic
imprinting or parental dominance, which is not necessarily gamete specific. The present paper aims to show
that such forms of species-specific imprinting occur in
plants in both F, hybrids and inbred lines.
22
J. S. Heslop-Harrison
Fig. 1. Morphological
characteristics of the
bases of leaves of (A)
Hordeum vulgare
(barley). (B) Secale
africanum, a wild rye
species and (C) the
intergeneric F| sexual
hybrid H. vulgarexS.
afncanum. The hybrid
resembles the S.
afncanum parent for
characteristics such as
the ligule (I) length,
auricle claw (au) size
and hairiness.
Parental dominance in wide hybrids
In Fi hybrids between cereals
F[ hybrids are of both practical and research interest
because they are the source of new combinations of
genes. Within a species, two varieties may be intercrossed to produce a new variety combining the best
characteristics of both parents. Wide hybrids may be
made to transfer alien genes into a crop species or as a
preliminary to producing a polyploid plant. Fi hybrids
also exhibit characteristics referred to as "hybrid
vigour," which is important for increasing yield and
yield stability.
Finch and Bennett (1980) made intergeneric hybrids
between various barley {Hordeum) and rye {Secale)
species. The F[ hybrid H. vulgarexS. africanum does
not resemble an intermediate between the two parents
but resembles the Secale parent for many different
characters (Bennett, 1984; Heslop-Harrison and Bennett, 1984; Fujigaki and Tozu, 1987). Fig. 1 shows
photographs of a leaf base of the parental species and a
new hybrid between H. vulgare cv. Tuleen 346 (Finch
and Bennett, 1982) and S. africanum, made following
the techniques of Finch and Bennett (1980). The ligule
and auricle characteristics of the hybrid resemble the S.
africanum parent more than the H. vulgare parent.
Fig. 2 shows leaf surface peels from representative
individuals of the three plants, again with the hybrid
resembling the S. africanum parent more than the H.
vulgare in hairiness and cell form. Other characteristics
include those of the perenniality (the hybrid and S.
africanum are perennial, while the barley flowers and
dies in one growing season) and cold hardiness (the
hybrid and S. africanum are extremely sensitive to cold
temperatures). Thus, the resemblance is not only for
morphological but some physiological characteristics.
Fig. 2. Leaf surface
peels from the lower
surface of leaves of (A)
H. vulgare. (B) the
hybrid H. vulgarexS.
africanum and (C) S.
africanum. As in Fig. 1,
the hybrid resembles the
S. africanum parent
more than the H.
vulgare parent.
n
Gene expression and parental dominance in hybrid plants
In this hybrid, the male parent was S. africanum, and
we have not succeeded in making the cross in the
reverse direction. The difficulty is probably related to
the breeding methods of the plants, since the rye species
is outbreeding and produces large amounts of vigourous
pollen, in contrast to the largely self-pollinating barley.
Nevertheless, although direct evidence is as yet limited,
it might be expected that the phenotypic dominance
observed is due to genotypic effects rather than a
consequence of the parental origin of the gametes.
Correlations of phenotype with chromosome position
The physical positioning of the chromosomes in the
hybrid H. vulgarexS. africanum has been investigated
by reconstructing metaphases from sets of electron
micrographs of serial sections. These results have
shown that the chromosomes originating from the two
parental genomes are not randomly positioned within
the hybrid, but tend to be spatially separated with those
from one species lying in a peripheral domain, while
those from the other are more central. In the H.
vulgarexS. africanum hybrid, the chromosomes of H.
vulgare origin tend to be central in the nucleus, while
those of 5. africanum origin are peripheral (Bennett,
1982, 1984; Finch and Bennett, 1981; Bennett, 1988;
Heslop-Harrison and Bennett, 1984). However, at
metaphase, the chromosomes are generally inactive in
gene expression and DNA replication, so it is necessary
to know if the genomes are also spatially separated at
interphase, when the chromosomes are active in gene
expression and DNA replication.
Jn hybrids where the DNA from the two parents is
sufficiently different, the chromosomes of the two
genomes can be distinguished at all stages of the cell
cycle by a method using in situ hybridization of total
genomic DNA. The basis of the technique is illustrated
in Fig. 3, which shows Southern transfers of restriction
enzyme digests of DNA from two parents and an
intergeneric hybrid. These have been hybridized with
labelled genomic DNA from one of the parents, H.
chilense, alone (Fig. 3A), or in the presence of a large
excess of unlabelled genomic DNA from the other
parent, S. africanum, to block sequences that are
common between the two genomes so the labelled
DNA cannot hybridize (Fig. 3B; see AnamthawatJ6nsson et al. 1990, for further details). The tracks with
DNA from the H. chilense parent show stronger
hybridization than the S. africanum tracks, and the
differentiation is emphasized in the blocked blot where
the two parents and hybrid can be distinguised clearly.
Fig. 4 shows the results following in situ hybridization
using genomic probe and blocking (Heslop-Harrison et
al. 1990; Schwarzacher et al. 1989) in the F, hybrid H.
vulgarexS. africanum. The micrographs show that the
two genomes, originating from different parents, tend
to be spatially separated both at prophase and during
interphase, as well as at metaphase. However, these
data are from spread material, where it is possible that
differential penetration of probe or detection reagents
could lead to the observed results. Fig. 5 shows a single
section through nuclei of the hybrid, which demon-
23
mn
\
Sa X
He
\
Sa
X He
B
Fig. 3. Luminographs showing sites of hybridization of
labelled total genomic Secale africanum DNA to Southern
transfers of size-fractionated restriction enzyme digests of
DNA from S. africanum (Sa), H. chilense (He) and the F,
hybrid between the two species (X). (A) Hybridized with
labelled H. chilense DNA alone; (B) hybridized with
labelled genomic H. chilense DNA in the presence of an
excess of unlabelled genomic DNA from S. africanum to
block non-specific hybridization of labelled probe (see
Anamthawat-J6nsson et al. 1990). The probe enables
discrimination between the two species and the hybrid, and
the specificity of probing is increased by the addition of the
blocking DNA. (\ lambda Hmdlll digest as size marker,
top to bottom 23.1, 9.4, 6.6, 4.4, 2.3 and 2.1 kb).
strates that the genome separation is also seen in
sectioned material where the three-dimensional structure of the cell has been preserved (Leitch et al. 1990).
Reconstructions now being made from such interphase
nuclei tend to confirm the impression of the spatial
separation of parental chromosome sets at interphase in
the wide hybrids (Leitch, Schwarzacher and HeslopHarrison, unpublished data).
The two hybrids H. vulgarexS. africanum and H.
chilensexS. africanum have been studied in greater
detail than others. However, further examples of
hybrids where the phenotype resembled the parent that
contributed the peripheral chromosomes in the metaphase have also been reported (Bennett, 1984).
Parental dominance in segregating populations after
hybridization
Another class of parental dominance occurs in the
hybrid between two species of cotton, Gossypium
hirsutum and G. barbadense. The characteristics and
behaviour of this hybrid were first described many years
ago (see Stephens, 1950 and Wallace, 1960, for
references). In summary, after interspecific crossing
and self-pollination of subsequent generations, the
phenotype of the progeny often reverted to resembling
one or other of the parental species showing that groups
of parental characters were preserved (Schwendiman,
1974). In more formal terms, the progenies of
interspecific hybrids do not give clear Mendelian ratios
for the segregation of genes that are known to be
24
J. S. Heslop-Harrison
allelomorphic on the basis of intraspecific tests. In some
cases, which are well documented and comprehensively
discussed by Stephens (1950), interspecific hybrids
followed by backcrossing to the female parent can give
a 'marked deficiency' in the expression of the gene from
the male parent. He goes on to report that "the
deficiency cannot be due to the effect of the introduced
gene itself because with successive backcrosses the
normal 1:1 segregation ratio tends to be more closely
approached." Cytoplasmic effects cannot play a major
role since there will be little introgression of the male
cytoplasm into the hybrids and backcrosses. Although
some results can be explained by the segregation of
expression modifiers, and by cryptic structural differentiation of chromosomes which leads to selection against
chromosome segments introduced from one parent, it
seems probable that some of the effects are the result of
the imprinting of genes, and certainly the area would be
worthy of further investigation.
Parental dominance within inbred cereal hybrids
The phenotype of first generation hybrids between
different cereals is discussed above. Intergeneric
hybrids between cereals can be made and the chromosome number doubled to give polyploid plants, which
are fertile and can be grown as a crop. One such crop is
Triticale (2n = 6\=42,xTriticosecaIe Witt.), a hybrid
between a tetraploid wheat (2n = 4x = 28; Triticum
durum) and rye, S. cereale (2n=2x=14). This crop is
widely grown, particularly in eastern Europe and
Canada, for use in animal feed and occasionally for
milling for flour. However, like many of the other
hybrids discussed above, it does not resemble a true
intermediate between the parental species, but has
many features of rye rather than wheat (see, e.g.
Percival, 1923).
Methylation and gene expression in Triticale
Other papers in this volume discuss the importance of
methylation, particularly of the cytidine nucleoside, in
the control of gene expression and perhaps genomic
imprinting (see also Michalowsky and Jones, 1989;
Holliday et al. 1990). The experiment to be described
here was initiated because of the potential importance
or correlation between DNA methylation and gene
expression - including the species-specific imprinting in the wide hybrids and hybrid crops discussed above.
The results obtained are preliminary, but worthy of
publication because of their surprising nature. If
methylation is important to imprinting in such hybrids,
then demethylation might allow expression of otherwise
supressed genes from one of the parents.
Demethylation has been correlated with transcriptional activation of genes in animals (Bird, 1986) and
plants (Flavell et al. 1986). During germination of wheat
seedlings, the amount of methylcytosine has been
reported to diminish by about 15%, and later during
development, new sequences are methylated (see
Vanyushin, 1984); although, in common with most
Fig. 4. In situ hybridization to chromosome spreads of the
hybrid H. vulgarexS. afrtcaiwm using the genomic probing
with blocking method (Schwarzacher et al. 1989; HeslopHarrison et al. 1990). (A.B) A telophase nucleus where the
chromosomes are not fully condensed and the major axes
can be followed. (CD) An interphase nucleus. (A,C)
After staining with the DNA-specific dye DAPI which
fluoresces blue when excited with ultra-violet light. It
shows all the chromatin in the nucleus. (B,D) Following in
situ hybridization with labelled S. africamim DNA in the
presence of unlabelled H. vulgare DNA. The sites of
hybridization of the labelled DNA were detected by blue
light excited yellow fluorescence, while unlabelled
chromatin fluoresces orange with the propidium iodide
counterstain. The micrographs show that the two genomes,
originating from different parents, tend not to be
intermixed both at telophase and interphase. and the DNA
originating from S. afncamnn tends to be peripheral.
Fig. 5. A single 0.25 //m thick section through a metaphase
(M). prophase (P) and interphase (1) nuclei of the hybrid
H. vulgareXS. africamim which demonstrates that
chromatin of different parental origin is not intermixed in
sectioned material where the three-dimensional structure of
the cell has been completely preserved (Leitch et al. 1990).
(A) The section stained with the DNA-specific dye DAPI,
showing that all chromatin fluoresces relatively uniformly.
(B) The same section after probing with labelled genomic
5. africanum DNA and detection of the sites of
hybridization with Texas Red (fluorescing red under green
light excitation). The probe hybridizes strongly to the
DNA of S. africanum origin, which occurs in domains that
are not intermixed with the DNA of H. vulgare origin
(compare A and B).
plants, a high proportion of the genome remains
methylated. The drug 5-azacytidine can be used to
demethylate DNA: it is incorporated into DNA during
replication instead of cytidine, but cannot be methylated because of a nitrogen atom at the 5' position
(Jones and Taylor. 1980). Almost complete demethylation of genomic DNA occurs when a small percentage
of substitution with azacytidine has occurred because of
the effect of the compound as a false substrate and
inhibitor of the methylases (see Parrow. Alestrom and
Gautvik, 1989).
Demethylation effects on Triticale
Seeds of the Triticale variety Lasko were treated with
various concentrations of azacytidine for up to two
days. The plants arising from the treated seeds were
referred to as the M| (first mutation) generation, and
the plants from these seeds were the M2 generation.
Young leaves were removed from plants of both
generations when they were about two months old for
DNA extraction and subsequent analysis of restriction
enzyme digests using modifications of standard methods
(Sharp et al. 1988. 1989). Total genomic DNA from
young leaves was digested to completion using a range
of methylation-sensitive and -insensitive restriction
enzymes including HpaW. Msp\ and Apa\. The nonradioactive chemiluminescence method, ECL (Amersham) was used for probe labelling, hybridization and
the detection of sites of probe hybridization, following
Gene expression and parental dominance in hybrid plants
Msp\
o
3
HpaW
EcoRI
EcoRU
Apa\
25
3
O
CONTROL
0.1 mM
0.5mM
1 OmM
ZOmM
1.0mM
20mM
Treatment
Fig. 6. Luminographs showing restriction enzyme digests of
DNA from azacytidine (AZC)-treated and control (Cont)
Triticale plants of the M2 generation. All five enzymes are
methylation sensitive. The Southern transfers were probed
with the wheat ribosomal DNA clone pTa71 (see text).
Additional lower molecular weight fragments are visible in
the Msp\ and Apa\ digests.
the manufacturer's instructions and methods described
by Anamthawat-J6nsson et al. (1990).
The luminographs in Fig. 6 show the lengths of
restriction fragments which are homologous to the
wheat ribosomal DNA clone pTa71 (Gerlach and
Bedbrook, 1979; kindly provided by R. B. Flavell and
M. O'Dell, IPSR Norwich, after recloning in pUC19).
Both the M| and M2 generations show similar patterns
of probe hybridization. Some remethylation of DNA is
apparent between the two generations, but additional,
short, fragments are found consistently from some of
the restriction digests of DNA from the azacytidinetreated plants. The digests with Mspl (recognition
sequence CCGG, but methylation sensitive and cleaving only when the outer C is unmethylated) and Apal
(recognition sequence GGGCCC, and cleaving only if
the internal C is unmethylated) show that there are
differences in methylation of particular cytidine residues between treated and untreated plants within the
ribosomal DNA repeat unit.
In summary, the luminographs show that the
azacytidine treatment is effective in the demethylation
of some cytidine residues in the Triticale DNA, and that
these differences are inherited through at least two
Table 1. The morphological characteristics of plants
that were scored
Frequency of plants with recurved flag leaves
Anlhocyanin coloration of auricles of flag leaf
Glaucosity of flag leaf sheath
Time of ear emergence
Length of flag leaf blade
Width of flag leaf blade
Density of stem neck hairs
Length (height) of plant
•I
i
i
CONTROL
0.1 mM
0.5mM
Treatment
CONTROL
Fig. 7. Results from the scoring of some of the
characteristics of the M| plants. All characters were scored
on coded plants using a relative scale system. Shaded bar
height represents the mean; and lines above and below, the
standard deviation of the character. (A) Plant height from
1 (short) to 4 (tall). (B) Maturity from 1 (early to ripen) to
6 (late). (C) Number of tillers from 1 (few) to 6 (many).
There are large differences between the treated and control
plants, but few significant differences between the four
treatments.
2S
J. S. Heslop-Harrison
Fig. 8. Photographs of two pots, each containing three glasshouse-grown Triticale plants, which illustrate some of the
characteristics scored in Fig. 7 in the M) generation. (A) M? plant; the seed of the parent of these plants was treated with
azacytidine. (B) Control; as (A) but treated with water only.
generations. Inheritance of methylation patterns
through generations has been observed in the human
(e.g. Silva and White, 1988); in the Triticale experiment, future work will aim to find other methylation
differences, and to examine further plant generations.
The substantial overall level of methylation observed in
the Southern blots is expected, since remethylation of
the DNA will occur over the first few cell cycles after
removal or degradation of the azacytidine (Gruenbaum
et al. 1981); clearly not all sites in the azacytidinetreated DNA are remethylated to the level observed in
the controls in either the Mi or M2 generation.
Morphological analysis of plants
When the earliest plants were beginning to ripen,
various morphological characteristics shown in Table 1
were scored (without knowledge of the treatment of the
plant), following the Draft Guide-lines for the Conduct
of Tests for Distinctness, Uniformity and Stability for
Triticale (IUPOV, 1988). Visual assessment of other
field characteristics given in the table VII of IUPOV
(1988) showed that there was little or no variation
between the plants. For example, both treated and
control plants had similar semi-erect growth habits,
very weak or no anthocyanin coloration in awns or
anthers, and similar ear lengths.
Results from the scoring of some of the characteristics of the M| plants are shown in Fig. 7. When
compared with control plants, the azacytidine-treated
plants showed some significant differences in ripeness,
height and number of ears or tillers. Different times and
chemical concentrations made relatively little difference to the plant performance (Fig. 7). Fig. 8 shows
photographs of two plants of the glasshouse-grown M2
generation plants, which illustrate some of the characteristics scored in Fig. 7 in the M, generation.
The experiments were carried out on a variety of
Gene expression and parental dominance in hybrid plants
Triticale, Lasko, which has passed standard tests for
stability and uniformity of the crop, and hence would be
expected to show negligible plant-to-plant variation.
The azacytidine treatment was able to induce a range of
new characteristics, presumably by enabling or inducing
the expression of genes that were repressed in the
hybrid. We do not know whether genes or control
regions belonging to the parental genome which is less
expressed are methylated more than those of the
expressed genome; nor do we know the parental origin
of the genes that are responsible for the altered
characteristics. However, if the supression of blocks of
genes originating from one or other parent were
involved, then the system would be a good example of
genomic imprinting.
Further verification and experimental work
The number of plants grown in the glasshouse was
small, and their performance may not be a good guide
to field performance, so detailed conclusions must await
the scoring of larger numbers of plants grown outdoors.
It will be important to study the heritability of the new
characteristics (when the treated and control plants are
used as both male and female parents), the methylation
patterns in sequences other than the ribosomal RNA
genes, the cytology of the plants and nucleolar
expression patterns. While mutagenesis induced by the
azacytidine cannot be ruled out, it is unlikely that such
consistent DNA restriction fragment differences would
be observed (Fig. 6 and additional data not shown), and
unlikely that the morphological characteristics of many
different plants would be consistent (Figs 7 and 8). It is
conceivable that the range of variability induced by
azacytidine is similar to that generated when plants
(particularly of the Solanaceae) are regenerated from
somatic cells. If so, then demethylation and activation
of repressed genes may be involved in both systems.
Prospects
It will be important to examine the experimental wide
hybrids discussed in the first section of the present work
to see if there are any differences in the methylation of
the two genomes. Perhaps gene expression, and hence
the morphology of the hybrids, can be altered by
demethylation; such changes may have effects on
chromosome and genome disposition. Conversely, it
will be important to examine aspects of chromosome
and interphase gene disposition within the methylated
and demethylated Triticales to see if there are any
changes that correlate with the different expression
patterns. Finally, investigation of the heritability
patterns of the methylation polymorphisms in the two
Triticale lines may give particularly significant results.
The phenotype and methylation patterns of the progeny
will point to any imprinting phenomena that correlate
with parental methylation pattern, while excluding
genetic differences between the parents.
I thank BP Venture Research Unit for support of this work,
Mrs Julia Coates for much help with the Triticale azacytidine
27
experiment, and Mr Phil Webb for information on breeding of
Triticale and the supply of seed. I also thank my colleagues in
the Karyobiology Group (Dr Andrew Leitch, Dr Trude
Schwarzacher and Mrs Kesara Anamthawat-J6nsson) and
Professor MD Bennett for assistance with the nuclear
organization work.
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