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Plant Molecular Biology 52: 401–414, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
401
Allopolyploidy alters gene expression in the highly stable hexaploid wheat
Ping He, Bernd R. Friebe, Bikram S. Gill and Jian-Min Zhou
∗
Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA (
∗
author for correspondence;
e-mail jzhou@ksu.edu)
Received 1 September 2002; accepted in revised form 13 December 2002
Key words: epigenetics, gene silencing, genome, polyploidy, wheat
Abstract
Hexaploid wheat (Triticum aestivum) contains triplicated genomes derived from three distinct species. To better
understand how different genomes are coordinated in the same nucleus of the hexaploid wheat, we globally
compared gene expression of a synthetic hexaploid wheat with its diploid (Aegilops tauschii) and tetraploid (T.
turgidum) parents by cDNA-AFLP display. The results suggested that the expression of a significant fraction
of genes was altered in the synthetic hexaploid; most appeared to be diminished and some were activated. We
characterized nine cDNA clones in details. Cytogenetic as well as genomic sequence analyses indicated that the
gene silencing was not due to chromosome/DNA loss but was caused by gene regulation. Northern and RT-PCR
divided these genes into three groups: (I) four genes were down-regulated nonspecifically, likely involving both
parental orthologues; (II) four genes were down-regulated in an orthologue-dependent manner; (III) one gene was
activated specifically in the synthetic hexaploid wheat. These genes were often altered non-randomly in different
synthetic hexaploids as well as natural hexaploid wheat, suggesting that many of the gene expression changes were
intrinsically associated with polyploidy.
Introduction
Polyploidization plays an important role in plant evo-
lution. According to Averett (1980), at least 70% of
angiosperm species have undergone a polyploidiza-
tion event in their evolutionary history. In addition
to well-established polyploids such as wheat, oat,
tobacco, potato, banana, sugarcane, cotton, and cer-
tain Brassica species, RFLP studies in maize, soy-
bean, and many classical diploid Brassica species
have detected genome-wide duplications, suggesting
that these plants were ancient polyploids (reviewed by
Wendel, 2000). Comparative genomic studies at the
nucleotide and amino acid sequence levels suggested
that the Arabidopsis and tomato genomes were dupli-
cated at least once (Ku et al. , 2000; Blanc et al., 2000).
Even Saccharomyces cerevisiae may be a segmental
tetraploid (Wolfe and Shields, 1997).
The nucleotide sequence data reported will appear in the
EMBL/GenBank Nucleotide Sequence Databases under the acces-
sion numbers 00000–00000.
After the initial genome doubling, the newly
formed polyploid undergoes a speciation process that
is driven by a combined force of chromosomal re-
patterning, gene deletion, mutation, suppression of
gene expression (silencing), or acquisition of new
gene function (for review see Wendel, 2000). Several
studies in Brassica (Song et al., 1995) and wheat (Liu
et al., 1998; Ozkan et al., 2001; Shaked et al., 2001)
suggested that genomic changes such as genomic
DNA elimination occur rapidly during the formation
of these allopolyploid species. Three recent reports
indicate that allopolyploidy in Arabidopsis (Comai
et al., 2000; Lee and Chen, 2001; Madlung et al.,
2002) and Triticeae (Kashkush et al. , 2002) is as-
sociated with gene silencing. These demonstrate that
substantial molecular changes can occur early during
polyploidization.
However, newly synthesized allotetraploid and
allohexaploid cotton did not exhibit rapid genomic
changes (Liu et al., 2001). Earlier studies on isozymes
in hexaploid wheat suggested that gene silencing was
402
rare in hexaploid wheat (Hart, 1979, 1996). These
discrepancies call for more investigations before ge-
nomic changes and gene silencing can be generalized.
In addition, mechanisms underlying these changes are
poorly understood. For example, McClintock (1984)
proposed that the merging of two distinct genomes in
the same nucleus during the formation of an allopoly-
ploid presents a major ‘genomic shock’ to which
plants respond with a variety of genomic restructuring.
Newly synthesized allopolyploids often are unstable,
exhibiting aberrant meiosis that leads to progeny with
a wide range genome restructuring including dele-
tions, translocations, inversions, and duplications of
chromosomal segments or entire chromosomes. It is
not known whether the genome instability triggers
the genomic sequence elimination and gene silencing
observed in new synthetic amphiploids. Indeed, the
materials used in these molecular studies are known
to display genome instability (Comai et al., 2000;
Kashkush et al., 2002).
One of the best-known allpolyploid complexes ex-
ists in the Triticeae. Among the roughly 30 species of
Aegilops and Triticum, 75% are natural allotetraploids
or allohexaploids (Sakamoto, 1973; Gill and Friebe, in
press). One of the most remarkable polyploids is the
young hexaploid Triticum aestivum, also called com-
mon wheat or bread wheat. T. aestivum has a genome
composition of AABBDD that arose by two rounds of
genome duplications. The first round of genome dou-
bling produced T. turgidum, a tetraploid wheat with
a genome composition of AABB in which T. urartu
(AA) donated the A genome (Dvorak, 1998; Gill and
Friebe, in press). The B genome donor is extinct, but
it was thought to be an ancestor of Aegilops spelto ides
(SS). T. turgidum can no longer be synthesized, be-
cause numerous differences exist between the B and S
genomes (Dvorak, 1998). Hybrids between T. urartu
and Ae. speltoides are highly unstable (B.R. Friebe and
B.S. Gill, unpublished results). Fossil evidence and
molecular studies demonstrate that the first T. aestivum
plant arose about 8000 years ago upon a hybridization
between T. turgidum and A. tauschii (DD) (Heun et al.,
1997; Huang et al., 2002 and the references cited
therein). Triticum turgidum and A. tauschii can be
readily crossed to produce synthetic T. aestivum that
is identical to the natural common wheat. The pairing
between homoeologous chromosomes is completely
suppressed in T. aestivum due to the presence of
two major pairing homoeologous genes Ph1 and Ph2
(McFadden and Sears, 1946), resulting in typical dis-
omic meiosis. This is distinct from other allopolyploid
plants that display genomic instability because of mis-
pairing of homoeologous chromosomes (Song et al.,
1995; Comai et al., 2000; Kashkush et al., 2002).
Thus, hexaploid wheat provides a unique system for
the investigation of the polyploidization process.
To determine the effect of allopolyploidy on gene
expression in the absence of genome instability, we
compared gene expression profiles in synthetic and
natural hexaploid wheat with parental A. tauschii and
T. turgidum lines. Our results indicate that a signifi-
cant fraction of parental genes was suppressed in the
hexaploid, while a smaller number of genes were ac-
tivated. Four of the five genes tested were suppressed
in both synthetic and natural hexaploid wheat, indica-
tive of changes intrinsically associated with hexaploid
wheat.
Materials and methods
Plant materials
The plant materials used for this study include two
independent synthetic hexaploids (in the 6th-7th gen-
erations), their parental lines and Chinese Spring (CS).
The first synthetic hexaploid (AABBDD, accession
number TA4152L3) was derived from the parental
lines Aegilops tauschii (genome DD, accession num-
ber TA1651) and the Mexican durum wheat cultivar
Altar 84 (Triticum turgidum, genome AABB, acces-
sion number TA2970). Both parents were maintained
by inbreeding and were expected to be homozy-
gous, as T. turgidum and A. tauschii are strictly self-
pollinating species. The second synthetic hexaploid
(accession number TA4152L26) was derived from a
cross between the parental lines A. tauschii (acces-
sion number TA2454) and the Mexican durum wheat
Aco89 (T. turgidum, accession number TA4185). The
synthetic hexaploids were produced by Mujeeb-Kazi
at CIMMYT and maintained in the Wheat Genetics
Resource Center (WGRC) at Kansas State University.
The A. tauschii lines had been self-pollinated for nu-
merous generations in WGRC prior to the crosses with
Mexican durum wheat. All seeds used in this study
were obtained from WGRC. Plants were grown in a
growth chamber at 20
◦
C.
Cytogenetic procedures
C-banding and chromosome identification were ac-
cording to Gill (1991). Meiotic metaphase I pair-
403
Figure 1. Cytogenetic comparisons of synthetic hexaploid with common wheat cultivar Opata. A. Comparison of C-banded mitotic metaphase
chromosomes of the synthetic hexaploid (shown on the right) with the corresponding chromosomes of Opata (shown on the left); note that
the overall chromosome morphology and C-banding patterns of the chromosomes of synthetic hexaploid wheat are very similar to the wheat
cultivar Opata. B. C-banded meiotic metaphase I chromosomes of the F
1
hybrid TA4152L3 × Opata; note that the A-, B-, and D-genome
chromosomes of the synthetic hexaploid show normal diploid-like bivalent pairing with the corresponding chromosomes of cv. Opata ruling
out any structural rearrangements during the formation of synthetic hexaploid.
ing was analyzed in C-banded pollen mother cells
(PMCs).
RNA extraction, cDNA synthesis and cDNA-AFLP
display
Total RNA was extracted from leaves of one-month
old seedlings. mRNA was isolated from 300 µgof
total RNA by using the polyATtract mRNA isolation
system (Promega, Madison, WI). First- and second-
strand cDNA was synthesized from 4 mg µRNA by
using a cDNA synthesis kit (Clontech, Palo Alto, CA).
The cDNA was then digested with MseIandApoI,
and subjected to AFLP analysis according to Bachem
et al. (1996) and Durrant et al. (2000). Sequences of
the adaptors, pre-amplification primers and selective
amplification primers used for AFLP reaction were ac-
cording to Durrant et al. (2000). cDNA-AFLP display
products were resolved on a 6% denaturing polyacry-
404
Figure 2. cDNA-AFLP display of gene expression in synthetic wheat. A. A typical cDNA-AFLP gel picture displaying cDNA from A. tauschii
(DD), T. turgidum (AABB), the synthetic hexaploid (AABBDD), and parental cDNA mixed at an 1:1 ratio. mRNA was isolated from three
independent sets of plant materials (I, II, and III). Arrow heads indicate differentially expressed bands. B. A close-up view of three differentially
expressed bands.
lamide gel, and radiolabeled DNA fragments were
visualized by autoradiography.
Differentially expressed bands were excised from
the gel by aligning with markings on the X-ray film
and eluted in 100 µlH
2
O for at least one hour.
The eluted DNA was PCR-amplified with AFLP pre-
amplification primers. The PCR products were cloned
with the TOPO TA cloning kit (Invitrogen, Carlsbad,
CA) and sequenced. The confirmed cDNA sequences
were compared with the GenBank database by using
BlastX and BlastN.
RNA and DNA gel blot analyses
Total RNA (10 µg) was separated on a denatur-
ing agarose gel, transferred onto a nylon membrane
(Hybond-N
+
; Amersham, UK). RNA gel blots were
performed in NorthernMax Prehyb/Hyb buffer (Am-
bion, Austin, TX) at 42
◦
C overnight. Southern blots
were hybridized to radiolabeled probes in 6× SSPE at
65
◦
C and washed to 0.3× SSC at 65
◦
C (Sambrook
et al. 1989). The probes for AFLP-1, -3, -5, -9, -10,
and -33 were PCR-amplified from plasmid clones by
using T7 and M13 reverse primers, and the PCR prod-
ucts were radiolabeled with the DecAprime random
priming DNA labeling kit (Ambion).
Reverse transcriptase (RT)-PCR and genomic PCR
analyses
Total RNA (5 µg) was treated with RQ1 RNase-
Free DNase (Promega, Madison, WI), and cDNA
was synthesized by using AMV reverse transcrip-
tase and RNaseH in a final volume of 25 µl. One
µl of cDNA or 100 ng genomic DNA template was
PCR-amplified with 35 cycles of 94
◦
C for 30 s,
60
◦
C for 30 s, and 72
◦
C for 30 s. Sequences
of specific primers for AFLP-3, 9, 10, 11, 23,
33, 36 and the actin cDNA are as follows: AFLP-
3, forward 5
-CATGGGTATTAGGAGTTG-3
and re-
verse 5
-ATGTGTCTGAATATCTCT-3
; AFLP-9, for-
ward 5
-GCAATGGTAGGACAGATC-3
and reverse
5
-CATCATTTATCCAGAAAC-3
; AFLP-10, for-
ward 5
-TATAGTAGCCAAATACAC-3
and reverse
5
-TCATCGTGACTAATAATG-3
; AFLP-11, for-
ward 5
- CAAAATCCGACCACAGCA-3
and reverse
5
-CACTGATTTGTTTAGTTC-3
; AFLP-23, for-
ward 5
-TAGTGAAGAGCGGCCATG-3
and reverse
5
-CTCACGGCACCTTCTGAA-3
; AFLP-33, for-
ward 5
-TGAAGAGAGAGGTACTTG-3
and reverse
5
-CTGGATCTGCTGCTTGAC-3
; AFLP-36, for-
ward 5
-CTTTTACCTGGTAACTGG-3
and reverse
5
-ATCTCAAGTACCAGCATG-3
; actin, forward
5
-GGCACACTGGTGTCATGG-3
and reverse 5
-
405
CTCCATGTCATCCCAGTT-3
. Each forward primer,
except for AFLP-33, was end-labeled with [γ -
33
P]ATP by T4 kinase (New England Biolabs, Bev-
erly, MA). After amplification, PCR products were
resolved in a 1% agarose gel or a 6% polyacrylamide
sequencing gel.
Competitive RT-PCR was carried out according to
Imaizumi et al. (2000). The firefly luciferase gene
(Promega) was amplified as a DNA competitor with
long primers containing both the LUC and AFLP-
23 cDNA sequences. A common forward primer, 5
-
TAGTGAAGAGCGGCCATGCTGGAGAGCAACT-
GCAT-3
, and two specific reverse primers, 5
-
GTGGACGATTCTCTGCCAGAGTTCATGATCAG-
TGC-3
(AFLP-23D) and 5
-GTGGATGATTCTCT-
GCCGGAGTTCATGATCAGTGC-3
(AFLP-23A),
were used to amplify competitors for AFLP-23D
and AFLP-23A, respectively. The competitors were
added at concentrations from 10
−4
to 10
2
pg to
RT reactions prior to PCR amplification with 35
cycles of 94
◦
C for 30 s, 62
◦
C for 30 s,
and 72
◦
C for 30 s. AFLP-23D and AFLP-23A
were amplified with a common forward primer, 5
-
TAGTGAAGAGCGGCCATG-3
, and two specific
reverse primers, 5
-GTGGACGATTCTCTGCCA-3
and 5
-GTGGATGATTCTCTGCCG-3
, respectively.
PCR products were separated in a 1.5% agarose gel.
Results
Characterization of synthetic hexaploid wheat
Most of the experiments described in this re-
port, unless indicated otherwise, used the synthetic
hexaploid (accession number TA4152L3), its parental
lines A. tauschii (accession number TA1651) and
T. turgidum Altar 84 (accession number TA2970). This
synthetic line has well documented pedigree and has
been used widely for the generation of an extensive
genetic linkage map of common wheat (Nelson et al.,
1995). The formation of hexaploid wheat through the
hybridization of T. turgidum and A. tauschii was first
established by McFadden and Sears (1944, 1946) and
is now a routine practice that produces fully fertile
plants. To demonstrate the presence of 21 chromo-
somes from the A, B, D genomes in the synthetic
hexaploid wheat (TA4152L3) and determine their sim-
ilarities to those in known common wheat (cv. Opata),
we performed C-banding analysis. This technique pro-
duces a diagnostic pattern of dark and light bands
along the chromosomes that is chromosome-specific
and permits identification of all 21 chromosome pairs
of wheat (Gill, 1991). The C-banding pattern of the
synthetic hexaploid chromosomes was highly similar
to the corresponding chromosomes of Opata (Fig-
ure 1A) and other wheat cultivars (Friebe and Gill,
1994). Only 16 polymorphic differences in C-banding
patterns were observed between the A-, B-, and D-
genome chromosomes of the synthetic hexaploid and
Opata. Similar polymorphic differences in C-banding
patterns were commonly observed between differ-
ent wheat cultivars (Friebe and Gill, 1994). The C-
banding patterns of the D-genome chromosomes of
the synthetic hexaploid are similar to the correspond-
ing chromosomes of the D genome donor species
Aegilops tauschii (Friebe et al., 1992). The presence of
a prominent telomeric C-band in the short arm of chro-
mosome 5D in the synthetic hexaploid indicates the
presence of a large number of 18–26S rDNA repeats in
this region which is characteristic of A. tauschii.How-
ever, the copy number of these repeats is drastically
reduced in T. aestivum cultivars (Mukai et al., 1991).
Some of the chromosomes in the haploid comple-
ment appeared to be larger than their homoeologous
chromosomes in the natural hexaploid, although the
significance is unknown.
The presence of Ph1 and Ph2 genes enables true
disomic chromosomal pairing in all T. aestivum lines,
including synthetic hexaploid (McFadden and Sears,
1946). To further verify the integrity of the chromo-
some complement of the synthetic hexaploid, meiotic
metaphase I pairing was analyzed in the F
1
testcross of
TA4152L3 × Opata. The chromosomes showed nor-
mal diploid-like bivalent pairing in 47 of the 55 pollen
mother cells (PMCs) analyzed (21
II
, 0.85; Figure 1B).
In four PMCs the chromosomes paired as 20 bivalents
plus 2 univalents (20
II
+ 2
I
, 0.07; data not shown)
and in six PMCs the chromosomes paired as 19 bi-
valents plus one quadrivalent (19
II
+ 1
IV
, 0.11; data
not shown). The C-banding pattern of the occasional
quadrivalent suggested that Opata and the synthetic
hexaploid differ by one reciprocal translocation proba-
bly involving a pair of A-genome chromosomes. This
is not unexpected, because translocation differences
are common between different hexaploid wheat cul-
tivars (Friebe and Gill, 1994). Thus, the cytogenetic
data revealed no major chromosomal rearrangements
between the chromosome complement of the synthetic
hexaploid and common wheat cultivars. Most impor-
tantly, no mispairing between homoeologous chromo-
somes was observed, demonstrating a disomic meiotic
406
behavior of synthetic hexaploid chromosomes. Taken
together, we conclude that the synthetic hexaploid
behaved like a true diploid and its chromosome com-
plement was intact.
The synthetic lines were completely stable, and
their morphologies were highly similar to common
wheat except for spike traits such as fragile rachis and
tough glumes (tg) that were inherited from A. tauschii.
Mutations at these loci occurred during domestica-
tion leading to tough spike and free threshing spike
of modern bread wheat. The synthetic hexaploid
plants were fully fertile and exhibited no lethality.
The synthetic hexaploid seedlings were more vigorous
and larger than both parents. Unlike the A. tauschii
parental lines that developed trichomes on leaves, the
two T. turgidum parents and the synthetic hexaploids
showed no trichome development (data not shown).
Spikes of the synthetic hexaploid were more similar to
T. turgidum than the A. tauschii parent although they
show longer rachis internodes. The durum parent was
glaucous, whereas the A. tauschii parent and synthetic
hexaploid were completely non-glaucous. Chromo-
some 2D is known to carry W 2
I
, a homoeoallelic sup-
pressor of the glaucousness gene W 1 on 2B (McIntosh
et al. 1998). The synthetic hexaploid also resembled
the T. turgidum parent in its spring-type flowering be-
havior, presumably because of the dominant Vrn genes
in the durum wheat.
Suppressed gene expression in synthetic hexaploid
wheat
To globally compare gene expression between
hexaploid wheat and its D-genome and AB-genome
donors, we used cDNA-AFLP display (Bachem et al.,
1996) to examine gene expression profiles in the
synthetic hexaploid (TA4152L3) and the parental
lines A. tauschii (TA1651) and T. turgidum Altar 84
(TA2970) at the seedling stage. To ensure that the
observed differential gene expression between syn-
thetic hexaploid and parental lines was not caused by
sampling variations, RNA samples from three sets of
seedlings that were grown independently were used
for cDNA-AFLP display. Figure 2 shows autoradi-
ograms of typical cDNA-AFLP gels. Each cDNA frag-
ment (band) represents a unique mRNA species. The
expression pattern was highly reproducible among
three replicates. Only bands detected by all three
replicates were scored. On average, each primer com-
bination displayed ca. 50–70 cDNA fragments ranging
from 50 bp to 400 bp in either parent. These frag-
Figure 3. Northern analysis of group I gene expression. Total RNA
(10 µg/lane) from A. tauschii (DD), durum wheat (AABB) and the
synthetic hexaploid (AABBDD) was fractionated on a denaturing
agarose gel, transferred onto nylon membranes, and replicated fil-
ters were hybridized with the indicated probes. Ethidium bromide
staining of rRNA indicates equal loading of RNA.
ments were highly polymorphic between the tetraploid
(AABB) and diploid (DD) parents. Parental bands
were completely additive in the mixed cDNA con-
trol, indicating that the presence of parental cDNA
species did not result in a competition in PCR reac-
tion (Figure 2A). About 64% of the fragments detected
in the D parent were absent in the AB parent, and
vice versa. By using 47 primer combinations, 2800
bands were scored, among which 1050 were unique
to the D-genome parent, 1150 were unique to the
AB-genome parent, and 600 were shared fragments
(Table 1). Only polymorphic cDNA fragments were
used for hexaploid-parent comparison. Monomorphic
cDNA fragments were not informative and were thus
ignored. For most polymorphic cDNA species, the
synthetic hexaploid and parental plants showed similar
expression. However, 168 of the 2200 polymorphic
fragments (7.7%) displayed altered expression in the
hexaploid line. One hundred twenty two D-genome
mRNA species (11.6% of the polymorphic D frag-
ments) and 38 AB-genome mRNA species (3.3% of
the polymorphic AB fragments) showed greatly re-
duced or no expression in the synthetic hexaploid.
Only eight mRNA species (0.4%) were more abundant
in the synthetic hexaploid compared with the parental
plants. These observations were consistent in all three
replicates.
407
Table 1. cDNA-AFLP display summary.
Origin
DD AABB total
Bands scored 1650 1750 2800
Bands polymorphic between two parents 1050 1150 2200
Bands reduced or missing in hexaploid 122 (11.6%)
a
38 (3.3%) 160 (7.3%)
Bands induced in hexaploid 8 (0.4%)
a
Percentage of reduced/induced bands was calculated based on polymorphic bands.
Characterization of differentially regulated genes
AFLP fragments were excised from the gel and cloned
into the TOPO TA cloning vector. Nine cDNA clones
were selected for further verification. cDNA-AFLP
suggested that eight of these genes were suppressed
and one was induced in the synthetic hexaploid. North-
ern analysis indicated that AFLP-3 and AFLP-33 were
highly expressed in the diploid parent A. tauschii
but reduced or completely silenced in the hexaploid
(Figure 3). AFLP-1 and AFLP-5 showed reduced
expression in the hexaploid. Although these differ-
ences are quantitative, they were verified in repeated
Northern analyses. Interestingly, very little orthol-
ogous transcripts were detected in the T. turgidum
parent, suggesting a suppressed expression of the or-
thologous genes. Alternatively, the probes might have
failed to cross-hybridize with orthologous genes, al-
though this is unlikely (see discussion). These genes
(AFLP-1, AFLP-3, AFLP-5, and AFLP-33) are desig-
nated group I genes. No cDNA clones isolated from T.
turgidum belonged to this group.
The other four putative down-regulated genes were
either not detected or showed similar transcript levels
in the hexaploid and parental plants in Northern analy-
sis. Figure 4A shows the Northern analysis of two
of these genes. Because Northern analysis does not
discriminate transcripts from different orthologues,
we could not conclude whether the genes examined
were not suppressed or they were suppressed in an
orthologue-specific manner in the synthetic hexaploid
plants. To determine if any of these genes were re-
duced in expression in an orthologue-specific man-
ner, we designed PCR primers according to the ends
of the cloned cDNA fragments and conducted RT-
PCR analysis. In the absence of reverse transcriptase,
no PCR products were detected, ruling out genomic
DNA contamination. RT-PCR products for three of
the four genes, AFLP-9, 11, 36 showed a size poly-
morphism between T. turgidum and A. tauschii ortho-
logues (Figure 4B). The expression of the respective
T. turgidum genes was dramatically reduced in the syn-
thetic hexaploid, while the expression of A. tauschii
orthologues was not affected in the hexaploid for
the three genes. Initial RT-PCR failed to produce
a cDNA length polymorphism for AFLP-23 (Fig-
ure 4B), and the parental and synthetic hexaploid lines
produced a similar level of total transcripts. We iso-
lated and sequenced a corresponding cDNA fragment
from T. turgidum plants homologous to AFLP-23. A
single T. turgidum cDNA species was detected, and it
contained seven single nucleotide substitutions com-
pared with AFLP-23 that is of A. tauschii origin. The
T. turgidum gene was carried by the A genome (see
Figure 5B) and is thus called AFLP-23A, whereas
the D genome copy is designated AFLP-23D. Re-
spective gene-specific primers were designed for the
AFLP-23A and AFLP-23D transcripts. Competitive
RT-PCR (Imaizumi et al., 2000) showed that AFLP-
23D gene expression was reduced ca. 10-fold in the
synthetic hexaploid plants compared to the A. tauschii
parent, whereas the AFLP-23A gene was not affected
in the hexaploid wheat (Figure 4C). Thus, the four
genes (AFLP-9, -11, -23, and -36) had undergone
selective suppression, in which only one of the or-
thologous genes showed reduced expression in the
hexaploid compared with its expression in parental
plants. An alternative interpretation is that competition
in PCR might have caused differential amplification
of orthologous transcripts for genes corresponding to
AFLP-9, -11, and -36. However, we view this as
unlikely for the following reasons. First, at least for
AFLP-36, both parental orthologues were amplified
equally when genomic DNA was used as template
(Figure 5). Secondly, a primer bias toward the con-
stitutively expressed orthologues is unlikely, because
primers were designed for perfect base-pairing with
the down-regulated transcripts. A remote possibility
may be that unforeseen factors could have impeded
the amplification of the AFLP-9 and AFLP-11 cDNAs
408
Figure 4. Northern and RT-PCR analyses of group II gene expression. A. Northern analysis of AFLP-9 and AFLP-23 in synthetic hexaploid
(AABBDD) and parental lines (AABB and DD). RNA blots were hybridized with cloned cDNA fragments as indicated. B. RT-PCR analysis of
group II gene expression. Radiolabeled RT-PCR products were fractionated through a sequencing gel, and the gel was exposed to X-ray film.
Actin cDNA was amplified as a control for a constitutively expressed gene. Arrows indicate differentially expressed orthologues. For control,
PCR reactions without reverse transcriptase were performed to rule out possible genomic DNA contamination (w/o RT). C. Competitive RT-PCR
analysis of AFLP-23 expression. Top panel, amplification with the primers and competitor designed for AFLP-23D; lower panel, amplification
with the primers and competitor designed for AFLP-23A. Triangles indicate minimal concentrations of competitors that inhibit the amplification
of target transcripts. The numbers indicate the amount of competitor DNA used in the assay. PCR without reverse transcriptase did not produce
any products (not shown). Lane M contains DNA markers.
in the RT-PCR experiments. Given consistent results
from both cDNA-AFLP and RT-PCR experiments, we
conclude that overall these four genes were selectively
down-regulated in the synthetic hexaploid wheat. We
designate these as group II genes. They include one
A. tauschii and three T. turgidum genes.
We next examined whether the orthologous tran-
script species detected for group II genes were en-
coded by homoeologous chromosomes. PCR primers
for AFLP-9 and -11 failed to amplify a product from
any genomic DNA templates, perhaps because of the
presence of introns in the templates. Amplification
of genomic DNA from the synthetic hexaploid, its
parental plants, and Chinese Spring with AFLP-36
primers produced PCR products identical to corre-
sponding RT-PCR products (Figure 5). The AFLP-36
409
Figure 5. Mapping of group II gene orthologues by using nulli-tetras lines. A. mapping of AFLP-36 orthologues. B. mapping of AFLP-23
orthologues. Genomic DNA from nulli-tetras lines (N1A represents nullisomic for chromosome 1A, etc.) was PCR-amplified with appropriate
primers. A. tauschii (D), T. tur gidum (AB) and the wild-type Chinese Spring (ABD) were used as controls. Triangles indicate nulli-tetras lines
lacking one of the orthologous bands.
orthologues were placed on chromosomes by using
Chinese Spring nullisomic-tetrasomics (nulli-tetras;
Sears, 1966) lines (Figure 5). Of the two PCR products
detected, the A. tauschii (DD) origin fragment (the
upper band) was missing in nullisomic 1D, whereas
T. turgidum origin fragment (the lower band) was
missing in nullisomic 1A. Thus, the orthologous genes
detected by PCR were AFLP-36A and AFLP-36D that
were mapped to homoeologous chromosomes 1A and
1D, respectively. A similar method was used to map
AFLP-23. The two orthologues for AFLP-23, AFLP-
23A and AFLP-23D were mapped to chromosomes
7A and 7D, respectively. AFLP-36A and AFLP-23D
were selectively suppressed in the synthetic hexaploid
compared with their respective donor plants. Interest-
ingly, the AFLP-23B and AFLP-36B homoeoalleles
were not amplified with the primers used, indicating
that either the primers were too specific for the amplifi-
cation of B homoeoalleles or that the B homoeoalleles
had been deleted. Nucleotide sequences of AFLP-
23A and AFLP-23D, and AFLP-36A and AFLP-36D
were 99% and 97% identical, respectively, with a few
base pairs of insertions/deletions or single base pair
substitutions.
Genes specifically activated in the synthetic
hexaploid line were designated group III genes.
cDNA-AFLP results suggested that these genes were
rare. AFLP-10 was the only gene of this group to
be examined in detail. Northern analysis using the
cloned AFLP-10 fragment as a probe indicated that
transcripts accumulated at a very low level in the
two parental lines but were abundant in the synthetic
hexaploid plants (Figure 6A). PCR primers were de-
signed from the AFLP-10 cDNA ends for RT-PCR and
genomic PCR analyses. In addition to the activated
allele in the hexaploid, RT-PCR revealed an orthol-
ogous transcript species that accumulated equally in
A. tauschii and the synthetic hexaploid. Comparison
of genomic PCR products with RT-PCR indicated that
the strong expression of AFLP-10 in the hexaploid
plants was a result of specific activation of a previously
silenced T. turgidum gene (Figure 6B). The weak hy-
bridization signal in T. turgidum detected by Northern
analysis may be caused by the expression of a ho-
mologous gene. In fact, Southern analysis suggested
that AFLP-10 belongs to a small gene family (data not
shown).
Thus, of all the nine genes for which the expres-
sion could be detected by either Northern analysis
or RT-PCR, the altered gene expression in synthetic
hexaploid wheat was confirmed. The results validate
our conclusion from the cDNA-AFLP display experi-
ment. Table 2 summarizes the expression analysis and
sequence homologies of the nine genes with known
proteins or DNA sequences in the database. Seven
cDNA clones shared significant homology with known
sequences with a wide range of functions. The homol-
ogy of AFLP-33 to histone H2A promoter sequence
is not understood, but this sequence is apparently
transcribed.
Alteration of gene expression is nonrandom and
occurs in the natural hexaploid wheat
We next tested if any of the gene silencing or activa-
tion described above was genotype-specific. Besides
the synthetic and parental lines used for cDNA-AFLP
410
Table 2. Summary of three groups of differentially expressed genes.
Clone Expression pattern Orthologue expression Putative function
a
E-value
Group I
AFLP-1 DD>AABBDD Low in 4× and 6× D83391 Zea mays
uroporphyrinogen III
methylase 8
e–50
AFLP-3 DD>AABBDD Low in 4× and 6× Unknown
AFLP-5 DD>AABBDD Low in 4× and 6× AY054525 A. thaliana
ABC transporter 7
e–84
AFLP-33 DD>AABBDD Low in 4× and 6× X94693 T. aestivum
histone H2A promoter 7
e–38
Group II
AFLP-9 AABB>AABBDD Not silenced AY063063 A. thaliana
Phe-tRNA synthetase 1
e–57
AFLP-11 AABB>AABBDD Not silenced X75089 T. aestivum
petF gene for ferredoxin 9
e–29
AFLP-23 DD>AABBDD Not silenced AF323103 M. truncatula
protein phosphatase 6
e–23
AFLP-36 AABB>AABBDD Not silenced Unknown
Group III
AFLP-10 AABBDD-specific Not affected U32429 T. aestivum
sulfur-rich/thionin-like
protein 4
e–30
a
Putative gene function as suggested by BLASTX searches.
Figure 6. Activation of a group III gene in synthetic hexaploid
wheat. A. Northern analysis of transcripts in synthetic hexaploid
(AABBDD), A. tauschii (DD) and T. turgidum (AABB). Ethid-
ium bromide staining of rRNA indicates equal loading of RNA.
B. RT-PCR (left) and genomic DNA PCR (center) of the AFLP-10
gene. PCR reactions with RNA but no reverse transcriptase were
performed to rule out possible genomic DNA contamination in
RT-PCR (right).
display, we compared a second synthetic hexaploid
line (AABBDD; accession number TA4152 L26) and
its parental lines A. tauschii (DD; accession number
TA2454) and Mexican durum wheat cultivar Aco89
(AABB; accession number TA4185). In addition, we
also examined gene expression in Chinese Spring
(CS), a natural hexaploid wheat. Figure 7A indi-
cates that the two A. tauschii accessions showed
a high level of transcripts corresponding to AFLP-
3 and AFLP-33, while the two durum wheat and
synthetic hexaploid produced little detectable tran-
scripts. Thus, the genes corresponding to AFLP-3
and AFLP-33 were silenced in the hexaploid wheat
in a genotype-independent manner. Similarly, RT-PCR
showed that the T. turgidum transcripts correspond-
ing to AFLP-9 and AFLP-36 were reduced in both
synthetic hexaploid lines compared with the tetraploid
parents (Figure 7B). These genes were also down-
regulated in Chinese Spring (Figure 7). Assuming that
the expression of 10% of the genes is randomly altered
in a hexaploid wheat, observing such a change for
any given gene in three independent hexaploid wheat
genotypes will have a probability of 0.001. Observing
411
such a change in the same four of nine genes exam-
ined in all three wheat lines will have a probability
of 2.6 × 10
−11
. The probably will be even lower if
the frequency of gene expression alteration is less than
10%. Therefore, we conclude that a significant portion
of the genes are down-regulated non-randomly in syn-
thetic as well as natural hexaploid wheat. In contrast,
the activation of AFLP-10 in the hexaploid wheat was
specific to TA4152 L3 (Altar84 × TA1641), but
Gene deletions are not the cause of decreased gene
expression
Most of the down-regulated not in TA4152L26 (Aco89
× TA2454) or Chinese Spring (Figure 7A). genes de-
scribed in this report showed a reduction rather than
a complete loss of transcripts, suggesting that re-
duced gene expression level instead of gene deletion
was the cause of silencing. Genomic PCR and South-
ern analyses were conducted to further test whether
the synthetic hexaploid carried deletions or chromo-
some rearrangements at the differentially expressed
loci. Genomic DNA from the synthetic hexaploid
(AABBDD; accession number TA4152L3), the durum
(AABB; accession number TA2970), and A. tauschii
(DD; accession number TA1651) parents was PCR-
amplified with the primers derived from AFLP-3 and
AFLP-33 (Figure 8). The parental (A. tauschii)ge-
nomic fragments for both genes were present in the
synthetic hexaploid. We did not detect a genomic se-
quence from the durum parent with the PCR primers
used, which maybe caused by the primer specificity
or gene deletion in the durum plant. Similarly, South-
ern analyses of AFLP-5 and AFLP-9 showed that all
parental genomic fragments were accounted for in the
synthetic hexaploid, and no new DNA fragments were
detected. Thus, at least with the four genes examined,
we did not find any evidence of genomic sequence
changes.
Discussion
Early studies of isozymes in hexaploid wheat indi-
cated that most enzymes examined showed similar
expression from three orthologues, and gene silencing
was rare (Hart, 1979, 1996). However, the expression
of high-molecular-weight (HMW) glutenin genes en-
coded by the A-genome, but not B- or D-genomes,
is frequently suppressed in tetraploid and hexaploid
wheat (Galili and Feldman, 1983, 1984; Feldman et al.
1986). A caveat with these results is that the results
may be biased toward the enzymes or storage proteins
chosen in the studies. Our cDNA-AFLP display com-
bined with Northern and RT-PCR analyses provide a
better assessment of gene expression in the hexaploid
wheat. Up to 7.7% of the parental cDNA-AFLP frag-
ments were altered in the synthetic line, suggesting
that a significant fraction of the genome is altered.
Northern and RT-PCR results with the selected genes
were completely consistent with cDNA-AFLP results,
indicating that the cDNA-AFLP results were highly
reliable. It is interesting that A. tauschii genes were
affected much more frequently than T. turgidum genes,
although the latter carried twice as many genes. This
correlated with the gross morphology of hexaploid
wheat that is more similar to Triticum than Aegilops.
A significant fraction of these genes was also down-
regulated in Chinese Spring. Thus, we conclude that
the alteration of gene expression is frequent in both
synthetic and natural hexaploid wheat.
Several possibilities can be proposed for the altered
gene expression in the hexaploid wheat. Allopoly-
ploidy may impact gene expression by: increased
genome size and gene number; interaction of different
genomes in the same nucleus; interaction of the nu-
clear genome with a new cytoplasmic genome (Gill,
1991).
Increased ploidy is known to cause both gene si-
lencing and gene activation in yeast (Galitski et al.,
1999) and gene silencing in Arabidopsis (Mittelsten-
Scheid et al., 1996; Lee and Chen, 2001; Madlung
et al., 2002) and maize (Guo et al., 1996). Group I
gene silencing appears to be associated with tetraploid
and hexaploid plants. All group I genes are expressed
in A. tauschii, but few orthologous transcripts were
detected in T. turgidum and T. aestivum (Figure 3).
This suggests that the orthologous genes might have
been silenced in T. turgidum in the previous round of
polyploidization, presumably because of the increased
ploidy. It is unlikely that the probes might have failed
to cross-hybridize with orthologous genes. In fact, the
AFLP-5 and AFLP-33 probes detected homologous
sequences in T. turgidum in Southern blot analysis
(Figure 8; P. He and J.-M. Zhou, unpublished results).
A previous round of gene silencing in tetraploid wheat
might explain the relatively low frequency of AB-
origin genes being affected in hexaploid wheat (Ta-
ble 1). Alternatively, the preferential down-regulation
of D genome genes may reflect the incompatibility be-
tween the A. tauschii nuclear genome and T. turgidum
cytoplasm. It is unlikely, however, that T. turgidum
412
Figure 7. Gene expression changes are nonrandom and occur in a natural hexaploid. A. Northern analysis of AFLP-3, AFLP-33, and AFLP-10
in the two synthetic hexaploid lines, their respective parental lines, and Chinese Spring. B. RT-PCR analysis of AFLP-9 and AFLP-36 in the two
synthetic hexaploid lines, their respective parental lines, and Chinese Spring. Actin cDNA was amplified as constitutive control. PCR reactions
without reverse transcriptase did not produce any products (not shown).
Figure 8. Lack of genomic changes at the differentially expressed loci. A. Genomic PCR of AFLP-3 and AFLP-33. PCR products were resolved
on an agarose gel (AFLP-33) or a sequencing gel (AFLP-3). B. Southern analysis of AFLP-5 and AFLP-9. Genomic DNA from the synthetic
hexaploid (AABBDD) and parental lines (AABB and DD) was digested with EcoRI before fractionation on an agarose gel. Southern blots were
hybridized with the indicated probes.
plants carried deletions at the orthologous loci, be-
cause homologous sequences of AFLP-5 and AFLP-
33 were detected in both A. tauschii and T. turgidum
(Figure 8; P. He and J.-M. Zhou, unpublished results).
In contrast to group I, group II gene expression
was affected for only one of the parental genes. Three
of the four group II genes were of the T. turgidum
origin. Therefore, it is unlikely that their down-
regulation is caused by a nuclear-cytoplasmic genome
interaction. These genes were down-regulated in an
orthologue-specific manner, and this can not be ex-
plained by the increased ploidy level. Instead, group
II genes may be regulated by interactions between
different subgenomes in the same nucleus. Loss and
activation of gene expression have been reported in
the newly synthesized allotetraploid Triticeae derived
from a cross between Aegilops sharonensis (S
sh
S
sh
)
and Triticum monococcum ssp. aegilopoides (A
m
A
m
).
413
Some of these alterations are accounted for by gene
loss (Kashkush et al., 2002). In contrast, gene loss did
not appear to be the cause of altered gene expression
in the synthetic hexaploid wheat. First, most of the
cDNA-AFLP fragments exhibited reduced, instead of
a lack of expression in the hexaploid plants. Second,
Southern blot and PCR analyses of genomic DNA in-
dicated that at least the five genes examined (AFLP-3,
-5, -9, -10, and -33) did not show detectable genomic
changes. It remains to be determined if rapid genomic
restructuring occur in the hexaploid wheat.
Acknowledgements
We thank Drs Scot Hulbert, Randall Warren, and
Xiaoyan Tang for critical reading of the manuscript.
We are also grateful to Dr Lili Qi for providing Chi-
nese Spring nullitetras genomic DNA. This work was
supported in part by Kansas State University Plant
Biotechnology Center and the Kansas Agricultural
Experimental Station (Contribution 02-359-J).
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