Abstract Background In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses. Results Evidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested. Conclusion Our results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.</p

A Bottley

Andrew Bottley

B Koukalova

E Risseeuw

EL Peredo

FM Morrish

H Hirochika

HD Jones

HJ Li

J Sambrook

J Wang

K Kashkush

K Mochida

KL Adams

Li Xiaoling

MA Matzke

Natalie H Chapman

PT Bednarek

R Hovav

Robert MD Koebner

SM Kaeppler

SS Yang

BMC Genetics

English

PubMed

Springer - Publisher Connector

Homoeologous gene silencing in tissue cultured wheat callus

Chapman Natalie H

Bottley Andrew

Koebner Robert MD

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BioMed CentralBMC Genetics
ssOpen AcceResearch article
Homoeologous gene silencing in tissue cultured wheat callus
Andrew Bottley*1, Natalie H Chapman2 and Robert MD Koebner
Address: 1Department of Pharmacy, University Nottingham, NG7 2RD, UK and 2Department of Plant Sciences, University of Nottingham, LE12 
5RD, UK
Email: Andrew Bottley* - andrew.bottley@nottingham.ac.uk; Natalie H Chapman - Natalie.Chapman@nottingham.ac.uk; 
Robert MD Koebner - mockbeggars@gmail.com
* Corresponding author    
Abstract
Background: In contrast to diploids, most polyploid plant species, which include the hexaploid
bread wheat, possess an additional layer of epigenetic complexity. Several studies have
demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-
genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be
tissue specific and may be linked to developmental or stress responses.
Results: Evidence was sought as to whether the frequency of homoeologous silencing in in vitro
cultured wheat callus differ from that in differentiated organs, given that disorganized cells are
associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-
PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of
expression of 20 homoeologous sets of single-copy genes known to be affected by this form of
silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.
Conclusion: Our results suggest that much of the homoeologous silencing observed in
differentiated tissues is probably under epigenetic control, rather than being linked to genomic
instability arising from allopolyploidization. This study reinforces the notion of plasticity in the
wheat epi-genome.
Background
In diploid species, the control of gene expression is largely
under the control of promoters and transcription factors,
whereas in polyploids such as hexaploid wheat or tetra-
ploid cotton, an additional layer of complexity is created
by epigenetic variation. Thus, there is a growing body of
evidence showing that each homoeologous gene copy
does not necessarily contribute equally to the global tran-
scriptome [1,2]. In hexaploid wheat, for example, one of
the three homoeologues normally present is not
expressed in the leaf for approximately 30% of single copy
genes, with a similar figure observed for roots [1]. This
form of silencing has been shown to be variety-dependent
[3]. In tetraploid cotton trichome cells, similarly around
30% of genes show a noticeable bias in expression level
towards one genome [4], and, during development, this
bias can be shifted. This form of transcriptional inactiva-
tion has been termed 'homoeologous gene silencing' and
can be either tissue-specific or associated with a develop-
mental process [5,6]. It is not known as yet how this
silencing is either imposed or maintained, although it has
been suggested to arise from genomic shock occurring at
or shortly after a polyploidization event [7]. However,
recent investigations based on synthetic Arabidopsis poly-
Published: 17 October 2008
BMC Genetics 2008, 9:65 doi:10.1186/1471-2156-9-65
Received: 9 June 2008
Accepted: 17 October 2008
This article is available from: http://www.biomedcentral.com/1471-2156/9/65
© 2008 Bottley et al; licensee BioMed Central Ltd. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 4
(page number not for citation purposes)
BMC Genetics 2008, 9:65 http://www.biomedcentral.com/1471-2156/9/65ploids have shown that when methylation maintenance is
compromised, some previously silenced genes become re-
activated, thus implicating epigenetic control [8].
In a disorganized callus grown in vitro, chromosomal dele-
tions, translocations, transposon movement and epige-
netic repatterning all occur at a much higher level than in
planta [9-12]. Among barley plants regenerated from tis-
sue culture, both DNA sequence mutations and changed
methylation patterns have been observed, with the latter
occurring much more often than the former [13]. Simi-
larly, in re-generated hop plants, "somaclonal" changes
are more frequently epigenetic rather than genetic [14]. If,
as has been suggested, methylation is responsible for
much of the observed silencing in polyploids [8], and
since callus cells are relatively demethylated compared to
differentiated cells in planta [12,15,16], then the level of
silencing present in callus cells would be predicted be
lower than in planta. Thus, in this study, we set out to
examine the pattern of homoeologous silencing in wheat
callus cells. Our aim was to establish whether the undiffer-
entiated state of these cells influenced the level of homoe-
ologous silencing of genes known to be affected in this
way in the root or the leaf.
Results and Discussion
As the process of tissue culture is known to induce
sequence re-arrangements and point mutations [17], we
first compared the SSCP profiles of amplified callus
genomic DNA (gDNA) to those of amplified cv. Florida
gDNA. No detectable evidence for such events within the
amplified sequences was noted in any of the material
tested. Of the seven homoeologous sets of single-copy
genes, in which at least one homoeologue is not expressed
in cv. Florida leaf or root [3], four showed expression of
all three homoeologues in the callus, while the other three
were not expressed at all (Table 1). For instance, the A
genome homoeologue of EST BE426364 is expressed in
callus, but not expressed in the root tissue of cv. Florida
and in ten of a sample of 16 other varieties [3]. In order to
increase the number of genes sampled, we sampled a fur-
ther nine genes, in which at least one homoeologue is not
expressed in at least one tissue of cv. Chinese Spring [1].
Of these, all homoeologues were expressed in the callus,
and the remaining one was not expressed. Among a fur-
ther four genes showing full expression in cv. Chinese
spring leaf and/or root were tested, all were also fully
expressed in the callus (Table 1).
Table 1: A comparison between; genes shown to be expressed in leaf and/or root tissue of Chinese Spring (CS) (Bottley et al, 2006), 
the presence or absence of homoeologue silencing in the cultivar Florida (Bottley et al, 2008) and the expression of the same genes in 
callus tissue.
Genbank id Expressed Callus Homoeologue 
silenced in CS
Tissue Homoeologues 
identifiable in CS
Homoeologue 
silenced in Florida
Putative function
BE399113 Y D/D LEAF/ROOT B D B Unknown
BE444894 Y D/B LEAF/ROOT A B D saline responsive OSSRIII 
protein
BF482273 Y D/B LEAF/ROOT B D Unknown
BF201235 N D LEAF A B D Rubisco subunit binding-
protein alpha subunit
BF473379 Y D LEAF B D D Unknown
BF478825 Y D LEAF A B D Unknown
BF484100 N D LEAF A B D B Unknown
BM138439 Y D ROOT A B D
BE443527 N B/B LEAF/ROOT A B D B Unknown
BE404371 Y B ROOT B D NADH glutamate 
dehydrogenase
BE495400 N B ROOT A B D A Unknown
BE499478 Y B ROOT B + B FAT domain-containing 
protein/
phosphatidylinositol 3- and 
4-kinase family protein
BF202681 Y B ROOT A B Unknown
BE426364 Y A/A LEAF/ROOT A D A glyceraldehyde-3-
phosphate dehydrogenase
BE591763 Y A LEAF A B D Unknown
BF202265 Y A LEAF A + Unknown
BE500510 Y - - -
BE591372 Y - - -
BE638105 Y - - -
BM136908 Y - - -
The symbol '-' denotes that the homoeologous gene set is not afflicted by silencing. EST sequences blasted against NCBI Nucleotide collectionPage 2 of 4
(page number not for citation purposes)
BMC Genetics 2008, 9:65 http://www.biomedcentral.com/1471-2156/9/65Homoeologous gene silencing, as understood to date,
refers to events in genetically predictable and stable
organs/tissues [reviewed by [18]]. This form of silencing
has been successfully detected in cDNA populations gen-
erated from global RNA extracted from a range of complex
multi-cell type organ such as a leaf, root, pistil, and spikes
at the bolting stage [19,1,2,6]. In the disorganized callus,
chromosomal deletions, translocations, transposon
movement and epigenetic repatterning are enhanced [9-
12]. DNA methylation levels in in vitro callus cells are
proven to be highly variable [20] and in many instances
lower than that in organized tissues, even though this dif-
ference, at a global level, is relatively small [12,18,16].
Demethylation has been demonstrated to relieve homoe-
ologous silencing in polyploid Arabidopsis [8]. Therefore
if methylation is responsible for this form of silencing in
wheat, one plausible hypothesis is that the demethylation
induced in the callus cells occurred at a relatively early
stage of callus development, since otherwise the majority
of cells would still be affected by silencing, and thus the
detection of de-silencing would be compromised. In
tobacco (Nicotiana tabacum) callus, rDNA genes become
demethylated as early as 14 days after callus induction
and a low level of methylation is stably maintained
throughout prolonged culture [16].
Conclusion
The pattern of homoeologous silencing in distinct tissues
of wheat [6,1] or single cells of cotton [4] suggests the evo-
lution of sub-functionalization of the expression of each
homoeologue within a particular cell type or organ [4].
This form of silencing is typically highly specific, heritable
and in some instances is associated with a particular
developmental phase [2,1,3] suggesting a model in which
silencing is strictly controlled. Therefore we propose that
the absence of cellular control lifts the suppression of
homoeologous expression, and we further speculate that
the most likely mechanism of suppression is methylation.
Methods
Plant materials, RNA extraction and cDNA synthesis
The bread wheat cv. Florida was selected on the basis that
it readily generates callus tissue when cultured in vitro
(Perry, personal communication). Calli were generated in
replicate from four individual scutella obtained from
mature seed, as described by [21]. Calli were split after 21
days in culture, and harvested after 42 days growth. The
tissue was snap frozen in liquid N2, and total RNA was
extracted using Trizol™ reagent (Sigma), following the
manufacturer's instructions. Crude RNA preparations
were treated with DNAse (Amersham Bioscience) and
phenol/chloroform extracted [22]. The presence/absence
of contaminating genomic DNA was tested by amplifica-
tion with a number of well-characterized PCR primers,
and the quantity and quality of RNA were compared after
agarose gel electrophoresis with a quantitative RNA stand-
ard (Ambion). cDNA was synthesised with Superscript II™
(Invitrogen), using oligo dT as the polyA primer and fol-
lowing the manufacturer's protocol. Newly synthesised
cDNA was again tested by amplifying with intron-span-
ning PCR primers, since this allows a distinction between
RNA-derived DNA and carry-through gDNA.
EST selection, primer design, PCR amplification and SSCP 
analysis
Unigene ESTs mapping exclusively to a set of homoeolo-
gous genes located on one of wheat chromosome groups
1, 2, 3 or 7 were selected from among those examined by
[1] on the basis that they generated clearly defined SSCP
profiles and showed some silencing in either cv. Florida or
cv. Chinese Spring [3,1]. Primer sequences were as
described in [1]. The identity of the EST loci is listed in
Table 1. cDNA was diluted 1:20, and 1 μl of this dilution
was used as template for a 10 μl PCR, together with 5 μl
Hotstar Master Mix™, 3.5 μl water and 0.25 μl of each
primer (10 mM concentration). The amplification pro-
gramme consisted of a 15 min/95°C Taq polymerase heat
activation, which also served to denature the template,
followed by 35 cycles of 95°C/30 s, 59°C/5 s and 72°C/
60 s, and completed with a single extension step of 10 min
at 72°C. Amplicons were electrophoretically separated by
SSCP and visualised by silver staining, as described else-
where [1].
Pattern analysis
SSCP profiles generated from genomic DNA were com-
pared with those derived from the equivalent cDNA tem-
plate. Where all homoeologous copies identified in the
gDNA template were also present in the cDNA profile, this
was taken to imply full expression. Where, despite the
known presence of a gDNA locus, no matching cDNA was
detected, the relevant locus was scored as 'silenced'.
Authors' contributions
AB carried out the molecular genetic studies and NC
helped in the drafting of the manuscript. RK conceived of
the study, and participated in its design and coordination.
All authors have read and approved the final manuscript.
Acknowledgements
We acknowledge the financial support of a BBSRC studentship. We partic-
ularly thank the US NSF wheat EST project http://wheat.pw.usda.gov/NSF/ 
for their generous assistance with access to the wheat EST database. We 
would also like to thank Dr Matthew Perry for his assistance and expertise 
in plant tissue culture.
References
1. Bottley A, Xia GM, Koebner RMD: Homoeologous gene silencing
in hexaploid wheat.  Plant J 2006, 47:897-906.
2. Adams KL, Cronn R, Percifield R, Wendel JF: Genes duplicated by
polyploidy show unequal contributions to the transcriptomePage 3 of 4
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and organ-specific reciprocal silencing.  Proc Natl Acad Sci USA
2003, 100:4649-4654.
3. Bottley A, Koebner RMD: Variation for homoeologous gene
silencing in hexaploid wheat.  Plant J 2008, 56:297-302.
4. Hovav R, Udall JA, Chaudhary B, Rapp R, Flagel L, Wendel JF: Parti-
tioned expression of duplicated genes during development
and evolution of a single cell in a polyploid plant.  Proc Natl Acad
Sci USA 2008, 105:6191-6195.
5. Yang SS, Cheung F, Lee JJ, Ha M, Wei NE, Sing-Hoi Sze, Stelly DM,
Thaxton P, Triplett B, Town CD, Jeffrey Chen Z: Accumulation of
genome-specific transcripts, transcription factors and phyto-
hormonal regulators during early stages of fiber cell develop-
ment in allotetraploid cotton.  Plant J 2006, 47:761-775.
6. Mochida K, Yamazaki Y, Ogihara Y: Discrimination of homoeolo-
gous gene expression in hexaploid wheat by SNP analysis of
contigs grouped from a large number of expressed sequence
tags.  Mol Genet Genomics 2004, 270:371-377.
7. Matzke MA, Mittelsten Scheid O, Matzke AJ: Rapid structural and
epigenetic changes in polyploid and aneuploid genomes.
Bioessays 1999, 21:761-767.
8. Wang J, Tian L, Madlung A, Lee H, Chen M, Lee JJ, Watson B, Kagochi
T, Comai L, Chen ZJ: Stochastic and epigenetic changes of gene
expression in Arabidopsis polyploids.  Genetics 2004,
167:1961-1973.
9. Risseeuw E, Franke-van Dijk ME, Hooykaas PJ: Gene targeting and
instability of Agrobacterium T-DNA loci in the plant
genome.  Plant J 1997, 11:717-728.
10. Li HJ, Guo BH, Li YW, Du LQ, Jia X, Chu CC: Molecular cytoge-
netic analysis of intergeneric chromosomal translocations
between wheat (Triticum aestivum L.) and Dasypyrum villo-
sum arising from tissue culture.  Genome 2000, 43:756-762.
11. Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M: Retro-
transposons of rice involved in mutations induced by tissue
culture.  Genetics 1996, 93:7783-7788.
12. Kaeppler SM, Phillips RL: Tissue culture-induced DNA methyla-
tion variation in maize.  Proc Natl Acad Sci USA 1993,
90:8773-8776.
13. Xiaoling Li, Xiaoming Yu, Ningning Wang, Qizhi Feng, Zhenying Dong,
Lixia Liu, Jinglin Shen, Bao Liu: Genetic and epigenetic instabili-
ties induced by tissue culture in wild barley (Hordeum
brevisubulatum (Trin.).  Plant Cell, Tissue and Organ Culture 2007,
90:153-168.
14. Peredo EL, Revilla MA, Arroyo-García R: Assessment of genetic
and epigenetic variation in hop plants regenerated from
sequential subcultures of organogenic calli.  J Plant Physiol 2006,
163(10):1071-9.
15. Morrish FM, Vasil IK: DNA Methylation and Embryogenic
Competence in Leaves and Callus of Napiergrass (Pennise-
tum purpureum Schum.).  Plant Physiol 1989, 90(1):37-40.
16. Koukalova B, Fojtova M, Lim KY, Fulnecek J, Leitch AR, Kovarik A:
Dedifferentiation of Tobacco Cells Is Associated with Ribos-
omal RNA Gene Hypomethylation, Increased Transcription,
and Chromatin Alterations.  Plant Physiol 2005, 139:275-286.
17. Bednarek PT, Orłowska R, Koebner RM, Zimny J: Quantification of
the tissue-culture induced variation in barley (Hordeum vul-
gare L.).  BMC Plant Biol 2007, 7:10.
18. Adams KL: Evolution of duplicate gene expression in polyploid
and hybrid plants.  J Hered 2007, 98:136-141.
19. Kashkush K, Feldman M, Levy A: Gene loss, silencing and activa-
tion in a newly synthesized wheat allotetraploid.  Genetics
2002, 160:1651-1659.
20. Kaeppler SM, Kaeppler HF, Rhee Y: Epigenetic aspects of soma-
clonal variation in plants.  Plant mol biol 2000, 43:179-188.
21. Jones HD, Doherty A, Wu H: Review of methodologies and a
protocol for the Agrobacterium-mediated transformation of
wheat.  Plant Methods 2005, 5:5.
22. Sambrook J, Russell DW: Molecular cloning: A laboratory manual 3rd
edition. Cold Spring Harbor Laboratory; 2001. Page 4 of 4
(page number not for citation purposes)


A: Dedifferentiation of Tobacco Cells Is Associated with Ribosomal RNA Gene Hypomethylation, Increased Transcription, and Chromatin Alterations. Plant Physiol

Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton.

Arroyo-García R: Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.

CC: Molecular cytogenetic analysis of intergeneric chromosomal translocations between wheat (Triticum aestivum L.) and Dasypyrum villosum arising from tissue culture. Genome

DW: Molecular cloning: A laboratory manual 3rd edition.

Epigenetic aspects of somaclonal variation in plants. Plant mol biol

Evolution of duplicate gene expression in polyploid and hybrid plants.

Franke-van Dijk ME, Hooykaas PJ: Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome. Plant J

Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics

JF: Partitioned expression of duplicated genes during development and evolution of a single cell in a polyploid plant. Proc Natl Acad Sci USA

Koebner RMD: Homoeologous gene silencing in hexaploid wheat.

Koebner RMD: Variation for homoeologous gene silencing in hexaploid wheat.

Ogihara Y: Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags. Mol Genet Genomics

Phillips RL: Tissue culture-induced DNA methylation variation in maize.

Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.). BMC Plant Biol

Rapid structural and epigenetic changes in polyploid and aneuploid genomes. Bioessays

Retrotransposons of rice involved in mutations induced by tissue culture. Genetics

Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods

Xiaoming Yu, Ningning Wang, Qizhi Feng, Zhenying Dong, Lixia Liu, Jinglin Shen, Bao Liu: Genetic and epigenetic instabilities induced by tissue culture in wild barley (Hordeum brevisubulatum (Trin.). Plant Cell, Tissue and Organ Culture

ZJ: Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids. Genetics

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Homoeologous gene silencing in tissue cultured wheat callus

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