Research

Meiotic chromosome stability of a newly formed allohexaploid

wheat is facilitated by selection under abiotic stress as a spandrel
Yao Bian1, Chunwu Yang1, Xiufang Ou1, Zhibin Zhang1, Bin Wang1, Weiwei Ma1, Lei Gong1, Huakun Zhang1,2
and Bao Liu1
1
Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun 130024, China; 2Department of Cell and Developmental Biology,
John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK

Summary
Authors for correspondence:
Polyploidy is a prominent route to speciation in plants; however, this entails resolving the
Huakun Zhang challenges of meiotic instability facing abrupt doubling of chromosome complement. This
Tel: +44 0740 5527566
issue remains poorly understood.
Email: Huakun.Zhang@jic.ac.uk

We subjected progenies of a synthetic hexaploid wheat, analogous to natural common
Bao Liu wheat, but exhibiting extensive meiotic chromosome instability, to heat or salt stress. We
Tel: +86 043185099367 selected stress-tolerant cohorts and generated their progenies under normal condition. We
Email: baoliu@nenu.edu.cn conducted fluorescent in situ hybridization/genomic in situ hybridization-based meiotic/mi-
Received: 23 March 2018 totic analysis, RNA-Seq and virus-induced gene silencing (VIGS)-mediated assay of meiosis
Accepted: 11 May 2018 candidate genes.

We show that heritability of stress tolerance concurred with increased euploidy frequency

New Phytologist (2018) 220: 262–277

doi: 10.1111/nph.15267
Key words: abiotic stress, gene expression,
meiosis, polyploidy, spandrel, speciation,
Triticum aestivum, virus-induced gene
silencing (VIGS).
due to enhanced meiosis stability. We identified a set of candidate meiosis genes with altered
expression in the stress-tolerant plants vs control, but the expression was similar to that of
common wheat (cv Chinese Spring, CS). We demonstrate VIGS-mediated downregulation of
individual candidate meiosis genes in CS is sufficient to confer an unstable meiosis phenotype
mimicking the synthetic wheat.

Our results suggest that heritable regulatory changes of preexisting meiosis genes may be
hitchhiked as a spandrel of stress tolerance, which significantly improves meiosis stability in
the synthetic wheat. Our findings implicate a plausible scenario that the meiosis machinery in
hexaploid wheat may have already started to evolve at its onset stage.

Hollister, 2015; Bomblies et al., 2016). These meiotic instabili-

Introduction
Polyploidy, or whole genome duplication (WGD), represents a
driving force in the evolution of all multicellular eukaryotes, and
is particularly prevailing in higher plants (Adams & Wendel,
2005; Doyle et al., 2008; Leitch & Leitch, 2008; Soltis et al.,
2009; Jiao et al., 2011; Madlung, 2013). Yet, there are critical
challenges associated with speciation via polyploidy (Ramsey &
Schemske, 1998; Comai, 2005; Otto, 2007), with meiosis insta-
bility standing out as the most critical problem that must be
resolved to ensure a successful speciation. This applies to both
autopolyploidy (i.e. WGD of a single species genome) or
allopolyploidy (i.e. WGD of two or more genomes brought into
a common nucleus/cytoplasm by interspecific hybridization). As
a highly conserved and intricate process, the meiosis machinery
has already evolved and been fine-tuned in the diploid parental
species (Mercier et al., 2015). Conceivably, it will be maladapted
to the abruptly doubled chromosome complements in a
polyploid genome, and may incur compromised pairing fidelity,
nonhomologous chromosome recombination, and imprecise
chromosome segregation (Cifuentes & Benavente, 2009;
ties will generate genetically unbalanced gametes, leading to
lower fertility and reduced fitness of the newly formed polyploids
relative to their diploid parent(s). This, coupled with the poten-
tial problem of minority cytotype disadvantage (Levin, 1975;
Husband, 2000), may have rendered most of the newly formed
polyploid individuals incapable of becoming a demographically
successful population under natural settings, an evolutionary
entity essential for speciation (Fowler & Levin, 2016); instead,
they are more likely ephemeral biological products, and hence
evolutionarily inconsequential.
Nevertheless, the ubiquitous and recurring nature of WGD
episodes in the evolutionary histories of all flowering plants and
the abundance of extant neopolyploid plant species (including
many crops) apparently with high fertility and fitness suggests
that, at least in some newly formed polyploids under certain cir-
cumstances, the meiosis machinery itself is evolvable to adapt to
handling a polyploid genome (Hollister, 2015; Bomblies et al.,
2016). Indeed, recent studies showed that a subset of core genes
involved in the meiosis machinery are under strong selection and
evolving rapidly (Hollister et al., 2012; Yant et al., 2013; Henry

262 New Phytologist (2018) 220: 262–277 Ó 2018 The Authors

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et al., 2014; Lloyd et al., 2014; Wright et al., 2015; Bomblies
et al., 2016). As such, established polyploid species often manifest
fully stable meiosis with diploid-like meiotic chromosome behav-
ior primarily due to genetically controlled cytological diploidiza-
tion (Riley, 1960; Sears, 1976; Cifuentes & Benavente, 2009).
Common or bread wheat (Triticum aestivum, genome
BBAADD) is a hexaploid species evolved via allohexaploidization
between a primitive domesticated subspecies of allotetraploid
emmer wheat, Triticum turgidum (such as dicoccon, spelta or
durum), and the diploid Tausch’s goatgrass, Aegilops tauschii
(Kihara, 1944; McFadden & Sears, 1946; Feldman et al., 1995;
Huang et al., 2002), which occurred only c. 8500 yr ago (Feld-
man et al., 1995; Huang et al., 2002). It has been widely assumed
that owing to the presence of the Pairing homeologs (Ph) genes
that function to ensure exclusive homologous pairing – that is,
prevent homeologous pairing (Griffiths et al., 2006; Greer et al.,
2012) in tetraploid wheat – meiosis is intrinsically stable in
hexaploid wheat, with chromosomes manifesting exclusive
diploid-like meiotic behavior and disomic inheritance. Although
this is the case in T. aestivum, the issue of whether the stable
meiosis seen in natural hexaploid wheat has been intrinsically so
upon allopolyploidization or entailed further evolution post-
allohexaploidization remains obscure.
We reported recently that newly synthesized allohexaploid
wheat did not manifest the expected stable meiosis as that of
T. aestivum; rather, it was associated with widespread organismal
whole-chromosome aneuploidy (Zhang et al., 2013). Thus,
newly formed allohexaploid wheat appears no different from
newly formed allopolyploids of other plant species – for example
Brassica (Xiong et al., 2011) and Tragopogon (Chester et al.,
2012) – with respect to numerical chromosome instability. Strik-
ingly, we found that numerical chromosome instability as a phe-
notype is transgenerationally persistent, and even continuous
selection for euploidy across multiple generations did not result
in ostensible increment of euploidy frequencies (Zhang et al.,
2013), suggesting that the refined meiosis machinery of
hexaploid wheat did not appear automatically upon allo-
hexaploidization. Furthermore, we found that the majority of the
aneuploid types in the newly formed allohexaploid wheat did not
compromise fitness, including seed setting under normal condi-
tions (Zhang et al., 2013). Together, these findings promoted us
to explore the potential solutions whereby possible meiosis insta-
bility at the initial stages of hexaploid wheat formation could be
attenuated towards the evolution of a stable karyotype.
Here, we report that self-propagated progeny cohorts of a syn-
thetic allohexaploid wheat, which survived a strong heat- or salt-
stress treatment, have, unexpectedly, manifested significantly
increased euploidy frequencies than their siblings that did not
undergo the stress. We document that this property is largely due
to improved diploid-like meiotic chromosome behavior, includ-
ing reduced incidents of univalent, early disjunction bivalents
and lagging chromosomes. Moreover, we show that both pheno-
types, stress tolerance and enhanced meiosis stability, are trans-
generationally heritable, but the latter being most likely a
spandrel of the former. We also provide evidence indicating that
moderate downregulation of either of two candidate meiosis
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Research 263
genes in natural common wheat, identified in comparative tran-
scriptome analysis, is sufficient to confer an unstable meiosis
manifestation that phenocopies that of the newly synthesized
hexaploid wheat.
Materials and Methods
Plant materials
The original seeds of synthetic allohexaploid wheat line 960
(genome BBAADD) at the S0 generation and its tetraploid parent
T. turgidum (genome BBAA) and diploid parent A. tauschii
(genome DD) were procured from Dr George Fedak. The plants
were then self-pollinated for six generations under normal condi-
tions in our laboratory. Seeds harvested from the 6th-selfed-
generation plants (hence representing the seventh generation) of
960 were used for the stress-selection experiments. The labora-
tory standard cultivar of common wheat, T. aestivum L. cv CS,
was also used.
Stress conditions and physiological measurements
For selection of heat-tolerant cohorts and tolerance assay of their
progenies, seedlings grown in the glasshouse under normal condi-
tions (c. 25°C in daytime and c. 15°C at night) to the trefoil stage
were transferred to a phytotron with the following parameters:
35°C : 30°C, day : night for 4 d, 38°C : 35°C, day : night for 1 d,
40°C : 35°C, day : night for 4 d. Seedlings grown under normal
conditions were used as the mock control. For selection of salt-
tolerant cohorts, we applied ½ Hogland solution containing
200 mmol l1 sodium chloride from seed germination to
seedling growth (c. 10 d). For assay of salt tolerance of progenies,
the solution was applied to seedlings at the trefoil stage. Seedlings
grown under ½ Hogland solution were used as the mock control.
The first leaf of sampled seedlings was used for physiological
measurements (Yang et al., 2014).
Cytological analysis of mitosis and meiosis
Mitosis analysis using genomic in situ hybridization (GISH) and
fluorescent in situ hybridization (FISH) was as described by
Zhang et al. (2013). For meiosis analysis, we fixed young inflores-
cences in Carnoy’s solution (3 : 1 ethanol : acetic acid) for 24 h at
room temperature. Anthers then were isolated and treated in
digesting enzymes (1% cellulose and 1% pectolase in
0.01 mol l1 citrate buffer) at 37°C for 30 min. After replacing
the enzyme solution with 75% ethanol, anthers were squashed in
10 ll 45% acetic solution on slides. The slides were subjected to
the FISH and GISH procedures as for mitosis (Zhang et al.,
2013).
RNA extraction, RNA sequencing, and transcriptome
analysis
All plants for RNA extraction were grown in the experimental
field at Changchun under normal conditions with season. The
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second leaves of trefoil-stage seedlings and anthers at four devel-
opmental stages were collected and frozen in liquid nitrogen.
Total RNAs were extracted by using the Trizol methods (Invitro-
gen) according to the manufacturer’s instructions. Quality and
quantity of RNAs were determined using an Agilent 2100 Bioan-
alyzer (Agilent Technologies, Waldbronn, Germany). The mes-
senger RNA libraries were constructed for leaves and each stage
of anthers and sequenced using Hiseq 2000 with standard proto-
cols. Two biological replicates were conducted for each sample
and sequenced in parallel. Low-quality reads (Q < 20) were
removed from the raw data by using the FASTX-TOOLKIT (http://
hannonlab.cshl.edu/fastx_toolkit/). The clean reads were aligned
to the common wheat (cv CS) genome sequence download from
IWGSC (https://www.wheatgenome.org/) using HISAT2 (v.2.0.1-
beta) (Kim et al., 2015). The steady state of transcript abundance
was normalized by using CUFFNORM (v.2.2.1), and the gene frag-
ments per kilobase of transcript per million mapped reads values
≥ 0.1 were defined as expressed genes. The differentially
expressed genes (DEGs) were filtered by using CUFFDIFF (v.2.2.1)
according to an adjusted P-value (< 0.01) at fold change ≥ 1.5
(Trapnell et al., 2012). Gene Ontology (GO) analysis of DEGs
was performed by hypergeometric distribution in R, with an
adjusted P-value (< 0.05). The wheat GO annotation file was
retrieved by searching the protein database of GenBank using the
BLAST2GO program (http://www.blast2go.com/).
Virus-induced gene silencing and real-time quantitative
reverse transcription PCR
The RNAs used for virus-induced gene silencing (VIGS) were
derived from three barley stripe mosaic virus (BSMV)-based plas-
mids. Plasmids pa and pb were common in all the experiments,
while pc was specific to each of the target genes (Supporting
Information Table S1). Plasmid pc multiple cloning site (MCS)
was used as a virus-only control with a 121 bp fragment derived
from the MCS. FES buffer was used as a negative control (abra-
sive agent). Uninfected CS was used as a mock control. The frag-
ments of antisense constructs (pc) for two targeted genes in CS
(Table S1) were designed from the SGN VIGS Tool website
(http://vigs.solgenomics.net/) with the following parameters: n-
mer size 21, fragment length 150, mismatches 0, and ‘Triticum
aestivum IWGSC2 26’ database. Each fragment was obtained by
annealing two complementary synthetic oligonucleotides with
restriction sites corresponding to PacI on the 50 end and NotI on
the 30 end of plasmid pc. The CS plants were grown under con-
trolled conditions (16 h 25°C : 8 h 15°C, day : night) and inocu-
lated before emergence of the flag leaf (c. 60 d after seed
germination) (Bhullar et al., 2014). The spikes were harvested
2 wk after inoculation, with half being fixed in Carnoy’s solution
for cytology and the other half to be used for RNA extraction
from anthers for real-time quantitative PCR analysis using gene-
specific primers (Table S1) designed by Primer Premier 5. A 1 lg
sample of RNA from triplicate samples of each plant was reverse
transcribed to complementary DNA using the SuperScript first-
strand synthesis kit (Invitrogen). PCR amplification was per-
formed with SYBR Green Real-Time PCR Master Mix reagent
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(Toyobo) on a StepOne Plus Real-Time PCR apparatus (Applied
Biosystems, Carlsbad, CA, USA ), and the amplification condi-
tions were 95°C for 1 min, followed by 40 cycles of 95°C for 5 s
and 60°C for 1 min. A tubulin gene was used to normalize the
expression data.
Statistics
Statistical tests for each comparison and graphical analysis were
executed in R (v.3.2.2) (R Core Team, 2012). An F-test was used
to test for differences in the ranges of SD, and the pairwise Stu-
dent’s t-test was used for comparisons between the tolerant group
and mock group, including physiological measurements,
euploidy ratio, and frequency of meiotic chromosome behavior.
A chi-squared test was used for assessing biases among different
subgenomes and chromosomes.
Accession numbers
Transcriptome data generated in this study are deposited in the
National Center for Biotechnology Information Sequence Read
Archive (accession no. SRP063352 for leaf data of CS and acces-
sion no. SRP146060 for all data of 960 lines and anther data of
CS).
Results
Selection, characterization, and propagation of heat- and
salt-tolerant plants from progenies of a synthetic
allohexaploid wheat
The study system concerns a synthetic allohexaploid wheat (960,
genome BBAADD) parented by a tetraploid wheat (T. turgidum,
genome BBAA) and Tausch’s goatgrass (A. tauschii, genome
DD), thus mimicking the natural allohexaploid common wheat,
T. aestivum, in genome composition (Fig. 1a). We subjected a
larger number of the 960 progenies to either heat or salt stresses,
with > 10 000 germinating seeds for each condition (Fig. 1b). In
parallel, > 2000 germinating seeds were grown under normal
conditions to be used as a mock control. We identified 64 and 72
individual seedlings as immediate survivors of the respective
stresses, which were designated heat-tolerant plants (H0) and salt-
tolerant plants (S0) (Fig. 1b). These surviving H0 and S0 plants
manifested little growth impairment under the respective stress
conditions, which were greatly outnumbered by nontolerant sib-
lings that showed severe growth retardation and persistent tissue
damage (Fig. 2a).
Both the tolerant seedlings (H0 and S0) and a fraction of non-
tolerant seedlings were transferred to the same normal condition
along with the mock control seedlings (M0). In contrast to the
nontolerant seedlings that all died after the transfer, some of the
tolerant seedlings survived. As expected, at adult stages, these
stress-survived plants manifested symptoms characteristic of
experiencing the abiotic stresses at their earlier growth/develop-
ment, such as smaller overall stature, reduced number of tillers,
and slightly decreased seed size, compared with the mock plants
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(a)

(b)

Fig. 1 Karyotypes and typical spike phenotypes of the synthetic allohexaploid wheat (960) along with its parental species and the experimental design. (a)
The spike morphology and sequential fluorescent in situ hybridization/genomic in situ hybridization (FISH/GISH)-based karyotypes of the parental
tetraploid, diploid, and 960 (bars: GISH, 10 lm; spikes, 3 cm). (b) Schematic illustration of the transgenerational propagation of 960 individuals used in this
study. The 64 (H0) and 72 (S0) cohorts tolerant to the heat and salt stresses respectively were separately selected from > 10 000 germinating seeds of the
7th-selfed generation progenies of 960. The selected cohorts were then propagated by selfing for three successive generations under normal conditions. At
each generation, tolerance to the heat or salt stress was assessed and somatic karyotyping was conducted together with plants of the mock control, which
were propagated in parallel. Anthers were sampled from individuals at the 3rd-selfed generation plants (mock control, M3; heat tolerant, H3; and salt
tolerant, S3) for meiotic and transcriptomic analyses.

(Fig. 2b). Some of the surviving plants produced seeds, making a
transgenerational investigation possible.
Seeds of H0, S0, and M0 were used to generate the next-
generation H1, S1, and M1 seedlings via selfing under normal
conditions (Fig. 1b), and which were found to show normal
growth and development, suggesting symptoms seen in the
immediately stressed seedlings were the result of physiological
damage. To test if the stress-tolerant phenotypes manifested by
the H0 and S0 plants were heritable, we subjected portions of
their progenies (H1 and S1) along with M1 under the same heat
and salt stresses (Fig. 1b). We measured several growth and physi-
ological parameters known to be associated with heat- and/or
salt-tolerance in wheat (Fig. 2) (Yang et al., 2014; Suzuki et al.,
2016). The results showed that progenies of the stress-tolerant
plants were also more tolerant to the respective stresses than those
of the mock control (Figs 2, S1). Plants of H1 and S1 along with
M1, all being grown under the same normal condition, were
selfed to generate the next generation (H2, S2, and M2), and
accordingly, H3, S3, and M3. These second- and third-generation
plants were again assessed for tolerance to the respective stresses
(Fig. 1b), and similar results to the first-generation plants (Fig. 2)
were obtained (Fig. S2).
The stress-tolerant plants manifest significantly increased
euploidy frequency than mock control
Our previous study indicated that progenies of 960 propagated
under normal condition harbor high frequencies (>50%) of
numerical chromosome variations in the form of whole-
chromosome aneuploidy (Zhang et al., 2013). In light of this, we
asked whether the chromosomal compositions of these stress-
tolerant H0 and S0 plants were altered or remained similar
(Fig. 1b). We karyotyped all the selected plants of H0 and S0
(n = 64 + 72 = 136), along with a comparable number (n = 110)
of the mock plants (Fig. 1b), by the sequential FISH/GISH pro-
tocol that enables reliable diagnosis for each of the 21 wheat
chromosome pairs (Zhang et al., 2013). Intriguingly, we found
that of the 64 H0 karyotyped plants, 51 (79.7%) were euploids
and only 13 (20.3%) were aneuploids; similarly, of the 72 S0
plants, 59 (81.9%) were euploids and only 13 (18.1%) were

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(a) (b)

(c) (d)

(e) (f) (g)

Fig. 2 Phenotypes and physiological parameters of the mock (M) and heat- (H) or salt-tolerant (S) individuals of the synthetic allohexaploid wheat
(960). (a) Plant phenotypes of seedlings of mock and heat- or salt-tolerant individuals of the S0 generation of 960 under normal and the stress
conditions (bars, 5 cm). (b) Plant and kernel phenotypes of mock and heat- or salt-tolerant adult individuals of the S0 generation of 960 after the
seedlings were transferred to normal conditions (bars: plants, 10 cm; kernels, 1 cm). (c) Cytomembrane permeability, (d) Chla content, (e) fresh
weight, (f) water content, and (g) sodium ion content, measured for the 1st-selfed generation plants of the mock control (M1) and heat- (H1) or
salt-tolerant (S1) plants under normal and the respective stress conditions. The values are means ( SE) of > 10 individuals, and asterisks denote
statistical differences between the stress-tolerant plants (H1/S1) vs the mock control plants based on pairwise Student’s t-tests: *, P < 0.05; **,
P < 0.01; ***, P < 0.001. Similar results were obtained for the 2nd- and 3rd-selfed generation plants for these measurements and shown in Supporting
Information Fig. S2.

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aneuploids (Table S2). By contrast, of the 110 karyotyped M0
plants, 39 (35.5%) were euploids and 71 (64.5%) were aneu-
ploids (Table S2), broadly consistent with the earlier results
(Zhang et al., 2013). In addition, the degrees of chromosome
number deviation were smaller in the stress-tolerant plants than
those of the mock control (Table S2).
The phenotype of enhanced euploidy frequency
manifested by the stress-tolerant plants is
transgenerationally heritable under normal condition
Given the foregoing results, an important question to ask was
whether the phenotype of enhanced euploidy frequencies
observed in the immediately selected plants (H0 and S0) could be
inherited by their organismal progenies that did not experience
the stresses. To address this, we karyotyped normally propagated
progenies of the selected stress-tolerant plants for three successive
generations (i.e. plants of H1/S1, H2/S2, and H3/S3), along with
the mock plants in parallel (Fig. 1b; Notes S1). Expectedly, the
mock plants of all three generations (M1, M2, and M3) showed
similar frequencies of euploidy (ranging from 33 to 37%), consis-
tent with our earlier results (Zhang et al., 2013).
Compared with the mock control, plants of both stress origins
at all three generations (S1–S3) showed significantly higher
euploidy frequencies (ranging from 73 to 93%, t-test,
P = 4.16 9 1010 for H vs M and P = 8.59 9 1010 for S vs M)
(Fig. 3). We noted, however, that there was a potential caveat in
the comparisons of the stress-tolerant plants vs mock plants in
euploidy frequencies of their progenies: the tolerant plants being
selected from the immediate stresses (H0/S0) already had higher
proportions of euploidy than those of the mock (M0) (Table S2).
Intuitively, a euploid individual likely generates more euploid
progenies than an aneuploid individual. Therefore, an additional
factor to consider was whether the higher euploidy frequencies
seen in the progenies (S1, S2 and S3) of the tolerant plants were
due to their being largely descendants of euploid progenitors
(Table S2). To take this potential contributing factor into consid-
eration, only progenies derived from euploid progenitors of each
of the preceding generations in the mock plants were used to
(a)
Fig. 3 Euploidy frequencies of the (a) heat-tolerant (H) and (b) salt-tolerant (S) plants relative to the mock control plants (M) of the synthetic allohexaploid
wheat (960) at three successive selfed generations (S1–S3), all being propagated under normal conditions (Fig. 1b). M(EU) refers to the euploidy
frequencies of those plants of the mock control, which were descended only from euploid mother plants at each of the preceding generations. The values
are means ( SE) of > 50 individuals, and P-values denoting statistical significances are based on pairwise Student’s t-tests.
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Research 267
retabulate the euploidy frequencies. The results showed that there
were indeed increases in the euploidy frequencies in progenies
that were descended only from euploidy for all three generations
of the mock plants, which ranged from 40 to 71% (Fig. 3a,b).
However, these increased euploidy frequencies are still signifi-
cantly lower than those of the tolerant plants (Fig. 3a,b, t-test,
P = 3 9 107 for H vs M and P = 5.99 9 107 for S vs M).
Together, it is clear that the stress-selected enhanced euploidy fre-
quency phenotype in 960 is stably inherited to progenies grown
under normal conditions, which mirrors heritability of the physi-
ological/morphological traits denoting enhanced stress tolerance
(Fig. 2).
Progenies of the stress-tolerant plants show substantially
reduced abnormal meiotic chromosome behavior than
those of mock control
Because all aneuploids of 960 are constitutive (i.e. organismal) –
that is, no somatic numerical chromosomal instability was
detected among the karyotyped cells within a given individual
(Zhang et al., 2013) – it is clear that the cause for numerical chro-
mosome variation is rooted to meiotic abnormality. To address
this issue, we conducted meiosis analysis on the progeny plants
already three generations away from the abiotic stresses to exclude
potential dragging physiological effects. Plants of H3 and S3,
along with the mock (M3), all being grown under normal condi-
tions, were used for meiosis analysis (Fig. 1b). We examined mei-
otic chromosome behavior at metaphase I (MI) and telophase I
(TI) by the sequential FISH/GISH protocol (Zhang et al., 2013).
As expected, normal pollen mother cells (PMCs) at MI are char-
acterized by 21 bivalents aligning on the equatorial plates with
seven bivalents from each of the three subgenomes (Fig. 4a,b).
We observed that some PMCs of all these plant groups showed
clear abnormality, including presence of univalent at MI
(Fig. 4c), early-disjunction bivalents at MI/anaphase I (Fig. 4d),
and lagging chromosomes at TI (Fig. 4e). At TI of normal
PMCs, each pair of homologous chromosomes showed syn-
chronous segregation, with the 21 chromosome pairs being
pulled to opposite poles (Fig. 4f).
(b)
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(a) (b)
(c) (d)
(e) (f)
(g) (h)
To explore possible differences in the occurrence of these mei-
otic chromosome abnormalities among the plant groups (H3, S3,
and M3), we quantified 2227 well-resolved PMCs taken from a
total of 32 individual plants (10 or 11 of each plant group). The
2227 PMCs included 870 at MI and 1357 at TI (Table 1;
Notes S2). We found that 30.2% and 39.2% of the MI PMCs in
H3 and S3 respectively harbored univalent(s); both were signifi-
cantly lower than the 55.2% univalent-containing PMCs found
in M3 (t-test, P = 9.99 9 104 for H3 vs M3, and
P = 4.49 9 103 for S3 vs M3), while the difference between H3
and S3 was statistically insignificant (t-test, P = 0.05122)
(Table 1). Similarly, 5.5% and 3.7% of the MI PMCs in H3 and
S3 respectively showed early disjunction bivalents, which were
also significantly lower than the 12% detected in M3 (t-test,
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Fig. 4 Representative meiotic chromosome
complements and chromosome/genome
biases of univalent frequency in individuals of
the synthetic allohexaploid wheat (960),
sampled from the third-generation selfed
heat-tolerant (H3), salt-tolerant (S3), and
mock control (M3) plants. (a) A fluorescent
in situ hybridization (FISH) image showing
21 bivalents at metaphase I using two
repetitive DNA probes, pSc119.2 (green) and
pAS1 (red). (b) The same complement was
probed by genomic in situ hybridization
(GISH) showing seven bivalents from the A
(green), B (blue), and D (red) subgenomes.
(c) A metaphase I chromosome complement
with 20 bivalents and two univalents (white
arrows), the identity of which could be
diagnosed by combining with FISH (inset).
(d) A metaphase I chromosome complement
with 20 regular bivalents and one early
disjunction bivalent (white arrows). (e) An
example of GISH-based complement at
telophase I with two lagging chromosomes
(white arrows). (f) A GISH-based example
showing normal chromosome complement at
telophase I. Bars, 10 lm. Chromosome and
subgenome biases for the occurrence of
univalents were quantified by sequential
FISH and GISH karyotyping, and are
presented in (g) and (h) respectively. The x-
axes in (g) and (h) refer to the seven
homeologous chromosome groups and the
three subgenomes respectively, while the y-
axes in (g, h) refer to the number of
metaphase I pollen mother cells with
univalent(s).
P = 0.036095 for H3 vs M3, and P = 0.010353 for S3 vs M3),
again, the difference between H3 and S3 was statistically insignifi-
cant (t-test, P = 0.215956) (Table 1). Consistent with differences
in the chromosome behavior at MI among the plant groups,
31.9% and 37.3% TI PMCs of H3 and S3 respectively were
found to contain lagging chromosomes, which were significantly
lower than 48.8% found in TI PMCs of M3 (t-test,
P = 5.00 9 104 for H3 vs M3, and P = 3.91 9 103 for S3 vs
M3); and again, the H3 vs S3 comparison was statistically
insignificant (t-test, P = 0.06314) (Table 1).
Based on the sequential FISH/GISH, we were able to unequiv-
ocally determine univalent identities for all the 21 chromosomes
constituting the three subgenomes of wheat (e.g. inset in Fig. 4c)
and quantify potential biases with respect to univalent
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Table 1 Meiotic chromosome behavior in the stress-tolerant and mock control individuals of the synthetic hexaploid wheat (960)
No. (%) of PMCs
Plant No. of PMCs No. (%) of PMCs with early disjunction
group at MI with univalent bivalent
H3 272 82 (30.2)*** 15 (5.5)
S3 406 159 (39.2)** 15 (3.7)*
M3 192 106 (55.2) 23 (12.0)
MI, metaphase I; TI, telophase I; PMC, pollen mother cell; statistical test for comparisons with M3 (t-test): *, P < 0.05; **, P < 0.01; ***, P < 0.001.
frequencies among the 21 chromosomes and the three
subgenomes. We found chromosomes of all three subgenomes
(A, B, and D) showed clear differences in their propensities to be
in a univalent state. Specifically, among the 21 chromosomes,
chromosome 4B showed the highest frequency of univalents (v2
test, P = 1.31 9 105), while no PMC was found to contain uni-
valents for chromosome 7D (Fig. 4g). If considering each
subgenome as a whole, subgenome B showed the highest fre-
quency of univalent formation, followed by subgenome A, while
subgenome D showed the lowest frequency among the three
subgenomes (v2 test, P < 2.20 9 1016 for subgenomes A vs B,
and D vs B, P = 0.002028 for subgenome A vs D) (Fig. 4h). Both
the chromosome and subgenome biases in forming meiotic uni-
valents were consistent with their biases towards whole-
chromosome aneuploidy detected in somatic cells (Zhang et al.,
2013), thus confirming our previously speculated causal relation-
ship between meiosis abnormality and organismal numerical
chromosome variation in this newly formed allohexaploid wheat
(Zhang et al., 2013).
Altered expression of meiosis-related genes in progenies of
the stress-tolerant plants relative to those of mock control
The foregoing results indicate that the occurrence of abnormal
meiosis was significantly attenuated in progenies of the abiotic
stress-tolerant plants, H3 and S3, compared with those (M3) of
the mock control (Table 1). We sought to investigate whether
expression of genes known or predicted to be critical in the meio-
sis machinery was altered in H3 and S3 relative to M3. First, we
addressed this issue by conducting a transcriptome-based com-
parative analysis of gene expression. As an additional control in
this experiment, the natural common wheat (cv CS) with default
normal meiosis was included.
Specifically, we conducted deep-sequencing of RNAs isolated
from four representative developmental stages of anthers corre-
sponding to pre-meiotic, meiotic I, meiotic II, and tri-nucleate
pollens (Deveshwar et al., 2011), from plant groups H3, S3, M3,
and CS (Fig. 1b), along with RNA-Seq of leaf (at trefoil stage) to
serve as a control for tissue specificity. The numbers of expressed
genes ranged from 54 475 to 62 831 across the RNA-Seq samples
(Table S3). We compared H3 vs M3, S3 vs M3, and CS vs M3,
for each anther stage and leaf, and identified larger and variable
numbers of DEGs among the comparisons (Table S4). A notable
feature is that the upregulated DEGs were markedly fewer than
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Research 269
Bivalent (mean  SE)
No. (%) of PMCs
Rods per Rings per No. of PMCs with lagging
PMC PMC at TI chromosome
4.8  0.3 15.7  0.5 370 118 (31.9)***
4.9  0.5 15.6  0.6 370 138 (37.3)**
4.5  0.1 15.6  0.1 617 301 (48.8)
downregulated DEGs for the three comparisons concerning 960;
that is, H3 vs M3 and S3 vs M3 (Table S4). Another notable
observation is that the numbers of DEGs involving different
genetic background comparisons (i.e. CS vs M3) were fewer than
those in the H3 vs M3 comparisons at some specific anther stages
(i.e. meiotic I, meiotic II and tri-nucleate pollen) (Table S4),
indicating the larger-effect nature of the genetic/epigenetic basis
that differentiates H3 and M3.
Because we were primarily interested in genes whose altered
expression would contribute to the enhanced meiosis stability in
progenies of the stress-tolerant plants vs those of the mock in
960, we selected a subset of the DEGs meeting the following
two criteria for further analysis: they were common DEGs in all
three pairwise comparisons, H3 vs M3, S3 vs M3, and CS vs M3;
they showed the same changing expression trend across H3, S3,
and CS relative to M3 (i.e. all being significantly up- or down-
regulated relative to M3). In total, a set of 1769 genes were iden-
tified as meeting both criteria. Among these, 279 unique genes
showed significantly higher expression levels in H3, S3, and CS
compared with M3 across all four stages (Fig. 5a), while 1490
unique genes showed significantly lower expression levels in H3,
S3, and CS relative to M3 across all four stages (Fig. 5b). A GO
analysis for these genes indicated that several functional cate-
gories were significantly enriched, including those involved in
metabolic and cellular processes, photosynthesis, and response to
stresses (Fig. 5c). However, no enrichment was found for genes
involved in meiosis, which might be due to the current poor
annotation of meiosis-related genes in wheat and/or only a small
proportion of these genes were differentially expressed in our
comparisons.
Recently, Yant et al. (2013) compiled a list of 71 candidate
meiosis genes in Arabidopsis thaliana. Based on this, we identified
442 homologous genes (Notes S3) in common wheat (cv CS)
based on its reference genome sequence at IWGSC (https://www.
wheatgenome.org/). We further securitized these genes and classi-
fied them into three classes: class I includes genes that were
expressed greater than or equal to fivefold higher in at least one
of the four anther stages than in leaves; class II includes genes that
were expressed at greater than or equal to twofold but less than or
equal to fivefold in at least one of the four anther stages than in
leaves; class III includes genes that were expressed in at least one
of the four anther stages but not expressed in leaves (Notes S3)
(Dukowic-Schulze et al., 2014). Consequently, the new list con-
tained 341 wheat candidate meiosis genes with 144, 75, and 122
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(a) (b) (c)

Fig. 5 Stratified genes that showed similar expression among the heat-tolerant (H3), salt-tolerant (S3), and common wheat (Triticum aestivum L., cv
Chinese Spring, CS) wheats but significantly up- or downregulated relative to the mock control M3 plants at each of the four representative meiotic stages
(pre-meiotic, meiotic I, meiotic II and tri-nucleate pollen), and their attended Gene Ontology (GO) enrichments. (a, b) Genes that showed common
upregulated and downregulated expression in the H3 vs M3, S3 vs M3 and CS vs M3 comparisons. The numbers of genes that are shared between or
among these stratified genes in anthers of the four stages or unique to a given stage are shown by the Venn diagrams. (c) Enriched GO terms and the
numbers of genes involved of the (a) upregulated and (b) downregulated stratified genes are illustrated.

in classes I, II, and III respectively (Notes S3). To test for the rep-
resentativeness of this gene list (341 genes in total), we searched
for the six known meiosis genes in wheat (TaRad50, TaASY1,
TaDMC1, TaRad51, TaMSH7 and TaNBS1) that were reported
to function in recombination and DNA repair during wheat
meiosis (Dong et al., 2002; Boden et al., 2007; Devisetty et al.,
2010; P
erez et al., 2011a,b). We found that all six genes were
included in our list (Notes S3), suggesting the meiotic candidate
gene list we compiled is representative.
We interrogated the expression of these 341 candidate meiosis
genes in the transcriptome data of the four meiosis stages (pre-
meiotic, meiotic I, meiotic II, and tri-nucleate pollen) in the H3,
S3, M3 and CS plant groups. We found that 93 of these candi-
date meiosis genes (44 in class I, 20 in class II, and 29 in class III)
were differentially expressed consistently in at least one of the
four stages between each of H3, S3, and CS vs M3 (Notes S3).
Further, we compared the two gene sets (1769 DEGs and 93 can-
didate meiosis genes) to assess whether any of the identified can-
didate meiosis genes were encompassed by the 1769 DEGs. In
total, 12 meiosis candidate genes were identified as DEGs in the
1769 gene list, which fell into the three classes (Table 2), defined
earlier. Of these 12 genes, seven (two in class I, four in class II,
and one in class III) showed upregulation, while five (one in
class I and four in class III) showed downregulation in the com-
parisons at one or more of the four anther stages analyzed
(Table 2).
Downregulation of each of two analyzed candidate meiosis
genes in natural common wheat (cv CS) phenocopies
meiosis instability of the synthetic hexaploid wheat
To experimentally validate whether the upregulated candidate
meiosis genes in the H3 vs M3, S3 vs M3 and CS vs M3 compar-
isons indeed contribute to the enhanced meiosis stability in pro-
genies of the stress-tolerant plants, we selected two genes and
downregulated their expression individually by the VIGS manip-
ulation in common wheat cv CS. The two target genes are
Traes_7DS_0DA047A5F (a homologue of PDS5) and
Traes_5DL_67A6B8CEB (a homologue of SMC6b) (Table 2).
PDS5 is reported as required for sister chromatid cohesion, regu-
lation of synaptonemal complex (SC) formation in fungi and ani-
mals (Panizza et al., 2000), and meiotic DNA repair in
A. thaliana (Pradillo et al., 2015). Coincidently, PDS5 is also one
of the meiosis-associated genes showing strong selection in
autotetraploid A. arenosa populations (Yant et al., 2013). SMC6
is a component of SMC5–SMC6 complex playing a prominent
part in DNA repair and DNA topology (Watanabe et al., 2009;
Verver et al., 2014).
The pc.PDS5-like and pc.SMC6b-like VIGS constructs were
used to inoculate CS separately, with pc.MCS, a 121 bp frag-
ment derived from the MCS of pBluescript K/S (Stratagene)
cloned into the BSMV pc vector, as a ‘virus-only’ control and
FES buffer as a negative control (Bennypaul et al., 2012). We

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Table 2 Annotated candidate meiotic genes in the stress-tolerant plant groups, which showed similar expression levels to common wheat (Triticum
aestivum L., cv Chinese Spring, CS) but were significantly different from the mock group plants of the synthetic hexaploid wheat

Expression fold
change (cf. M3) in
Gene
category Gene ID Anther stage H3 S3 CS Annotation in IWGSC Annotation in Arabidopsis thaliana

Class I Traes_3AL_E771E6430 Pre-meiotic 2.45 1.83 3.74 Sister chromatid cohesion Homologous to animal sister chromatid
protein PDS5 cohesion protein PDS5
Traes_2BL_F49E62B6E Tri-nucleate 2.34 2.33 2.40 Synaptonemal complex ZYP1b SC transverse filament protein
pollen protein 2
Traes_4BS_C203755F7 Pre-meiotic 0.04 0.27 0.23 Histone H2A H2AX Meiosis-specific histone
Meiotic I, II
Tri-nucleate
pollen
Class II Traes_1AL_696D9125F Pre-meiotic 1.77 1.65 1.83 Serine/threonine-protein ATM homologue of human ataxia
kinase ATM telangiectasia mutated
Traes_7BL_586233664 Pre-meiotic 2.43 1.94 4.89 Serine/threonine-protein GMI1 involved in somatic homologous
kinase tel1 recombination
Traes_7DS_0DA047A5F Pre-meiotic 1.93 1.72 2.60 Sister chromatid cohesion PDS5-like homologous to animal sister
protein PDS5 homologue B chromatid cohesion protein PDS5
Traes_5DL_67A6B8CEB Pre-meiotic 1.63 1.50 1.62 Structural maintenance of SMC6b SMC5/6 complex; sister
chromosomes 6A (SMC6a) chromatid alignment and homologous
recombination
Class III Traes_5AL_7D6F45F6B Pre-meiotic 1.91 2.20 3.44 Kinesin-related protein 4 TETRASPORE required for cytokinesis in pollen
Traes_5BL_5D8E3A685 Meiotic I 0.27 0.39 0.45 Unknown DYAD/SWI1 involved in sister chromatid
cohesion and meiotic chromosome organization
Traes_5BL_D5F9F2041 Meiotic II 0.04 0.49 0.27 PHD finger protein MMD1 DNA binding, male meiosis,
microsporogenesis
Traes_7DL_6511AD958 Meiotic I 0.01 0.06 0.47 Histone H2A HTA3 encodes HTA3, a histone H2A
Tri-nucleate
pollen
Traes_4DS_7394D3FBE Pre-meiotic 0.04 0.22 0.41 Histone H2A HTA3 encodes HTA3, a histone H2A
Meiotic I, II

obtained three virus-infected plants for each construct, which
manifested the typical photobleaching phenotype denoting suc-
cessful infection (Bennypaul et al., 2012), and which showed 25–
33% downregulation of the respective target genes (Fig. 6a,b).
We analyzed meiosis for each of these plants and found they all
manifested meiosis instability; that is, abnormal chromosome
behavior in certain proportions of MI and TI PMCs. This meio-
sis instability manifestation was highly similar to that of 960; that
is, with univalent and early-disjunction bivalents at MI and lag-
ging chromosomes at TI (Fig. 6c–f). Although variable, the three
downregulated pc.PDS5-like plants, on average, harbored univa-
lent and early-disjunction bivalents in 19.5% and 20.3% of the
MI PMCs examined respectively and lagging chromosomes in
34.1% of the TI PMCs (Table 3; Notes S4). Likewise, the three
downregulated pc.SMC6b-like plants harbored univalents in
22.3% and early-disjunction bivalents in 10.8% PMCs at MI
and lagging chromosomes in 28.74% PMCs at TI (Table 3). In
stark contrast, no such abnormal meiotic chromosome behavior
was observed in comparable numbers of PMCs from the mock-
inoculated plants (Table 3), indicating specific downregulation of
either of the two meiosis genes is causal for the induced meiosis
instability.
We further karyotyped 118 PMCs of the VIGS plants of CS
to investigate whether there were also subgenome and
chromosome biases in the occurrence of univalents. We found
clear biases both among the 21 chromosomes and across the three
subgenomes in terms of univalent frequencies (Fig. 6g,h).
Remarkably, these biases were also highly similar to those
observed in 960 (Fig. 4g,h); that is, chromosome 4B showed the
highest frequency of univalents among all 21 chromosomes (v2
test, P = 2.57 9 104), and subgenome B as a whole showed the
highest frequency of univalents compared with subgenomes A
and D (v2 test, P = 8.35 9 107) (Fig. 6g,h). In aggregates,
results of the VIGS-based experiments indicate that even a mod-
erate downregulation (by 25–33%) of each of the two meiosis
genes, Traes_7DS_0DA047A5F (a homologue of PDS5) and
Traes_5DL_67A6B8CEB (a homologue of SMC6b), is sufficient
to induce meiosis instability in CS, which phenocopies the syn-
thetic hexaploid wheat.
Discussion
With amassing of whole-genome sequences from diverse taxa, lit-
tle doubt remains regarding the important roles of polyploidy in
the evolution and speciation of higher plants. There are well-
defined properties of being polyploid, which may confer evolu-
tionary advantages (Comai, 2005; Chen, 2007; Leitch & Leitch,
2008; Soltis et al., 2009; Jackson & Chen, 2010; Parisod et al.,

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(a) (c) (d)

(e) (f)

(b)

(g) (h)

Fig. 6 Downregulation of two meiosis-related genes in separation by the virus-induced gene silencing (VIGS) approach in common wheat (cv Chinese
Spring, CS), and their meiosis analysis. Moderate but significant downregulation of each of the two genes, (a) Traes_7DS_0DA047A5F and (b)
Traes_5DL_67A6B8CEB, was validated by real-time quantitative RT-PCR analysis in all three independent plants for each gene relative to wild-type CS and
mock-manipulated CS plants (including FES-buffer-inoculated and virus-only multiple-cloning-site plants). Gene expression level (y-axes) was normalized
against a wheat tubulin gene using the DDCt method. The values are means ( SE) of three technical replicates, and asterisks denote statistical differences
based on pairwise Student’s t-tests: *, P < 0.05; **, P < 0.01. mRNA, messenger RNA. (c–f) Genomic in situ hybridization (GISH)-based analysis of meiotic
chromosome behavior at metaphase I and telophase I of the VIGS-manipulated CS plants. The chromosomes in green, blue, and red are A-, B- and D-
subgenome chromosomes. (c) A metaphase I complement with 21 regular bivalents. (d) A metaphase I complement with 20 bivalents and two univalents
(white arrows). (e) A normal telophase I complement. (f) A telophase I complement with two lagging chromosomes (white arrows). Bars, 10 lm.
Chromosome and subgenome biases of univalent occurrence among (g) the 21 chromosomes and (h) the three subgenomes in pollen mother cells (PMCs)
of the six VIGS-manipulated CS individuals. Identity of the univalents was diagnosed by sequential fluorescent in situ hybridization (FISH) and GISH
karyotyping. The x-axes in (g) and (h) refer to the seven homeologous chromosome groups and the three subgenomes respectively, while the y-axes refer
to the number of metaphase I PMCs with univalent(s).

2010; Van de Peer et al., 2017). Accumulating evidence indicates
that polyploidy can be intrinsically associated with adaptive fea-
tures due to physiological properties and genetic mechanisms,
such as better tolerance to biotic and abiotic stresses (Oswald &
Nuismer, 2007; Allario et al., 2013; Chao et al., 2013; Yang
et al., 2014). In comparison, less attention has been paid to the
genetic challenges facing neopolyploids; in particular, the issue of
how stable meiosis could be reestablished remains largely
unexplored.
Many studies have shown that, unlike established natural poly-
ploid species, neopolyploids often exhibit cytological
abnormalities, including aneuploidy and chromosome rearrange-
ments (Gaeta & Chris Pires, 2010; Xiong et al., 2011; Zhang
et al., 2013; Henry et al., 2014). Extensive aneuploidy was also
observed in all 16 newly synthesized independent allohexaploid
wheat lines studied (Zhang et al., 2013). This is surprising given
that all the tetraploid wheat parents used to construct these allo-
hexaploid lines harbored the pairing homeologous (Ph) genes,
including the potent Ph1 locus (Griffiths et al., 2006). Notably,
two additional features were found in these synthetic hexaploid
wheat lines: (1) aneuploidy is persistent, and even transgenera-
tional selection for euploidy did not result in appreciable increase

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Table 3 Chromosome behavior at metaphase I and telophase I in inoculated plants (pc.PDS5-like and pc.SMC6B-like), virus-only control plants
(pc.multiple cloning site, MCS), abrasive agent control plants (FES), and uninoculated control plants of common wheat (Triticum aestivum, cv Chinese
Spring, CS)

Bivalent (mean  SE)

No. (%) of PMCs
No. of PMCs No. (%) of PMCs No. (%) of PMCs with Rods per Rings per No. of PMCs with lagging
Plant at MI with univalent early disjunction bivalent PMC PMC at TI chromosome

pc.PDS5-like-1 37 7 (18.9) 12 (32.4) 2.1  0.2 18.4  0.3 69 17 (24.6)

pc.PDS5-like-2 92 19 (20.7) 14 (15.2) 1.8  0.2 18.8  0.2 82 25 (30.5)
pc.PDS5-like-3 53 10 (18.9) 7 (13.2) 2.1  0.2 18.6  0.2 108 51 (47.2)
Average 19.5%** 20.3%* 2.0  0.1 18.6  0.1 34.1%*

pc.SMC6B-like-1 46 11 (23.9) 6 (13.0) 2.6  0.2 18.0  0.3 141 38 (27.0)

pc.SMC6B-like-2 35 5 (14.3) 3 (8.6) 2.2  0.2 18.5  0.2 131 24 (18.3)
pc.SMC6B-like-3 28 8 (28.6) 3 (10.7) 2.5  0.3 18.1  0.3 149 61 (40.9)
Average 22.3%** 10.8%** 2.5  0.1 18.2  0.2 28.74%*

pc.MCS-1 54 0 0 2.0  0.2 19.0  0.2 70 8 (11.4)

pc.MCS-2 31 2 (6.45) 0 1.8  0.2 19.1  0.2 63 6 (9.5)
Average 3.23% 0 1.9  0.2 19.1  0.2 10.5%

FES-1 45 3 (6.7) 1 (2.2) 2.2  0.2 18.7  0.2 206 15 (6.8)

FES-2 60 5 (8.3) 0 2.1  0.1 18.8  0.1 134 10 (7.5)
Average 7.5% 1.1% 2.2  0.1 18.8  0.1 7.2%

CS-1 40 2 (5.0) 0 2.1  0.2 18.9  0.2 149 15 (10.1)

CS-2 43 2 (4.7) 1 (2.2) 2.1  0.2 19.0  0.2 185 12 (6.5)
Average 4.85% 1.1% 2.1  0.1 18.9  0.1 8.3%

MI, metaphase I; TI, telophase I; PMC, pollen mother cell; *, P < 0.05; **, P < 0.01 (t-test).

in euploidy frequencies; (2) the majority types of aneuploid indi-
viduals did not show compromised phenotypes related to organ-
ismal fitness, including seed production (Zhang et al., 2013).
Together, these results suggest that the immediately inherited Ph
genes from tetraploid wheat (T. turgidum), albeit functional, are
insufficient to ensure the exclusive diploid-like meiotic chromo-
some behavior characteristic of hexaploid common wheat. Here,
we confirmed this possibility by detailed meiotic analysis, which
showed that, although no multivalent occurred, high incidents of
univalent, early-disjunction bivalents and lagging chromosomes
were associated with 960. Together, this suggests that additional
modifications are needed for the evolution of stable meiosis in
T. aestivum.
Previous studies have shown that, similar to the situation in
most other plant taxa studied (Madlung et al., 2002; Comai,
2005; Salmon et al., 2005; Otto, 2007; Doyle et al., 2008; Jack-
son & Chen, 2010; Van de Peer et al., 2017), newly synthesized
allohexaploid wheat is associated with genetic, epigenetic, and
gene expression changes (Feldman et al., 1997; Shaked et al.,
2001; Adams, 2007; Ozkan€ et al., 2001; Zhao et al., 2011; Feld-
man & Levy, 2012; Li et al., 2014). In light of this, it is conceiv-
able that some of these rapidly occurring genetic or epigenetic
mutations and their downstream effects on gene expression in the
synthetic hexaploid wheat might be involved in the meiosis
machinery to fuel the evolution of a more stabilized meiosis phe-
notype. However, given the prior observation (Zhang et al.,
2013), listed as (2) earlier, an apparent conundrum is what would
be the driving force underlying the selection and eventual fixation
of the genetic/epigenetic variant that confers a stabilized meiosis
phenotype at the population level.
Here, we show that strong abiotic stresses of heat and salt treat-
ments, operating in separation, have enabled the selection of
cohorts from an early selfed generation (F7) of a synthetic allo-
hexaploid wheat 960 (Zhang et al., 2013), which showed herita-
ble tolerance to the respective stresses. Unexpectedly, these
selected plants also manifested significantly increased euploid fre-
quencies due to a more stable meiosis, and, importantly, this
property was transgenerationally heritable under normal growing
conditions. Given the low frequencies at which the stress-tolerant
cohorts were selected (c. 0.64% and 0.72% for heat and salt toler-
ance respectively), the following two possibilities can be ruled out
as a causation to the stress-tolerant phenotypes: being euploidy
per se; and allopolyploidy-induced regulatory rewiring of gene
expression as a result of aggregated cis/trans interactions (Zhuang
& Adams, 2007; Shi et al., 2012; Yoo et al., 2014). Both these
causes would have been reflected at much higher frequencies.
Thus, we suspect these stress-tolerant individuals were hypermor-
phic genetic or epigenetic variants existing in the F7 population;
these variants were selected out because they conferred tolerance
to the respective stresses. We consider the source of the variants
was likely the stochastic genetic/epigenetic changes that occurred
immediately in the newly formed hexaploids and remained segre-
gating in the selfed generations. The variants were cryptic under
normal conditions but became phenotypically revealing under

Ó 2018 The Authors New Phytologist (2018) 220: 262–277

New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com

274 Research
the stresses. We consider the variants were unlikely standing poly-
morphisms in the tetraploid and diploid parental plants used to
construct the synthetic wheat, because both parents are pure line
species and homogeneously less tolerant to the respective stresses
(Yang et al., 2014).
The most relevant question we asked was: Why would the
stress-tolerant plants concomitantly manifest a more stable meio-
sis phenotype? We consider the most plausible explanation for the
concurrence of the two seemingly disparate phenotypes (i.e. stress
tolerance and stabilized meiosis) is their connectivity in genetic
architecture (pleiotropy, linkage, or epistasis); as such, stabilized
meiosis was selected as a by-product or ‘spandrel’ of the stress tol-
erance (Gould & Lewontin, 1979; Barrett & Hoekstra, 2011;
Freeling, 2017). This appears de facto, because being euploidy per
se did not show a fitness gain under either normal or the stress
conditions we tested. This spandrel scenario would imply that, in
the initial stages of hexaploid wheat formation, owing to presence
of the Ph genes and genetic robustness of being hexaploidy
(Zhang et al., 2013), there was no immediate fitness advantage by
evolving a more stable meiosis per se. However, owing to its
genetic correlation with another trait that is adaptive under a
specific environmental context, a more stabilized meiosis was indi-
rectly selected and maintained. This scenario is consistent with
findings in other plant neopolyploids. For example, in newly syn-
thesized Brassica allotetraploid lines, aneuploidy frequencies
accrue (rather than diminish) with generations, and by the 10th
selfed generation nearly 95% of the karyotyped individuals were
aneuploids (Xiong et al., 2011). Likewise, even natural popula-
tions of the recently formed allotetraploid species in Tragopogon
still contained up to 69% aneuploid individuals (Chester et al.,
2012). Both studies suggest that many types of aneuploidies under
the polyploid genetic background did not compromise organis-
mal fitness, thus implying stable meiosis was not immediately
under selection. Recently, the importance of environmental fac-
tors on the evolution of meiosis stability has been emphasized (De
Storme & Geelen, 2014; Wright et al., 2015; Morgan et al.,
2017), but our results suggest the roles of environments on the
evolution of meiosis can be indirect.
The aforementioned allopolyploidization-induced rapid
genetic/epigenetic changes may bring about their effects via
altered gene expression rather than entailing altered gene function
(Doyle et al., 2008; Jackson & Chen, 2010; Buggs et al., 2011;
Feldman & Levy, 2012; Coate et al., 2014; Pfeifer et al., 2014;
Combes et al., 2015). This promoted us to explore whether there
was heritable alteration in the expression of genes related to meio-
sis in the stress-tolerant plants vs the control in 960, and their
expression in common wheat cv CS with default stable meiosis.
By setting rigorous criteria, we identified 12 putative meiosis-
related genes that showed significantly altered expression in the
stress-tolerant vs control comparisons but similar expression in
the stress-tolerant vs CS. To test whether the expression of these
genes was indeed functionally relevant to stable meiosis, we
selected two stress-tolerant vs control upregulated genes (homo-
logues of PDS5 and SMC6b) and artificially downregulated their
expression via VIGS in CS. Intriguingly, we observed compro-
mised meiosis stability in the VIGS-downregulated plants of CS
New Phytologist (2018) 220: 262–277
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New
Phytologist
for each of the two genes studied and which, remarkably, pheno-
copies features of the synthetic wheat, including both cytological
manifestations of meiosis instability and subgenome and chro-
mosome biases. It is thus tempting to speculate that an allo-
hexaploidization-induced regulatory change leading to altered
expression of critical meiosis genes has played an important role
in fine-tuning the meiosis machinery in hexaploid wheat, and the
selection of which was not because it was adaptive per se but
because it was connected in genetic architecture to stress tolerance
as a spandrel. This said, we certainly cannot rule out the possibil-
ity that the original founder parental stands leading to speciation
of T. aestivum have behaved differently (Mestiri et al., 2010; Li
et al., 2015; Jahier et al., 2017). Notwithstanding, considering
the climate and edaphic conditions in the most probable region
of T. aestivum speciation – that is, the Fertile Crescent in the
Near East (Salamini et al., 2002) – similar conditions to the
stresses we used might occur frequently.
In conclusion, our results suggest that, in addition to the Ph
genes, the stable meiotic chromosome behavior in T. aestivum
might have been tinkered from regulatory changes of exiting
meiosis genes as a spandrel of other adaptive traits at the onset of
its formation. Further studies are needed to identify the exact
genetic or epigenetic variant(s) underpinning the regulatory mod-
ifications of the meiosis-related genes, and the mechanistic basis
of their architectural connectivity with the truly adaptive traits
through which it was selected as a spandrel.
Acknowledgements
We thank Dr Sachin Rustgi for comments to this study. We ded-
icate this paper to the memory of the late Professor Diter von
Wettstein, who enthusiastically supported the initiation of this
project. This work was supported by the National Key Research
and Development Program of China (2016YFD0102003), the
National Natural Science Foundation of China (31290210 and
31670218), and the National Program for Introducing Talents
to Universities (B07017). The authors declare that no competing
interests exist.
Author contributions
B.L. conceived and supervised the study; Y.B., C.Y., X.O., B.W.
and W.M. carried out the experiments; Y.B., Z.Z., L.G. and
H.Z. analyzed the data; Y.B., H.Z. and B.L. interpreted the
results and wrote the manuscript, with contribution and approval
from all authors.
References
Adams KL. 2007. Evolution of duplicate gene expression in polyploid and hybrid
plants. Journal of Heredity 98: 136.
Adams KL, Wendel JF. 2005. Polyploidy and genome evolution in plants.
Current Opinion in Plant Biology 8: 135–141.
Allario T, Brumos J, Colmenero-Flores JM, Iglesias DJ, Pina JA, Navarro L,
Talon M, Ollitrault P, Morillon R. 2013. Tetraploid Rangpur lime rootstock
increases drought tolerance via enhanced constitutive root abscisic acid
production. Plant, Cell & Environment 36: 856–868.
Ó 2018 The Authors
New Phytologist Ó 2018 New Phytologist Trust

New
Phytologist
Barrett RD, Hoekstra HE. 2011. Molecular spandrels: tests of adaptation at the
genetic level. Nature Reviews Genetics 12: 767.
Bennypaul HS, Mutti JS, Rustgi S, Kumar N, Okubara PA, Gill KS. 2012.
Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and
meiotic tissues of wheat. Functional & Integrative Genomics 12: 143–156.
Bhullar R, Nagarajan R, Bennypaul H, Sidhu GK, Sidhu G, Rustgi S, von
Wettstein D, Gill KS. 2014. Silencing of a metaphase I-specific gene results in
a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene
mutations. Proceedings of the National Academy of Sciences, USA 111: 14187–
14192.
Boden SA, Shadiac N, Tucker EJ, Langridge P, Able JA. 2007. Expression and
functional analysis of TaASY1 during meiosis of bread wheat (Triticum
aestivum). BMC Molecular Biology 8: 65.
Bomblies K, Jones G, Franklin C, Zickler D, Kleckner N. 2016. The challenge
of evolving stable polyploidy: could an increase in “crossover interference
distance” play a central role? Chromosoma 125: 287–300.
Buggs RJA, Zhang L, Miles N, Tate JA, Gao L, Wei W, Schnable PS, Barbazuk
WB, Soltis PS, Soltis DE. 2011. Transcriptomic shock generates evolutionary
novelty in a newly formed, natural allopolyploid plant. Current Biology 21:
551–556.
Chao DY, Dilkes B, Luo H, Douglas A, Yakubova E, Lahner B, Salt DE. 2013.
Polyploids exhibit higher potassium uptake and salinity tolerance in
Arabidopsis. Science 341: 658–659.
Chen ZJ. 2007. Genetic and epigenetic mechanisms for gene expression and
phenotypic variation in plant polyploids. Annual Review of Plant Biology 58:
377–406.
Chester M, Gallagher JP, Symonds VV, Av CDS, Mavrodiev EV, Leitch AR,
Soltis PS, Soltis DE. 2012. Extensive chromosomal variation in a recently
formed natural allopolyploid species, Tragopogon miscellus (Asteraceae).
Proceedings of the National Academy of Sciences, USA 109: 1176–1181.
Cifuentes M, Benavente E. 2009. Complete characterization of wheat–alien
metaphase I pairing in interspecific hybrids between durum wheat (Triticum
turgidum L.) and jointed goatgrass (Aegilops cylindrica Host). Theoretical and
Applied Genetics 118: 1609–1616.
Coate JE, Bar H, Doyle JJ. 2014. Extensive translational regulation of gene
expression in an allopolyploid (Glycine dolichocarpa). Plant Cell 26: 136–150.
Comai L. 2005. The advantages and disadvantages of being polyploid. Nature
Reviews Genetics 6: 836–846.
Combes M-C, Hueber Y, Dereeper A, Rialle S, Herrera J-C, Lashermes P.
2015. Regulatory divergence between parental alleles determines gene
expression patterns in hybrids. Genome Biology and Evolution 7: 1110–1121.
De Storme N, Geelen D. 2014. The impact of environmental stress on male
reproductive development in plants: biological processes and molecular
mechanisms. Plant, Cell & Environment 37: 1–18.
Deveshwar P, Bovill WD, Sharma R, Able JA, Kapoor S. 2011. Analysis of
anther transcriptomes to identify genes contributing to meiosis and male
gametophyte development in rice. BMC Plant Biology 11: 78.
Devisetty UK, Mayes K, Mayes S. 2010. The RAD51 and DMC1 homoeologous
genes of bread wheat: cloning, molecular characterization and expression
analysis. BMC Research Notes 3: 245.
Dong C, Whitford R, Langridge P. 2002. A DNA mismatch repair gene links to
the Ph2 locus in wheat. Genome 45: 116–124.
Doyle JJ, Flagel LE, Paterson AH, Rapp RA, Soltis DE, Soltis PS, Wendel JF.
2008. Evolutionary genetics of genome merger and doubling in plants. Annual
Review of Genetics 42: 443–461.
Dukowic-Schulze S, Harris A, Li J, Sundararajan A, Mudge J, Retzel EF,
Pawlowski WP, Chen C. 2014. Comparative transcriptomics of early meiosis
in Arabidopsis and maize. Journal of Genetics and Genomics 41: 139–152.
Feldman M, Levy AA. 2012. Genome evolution due to allopolyploidization in
wheat. Genetics 192: 763–774.
Feldman M, Liu B, Segal G, Abbo S, Levy AA, Vega JM. 1997. Rapid
elimination of low-copy DNA sequences in polyploid wheat: a possible
mechanism for differentiation of homoeologous chromosomes. Genetics 147:
1381–1387.
Feldman M, Lupton FGH, Miller TE. 1995. Wheats. In: Smartt J, Simmonds
NW, eds. Evolution of crop plants, 2nd edn. Longman Scientific: London, UK,
184–192.
Ó 2018 The Authors
New Phytologist Ó 2018 New Phytologist Trust
14698137, 2018, 1, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.15267 by Cochrane Hungary, Wiley Online Library on [05/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Research 275
Fowler NL, Levin DA. 2016. Critical factors in the establishment of
allopolyploids. American Journal of Botany 103: 1236–1251.
Freeling M. 2017. Picking up the ball at the K/Pg boundary: the distribution of
ancient polyploidies in the plant phylogenetic tree as a spandrel of asexuality
with occasional sex. Plant Cell 29: 202–206.
Gaeta RT, Chris Pires J. 2010. Homoeologous recombination in allopolyploids:
the polyploid ratchet. New Phytologist 186: 18–28.
Gould SJ, Lewontin RC. 1979. The spandrels of San Marco and the Panglossian
paradigm: a critique of the adaptationist programme. Proceedings of the Royal
Society of London Series B: Biological Sciences 205: 581–598.
Greer E, Martin AC, Pendle A, Colas I, Jones AM, Moore G, Shaw P. 2012.
The Ph1 locus suppresses Cdk2-type activity during premeiosis and meiosis in
wheat. Plant Cell 24: 152–162.
Griffiths S, Sharp R, Foote TN, Bertin I, Wanous M, Reader S, Colas I, Moore
G. 2006. Molecular characterization of Ph1 as a major chromosome pairing
locus in polyploid wheat. Nature 439: 749–752.
Henry IM, Dilkes BP, Tyagi A, Gao J, Christensen B, Comai L. 2014. The BOY
NAMED SUE quantitative trait locus confers increased meiotic stability to an
adapted natural allopolyploid of Arabidopsis. Plant Cell 26: 181–194.
Hollister JD. 2015. Polyploidy: adaptation to the genomic environment. New
Phytologist 205: 1034–1039.
Hollister JD, Arnold BJ, Svedin E, Xue KS, Dilkes BP, Bomblies K. 2012.
Genetic adaptation associated with genome-doubling in autotetraploid
Arabidopsis arenosa. PLoS Genetics 8: e1003093.
Huang S, Sirikhachornkit A, Su X, Faris J, Gill B, Haselkorn R, Gornicki P.
2002. Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate
kinase of the Triticum/Aegilops complex and the evolutionary history of
polyploid wheat. Proceedings of the National Academy of Sciences, USA 99:
8133–8138.
Husband BC. 2000. Constraints on polyploid evolution: a test of the minority
cytotype exclusion principle. Proceedings of the Royal Society of London Series B:
Biological Sciences 267: 217–223.
Jackson S, Chen ZJ. 2010. Genomic and expression plasticity of polyploidy.
Current Opinion in Plant Biology 13: 153–159.
Jahier J, Coriton O, Deffains D, Arnaud D, Chalhoub B. 2017. Revisiting
meiotic pairing in wheat synthetics in relation to the evolution of the meiotic
system in wheat. Plant Systematics & Evolution 303: 1311–1316.
Jiao Y, Wickett NJ, Ayyampalayam S, Chanderbali AS, Landherr L, Ralph PE,
Tomsho LP, Hu Y, Liang H, Soltis PS, et al. 2011. Ancestral polyploidy in
seed plants and angiosperms. Nature 473: 97–100.
Kihara H. 1944. Discovery of the DD-analyser, one of the ancestors of Triticum
vulgare. Agriculture and Horticulture 19: 889–890.
Kim D, Langmead B, Salzberg SL. 2015. HISAT: a fast spliced aligner with low
memory requirements. Nature Methods 12: 357.
Leitch AR, Leitch IJ. 2008. Genomic plasticity and the diversity of polyploid
plants. Science 320: 481–483.
Levin DA. 1975. Minority cytotype exclusion in local plant populations. Taxon
24: 35–43.
Li A, Geng S, Zhang L, Liu D, Mao L. 2015. Making the bread: insights from
newly synthesized allohexaploid wheat. Molecular Plant 8: 847–859.
Li A, Liu D, Wu J, Zhao X, Hao M, Geng S, Yan J, Jiang X, Zhang L, Wu J,
et al. 2014. mRNA and small RNA transcriptomes reveal insights into dynamic
homoeolog regulation of allopolyploid heterosis in nascent hexaploid wheat.
Plant Cell 26: 1878–1900.
Lloyd AH, Ranoux M, Vautrin S, Glover N, Fourment J, Charif D, Choulet F,
Lassalle G, Marande W, Tran J, et al. 2014. Meiotic gene evolution: can you
teach a new dog new tricks? Molecular Biology and Evolution 31: 1724–1727.
Madlung A. 2013. Polyploidy and its effect on evolutionary success: old questions
revisited with new tools. Heredity 110: 99–104.
Madlung A, Masuelli RW, Watson B, Reynolds SH, Davison J, Comai L. 2002.
Remodeling of DNA methylation and phenotypic and transcriptional changes
in synthetic Arabidopsis allotetraploids. Plant Physiology 129: 733–746.
McFadden ES, Sears ER. 1946. The origin of Triticum spelta and its free-
threshing hexaploid relatives. Journal of Heredity 37: 107–116.
Mercier R, Mezard C, Jenczewski E, Macaisne N, Grelon M. 2015. The
molecular biology of meiosis in plants. Annual Review of Plant Biology 66:
297–327.
New Phytologist (2018) 220: 262–277
www.newphytologist.com

276 Research
Mestiri I, Chague V, Tanguy AM, Huneau C, Huteau V, Belcram H, Coriton
O, Chalhoub B, Jahier J. 2010. Newly synthesized wheat allohexaploids
display progenitor-dependent meiotic stability and aneuploidy but structural
genomic additivity. New Phytologist 186: 86–101.
Morgan CH, Zhang H, Bomblies K. 2017. Are the effects of elevated
temperature on meiotic recombination and thermotolerance linked via the axis
and synaptonemal complex? Philosophical Transactions of the Royal Society B:
Biological Sciences 372: 20160470.
Oswald BP, Nuismer SL. 2007. Neopolyploidy and pathogen resistance.
Proceedings of the Royal Society of London Series B: Biological Sciences 274: 2393–
2397.
Otto SP. 2007. The evolutionary consequences of polyploidy. Cell 131: 452–
462.
€
Ozkan H, Levy AA, Feldman M. 2001. Allopolyploidy-induced rapid genome
evolution in the wheat (Aegilops–Triticum) group. Plant Cell 13: 1735–1747.
Panizza S, Tanaka T, Hochwagen A, Eisenhaber F, Nasmyth K. 2000. Pds5
cooperates with cohesin in maintaining sister chromatid cohesion. Current
Biology 10: 1557–1564.
Parisod C, Holderegger R, Brochmann C. 2010. Evolutionary consequences of
autopolyploidy. New Phytologist 186: 5–17.
P
erez R, Cuadrado A, Chen IP, Puchta H, Jouve N, De Bustos A. 2011a. The
Rad50 genes of diploid and polyploid wheat species. Analysis of homologue
and homoeologue expression and interactions with Mre11. Theoretical and
Applied Genetics 122: 251–262.
P
erez R, Cuadrado A,
Jouve N, Bustos AD. 2011b. Characterization of the
Nbs1gene and analysis of the expression of homologous and homoeologous
MRN complex genes in meiocytes and somatic cells of different wheat species.
International Journal of Plant Sciences 172: 959–969.
Pfeifer M, Kugler KG, Sandve SR, Zhan B, Rudi H, Hvidsten TR,
International Wheat Genome Sequencing Consortium, Mayer KF, Olsen
OA. 2014. Genome interplay in the grain transcriptome of hexaploid bread
wheat. Science 345: 1250091.
Pradillo M, Knoll A, Oliver C, Varas J, Corredor E, Puchta H, Santos JL. 2015.
Involvement of the cohesin cofactor PDS5 (SPO76) during meiosis and DNA
repair in Arabidopsis thaliana. Frontiers in Plant Science 6: 1034.
R Core Team. 2012. R: a language and environment for statistical computing.
Computing 1: 12–21.
Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of polyploid
formation in flowering plants. Annual Review of Ecology & Systematics 29:
467–501.
Riley R. 1960. The diploidisation of polyploid wheat. Heredity 15: 407–429.
Salamini F, Ozkan H, Brandolini A, Sch€afer-Pregl R, Martin W. 2002. Genetics
and geography of wild cereal domestication in the Near East. Nature Reviews
Genetics 3: 429–441.
Salmon A, Ainouche ML, Wendel JF. 2005. Genetic and epigenetic
consequences of recent hybridization and polyploidy in Spartina (Poaceae).
Molecular Ecology 14: 1163–1175.
Sears ER. 1976. Genetic control of chromosome pairing in wheat. Annual Review
of Genetics 10: 31–51.
Shaked H, Kashkush K, Ozkan H, Feldman M, Levy AA. 2001. Sequence
elimination and cytosine methylation are rapid and reproducible responses of
the genome to wide hybridization and allopolyploidy in wheat. Plant Cell 13:
1749–1759.
Shi X, Ng DW, Zhang C, Comai L, Ye W, Chen ZJ. 2012. Cis- and trans-
regulatory divergence between progenitor species determines gene-expression
novelty in Arabidopsis allopolyploids. Nature Communications 3: 950.
Soltis DE, Albert VA, Leebens-Mack J, Bell CD, Paterson AH, Zheng C,
Sankoff D, de Pamphilis CW, Wall PK, Soltis PS. 2009. Polyploidy and
angiosperm diversification. American Journal of Botany 96: 336–348.
Suzuki N, Bassil E, Hamilton JS, Inupakutika MA, Zandalinas SI, Tripathy
D, Luo Y, Dion E, Fukui G, Kumazaki A. 2016. ABA is required for
plant acclimation to a combination of salt and heat stress. PLoS ONE 11:
e0147625.
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H,
Salzberg SL, Rinn JL, Pachter L. 2012. Differential gene and transcript
expression analysis of RNA-seq experiments with TopHat and Cufflinks.
Nature Protocols 7: 562.
New Phytologist (2018) 220: 262–277
www.newphytologist.com
14698137, 2018, 1, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.15267 by Cochrane Hungary, Wiley Online Library on [05/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
New
Phytologist
Van de Peer Y, Mizrachi E, Marchal K. 2017. The evolutionary significance of
polyploidy. Nature Reviews Genetics 18: 411–424.
Verver DE, Langedijk NSM, Jordan PW, Repping S, Hamer G. 2014. The
SMC5/6 complex is involved in crucial processes during human
spermatogenesis. Biology of Reproduction 91: 22.
Watanabe K, Pacher M, Dukowic S, Schubert V, Puchta H, Schubert I.
2009. The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6
complex promotes sister chromatid alignment and homologous
recombination after DNA damage in Arabidopsis thaliana. Plant Cell 21:
2688–2699.
Wright KM, Arnold B, Xue K, Surinova M, O’Connell J, Bomblies K. 2015.
Selection on meiosis genes in diploid and tetraploid Arabidopsis arenosa.
Molecular Biology and Evolution 32: 944–955.
Xiong Z, Gaeta RT, Pires JC. 2011. Homoeologous shuffling and
chromosome compensation maintain genome balance in resynthesized
allopolyploid Brassica napus. Proceedings of the National Academy of Sciences,
USA 108: 7908–7913.
Yang C, Zhao L, Zhang H, Yang Z, Wang H, Wen S, Zhang C, Rustgi S, von
Wettstein D, Liu B. 2014. Evolution of physiological responses to salt stress in
hexaploid wheat. Proceedings of the National Academy of Sciences, USA 111:
11882–11887.
Yant L, Hollister JD, Wright KM, Arnold BJ, Higgins JD, Franklin FC,
Bomblies K. 2013. Meiotic adaptation to genome duplication in Arabidopsis
arenosa. Current Biology 23: 2151–2156.
Yoo MJ, Liu X, Pires JC, Soltis PS, Soltis DE. 2014. Nonadditive gene
expression in polyploids. Annual Review of Genetics 48: 485–517.
Zhang H, Bian Y, Gou X, Zhu B, Xu C, Qi B, Li N, Rustgi S, Zhou H, Han F,
et al. 2013. Persistent whole-chromosome aneuploidy is generally associated
with nascent allohexaploid wheat. Proceedings of the National Academy of
Sciences, USA 110: 3447–3452.
Zhao N, Zhu B, Li M, Wang L, Xu L, Zhang H, Zheng S, Qi B, Han F, Liu B.
2011. Extensive and heritable epigenetic remodeling and genetic stability
accompany allohexaploidization of wheat. Genetics 188: 499–510.
Zhuang Y, Adams KL. 2007. Extensive allelic variation in gene expression in
Populus F1 hybrids. Genetics 177: 1987–1996.
Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 Seedlings of the 1st-selfed generation heat- or salt-tolerant
plants and those of the mock control plants, as well as the
tetraploid wheat and diploid parents of the synthetic
allohexaploids wheat (960) were grown under normal and the
respective stress conditions.
Fig. S2 Measurement of physiological parameters from the 2nd
and 3rd-selfed progenies of the stress-tolerant and mock control
plants under normal and the respective stress conditions.
Table S1 Primers used in qRT-PCR experiments for VIGS
Table S2 Karyotypes in the selected stress-tolerant plants and the
randomly chosen mock control plants
Table S3 Total number of the expressed genes at the four stages
of anther and leaf from the H3, S3, M3 and CS plants
Table S4 Differentially expressed genes (DEGs) identified in dif-
ferent comparisons from leaf and various stages of anther
Ó 2018 The Authors
New Phytologist Ó 2018 New Phytologist Trust
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New
Phytologist Research 277

Notes S1 Somatic karyotyping of three successive-generations
progenies of stress-tolerant plants and mock control plants of the
synthetic hexaploid wheat (960), respectively.
Notes S2 Meiosis analysis of H3, S3 and M3 plants of the syn-
thetic hexaploid wheat (960) at metaphase I and telophase I.
Notes S3 Expression levels and classification of 442 wheat
homologs of meiosis-related genes identified in Arabidopsis
thaliana.
Notes S4 Meiosis analysis of VIGS-manipulated common wheat
(cv CS) plants at metaphase I and telophase I.
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Ó 2018 The Authors New Phytologist (2018) 220: 262–277

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