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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: Huakun Zhang Tel: +44 0740 5527566 Email:
[email protected] Bao Liu Tel: +86 043185099367 Email:
[email protected] Received: 23 March 2018 Accepted: 11 May 2018
New Phytologist (2018) doi: 10.1111/nph.15267
Key words: abiotic stress, gene expression, meiosis, polyploidy, spandrel, speciation, Triticum aestivum, virus-induced gene silencing (VIGS).
Polyploidy is a prominent route to speciation in plants; however, this entails resolving the challenges of meiotic instability facing abrupt doubling of chromosome complement. This issue remains poorly understood. We subjected progenies of a synthetic hexaploid wheat, analogous to natural common wheat, but exhibiting extensive meiotic chromosome instability, to heat or salt stress. We selected stress-tolerant cohorts and generated their progenies under normal condition. We conducted fluorescent in situ hybridization/genomic in situ hybridization-based meiotic/mitotic analysis, RNA-Seq and virus-induced gene silencing (VIGS)-mediated assay of meiosis candidate genes. We show that heritability of stress tolerance concurred with increased euploidy frequency 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.
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 instability 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; Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
Hollister, 2015; Bomblies et al., 2016). These meiotic instabilities 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 potential 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 circumstances, 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 New Phytologist (2018) 1 www.newphytologist.com
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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 behavior primarily due to genetically controlled cytological diploidization (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 (Feldman 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 postallohexaploidization 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. Strikingly, we found that numerical chromosome instability as a phenotype 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 allohexaploidization. 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 conditions (Zhang et al., 2013). Together, these findings promoted us to explore the potential solutions whereby possible meiosis instability 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 synthetic allohexaploid wheat, which survived a strong heat- or saltstress 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, including reduced incidents of univalent, early disjunction bivalents and lagging chromosomes. Moreover, we show that both phenotypes, stress tolerance and enhanced meiosis stability, are transgenerationally 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 New Phytologist (2018) www.newphytologist.com
genes in natural common wheat, identified in comparative transcriptome 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 conditions in our laboratory. Seeds harvested from the 6th-selfedgeneration plants (hence representing the seventh generation) of 960 were used for the stress-selection experiments. The laboratory 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 conditions (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 salttolerant 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 inflorescences 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 Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
New Phytologist second leaves of trefoil-stage seedlings and anthers at four developmental stages were collected and frozen in liquid nitrogen. Total RNAs were extracted by using the Trizol methods (Invitrogen) according to the manufacturer’s instructions. Quality and quantity of RNAs were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The messenger RNA libraries were constructed for leaves and each stage of anthers and sequenced using Hiseq 2000 with standard protocols. 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.2.0.1beta) (Kim et al., 2015). The steady state of transcript abundance was normalized by using CUFFNORM (v.2.2.1), and the gene fragments 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/).
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(Toyobo) on a StepOne Plus Real-Time PCR apparatus (Applied Biosystems, Carlsbad, CA, USA ), and the amplification conditions 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 Student’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 accession no. SRP146060 for all data of 960 lines and anther data of CS).
Results 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 plasmids. 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 (abrasive agent). Uninfected CS was used as a mock control. The fragments 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: nmer 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 controlled conditions (16 h 25°C : 8 h 15°C, day : night) and inoculated 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 genespecific 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 firststrand synthesis kit (Invitrogen). PCR amplification was performed with SYBR Green Real-Time PCR Master Mix reagent Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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 salttolerant 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 siblings that showed severe growth retardation and persistent tissue damage (Fig. 2a). Both the tolerant seedlings (H0 and S0) and a fraction of nontolerant 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/development, such as smaller overall stature, reduced number of tillers, and slightly decreased seed size, compared with the mock plants New Phytologist (2018) www.newphytologist.com
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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 nextgeneration 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 physiological 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 New Phytologist (2018) www.newphytologist.com
(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 wholechromosome aneuploidy (Zhang et al., 2013). In light of this, we asked whether the chromosomal compositions of these stresstolerant 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 protocol 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 Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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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 aneuploids (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%), consistent 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 consideration, only progenies derived from euploid progenitors of each of the preceding generations in the mock plants were used to
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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 significantly 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 frequency phenotype in 960 is stably inherited to progenies grown under normal conditions, which mirrors heritability of the physiological/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 chromosome 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 conditions, were used for meiosis analysis (Fig. 1b). We examined meiotic 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 characterized 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 synchronous segregation, with the 21 chromosome pairs being pulled to opposite poles (Fig. 4f).
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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. New Phytologist (2018) www.newphytologist.com
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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 xaxes in (g) and (h) refer to the seven homeologous chromosome groups and the three subgenomes respectively, while the yaxes in (g, h) refer to the number of metaphase I pollen mother cells with univalent(s).
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To explore possible differences in the occurrence of these meiotic 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 significantly 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, Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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P = 0.036095 for H3 vs M3, and P = 0.010353 for S3 vs M3), again, the difference between H3 and S3 was statistically insignificant (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 unequivocally 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 New Phytologist (2018) www.newphytologist.com
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8 Research Table 1 Meiotic chromosome behavior in the stress-tolerant and mock control individuals of the synthetic hexaploid wheat (960)
Plant group
No. of PMCs at MI
No. (%) of PMCs with univalent
No. (%) of PMCs with early disjunction bivalent
H3 S3 M3
272 406 192
82 (30.2)*** 159 (39.2)** 106 (55.2)
15 (5.5) 15 (3.7)* 23 (12.0)
Bivalent (mean SE) Rods per PMC
Rings per PMC
No. of PMCs at TI
No. (%) of PMCs with lagging chromosome
4.8 0.3 4.9 0.5 4.5 0.1
15.7 0.5 15.6 0.6 15.6 0.1
370 370 617
118 (31.9)*** 138 (37.3)** 301 (48.8)
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 univalents for chromosome 7D (Fig. 4g). If considering each subgenome as a whole, subgenome B showed the highest frequency 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 univalents were consistent with their biases towards wholechromosome aneuploidy detected in somatic cells (Zhang et al., 2013), thus confirming our previously speculated causal relationship 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 meiosis machinery was altered in H3 and S3 relative to M3. First, we addressed this issue by conducting a transcriptome-based comparative 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 corresponding 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 New Phytologist (2018) www.newphytologist.com
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 downregulated relative to M3). In total, a set of 1769 genes were identified 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 categories 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 classified 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 contained 341 wheat candidate meiosis genes with 144, 75, and 122 Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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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 representativeness 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; Perez 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 (premeiotic, meiotic I, meiotic II, and tri-nucleate pollen) in the H3, S3, M3 and CS plant groups. We found that 93 of these candidate 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 candidate meiosis genes) to assess whether any of the identified candidate 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 comparisons at one or more of the four anther stages analyzed (Table 2). Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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 comparisons indeed contribute to the enhanced meiosis stability in progenies of the stress-tolerant plants, we selected two genes and downregulated their expression individually by the VIGS manipulation 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, regulation of synaptonemal complex (SC) formation in fungi and animals (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 fragment 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 New Phytologist (2018) www.newphytologist.com
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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
Traes_2BL_F49E62B6E
2.34
2.33
2.40
Homologous to animal sister chromatid cohesion protein PDS5 ZYP1b SC transverse filament protein
0.04
0.27
0.23
Traes_1AL_696D9125F
Tri-nucleate pollen Pre-meiotic Meiotic I, II Tri-nucleate pollen Pre-meiotic
Sister chromatid cohesion protein PDS5 Synaptonemal complex protein 2 Histone H2A
1.77
1.65
1.83
Traes_7BL_586233664
Pre-meiotic
2.43
1.94
4.89
Traes_7DS_0DA047A5F
Pre-meiotic
1.93
1.72
2.60
Traes_5DL_67A6B8CEB
Pre-meiotic
1.63
1.50
1.62
Serine/threonine-protein kinase ATM Serine/threonine-protein kinase tel1 Sister chromatid cohesion protein PDS5 homologue B Structural maintenance of chromosomes 6A (SMC6a)
Traes_5AL_7D6F45F6B Traes_5BL_5D8E3A685
Pre-meiotic Meiotic I
1.91 0.27
2.20 0.39
3.44 0.45
Kinesin-related protein 4 Unknown
Traes_5BL_D5F9F2041
Meiotic II
0.04
0.49
0.27
PHD finger protein
Traes_7DL_6511AD958
Meiotic I Tri-nucleate pollen Pre-meiotic Meiotic I, II
0.01
0.06
0.47
Histone H2A
ATM homologue of human ataxia telangiectasia mutated GMI1 involved in somatic homologous recombination PDS5-like homologous to animal sister chromatid cohesion protein PDS5 SMC6b SMC5/6 complex; sister chromatid alignment and homologous recombination TETRASPORE required for cytokinesis in pollen DYAD/SWI1 involved in sister chromatid cohesion and meiotic chromosome organization MMD1 DNA binding, male meiosis, microsporogenesis HTA3 encodes HTA3, a histone H2A
0.04
0.22
0.41
Histone H2A
HTA3 encodes HTA3, a histone H2A
Traes_4BS_C203755F7
Class II
Class III
Traes_4DS_7394D3FBE
obtained three virus-infected plants for each construct, which manifested the typical photobleaching phenotype denoting successful 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 meiosis instability manifestation was highly similar to that of 960; that is, with univalent and early-disjunction bivalents at MI and lagging chromosomes at TI (Fig. 6c–f). Although variable, the three downregulated pc.PDS5-like plants, on average, harbored univalent 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 mockinoculated 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 New Phytologist (2018) www.newphytologist.com
H2AX Meiosis-specific histone
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 moderate 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 synthetic hexaploid wheat.
Discussion With amassing of whole-genome sequences from diverse taxa, little doubt remains regarding the important roles of polyploidy in the evolution and speciation of higher plants. There are welldefined properties of being polyploid, which may confer evolutionary advantages (Comai, 2005; Chen, 2007; Leitch & Leitch, 2008; Soltis et al., 2009; Jackson & Chen, 2010; Parisod et al., Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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(e)
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(g)
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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 Dsubgenome 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 features 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 polyploid species, neopolyploids often exhibit cytological Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
abnormalities, including aneuploidy and chromosome rearrangements (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 allohexaploid 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 transgenerational selection for euploidy did not result in appreciable increase New Phytologist (2018) www.newphytologist.com
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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 with lagging chromosome
No. of PMCs at MI
No. (%) of PMCs with univalent
No. (%) of PMCs with early disjunction bivalent
Rods per PMC
Rings per PMC
No. of PMCs at TI
pc.PDS5-like-1 pc.PDS5-like-2 pc.PDS5-like-3 Average
37 92 53
7 (18.9) 19 (20.7) 10 (18.9) 19.5%**
12 (32.4) 14 (15.2) 7 (13.2) 20.3%*
2.1 0.2 1.8 0.2 2.1 0.2 2.0 0.1
18.4 0.3 18.8 0.2 18.6 0.2 18.6 0.1
69 82 108
17 (24.6) 25 (30.5) 51 (47.2) 34.1%*
pc.SMC6B-like-1 pc.SMC6B-like-2 pc.SMC6B-like-3 Average
46 35 28
11 (23.9) 5 (14.3) 8 (28.6) 22.3%**
6 (13.0) 3 (8.6) 3 (10.7) 10.8%**
2.6 0.2 2.2 0.2 2.5 0.3 2.5 0.1
18.0 0.3 18.5 0.2 18.1 0.3 18.2 0.2
141 131 149
38 (27.0) 24 (18.3) 61 (40.9) 28.74%*
pc.MCS-1 pc.MCS-2 Average
54 31
0 2 (6.45) 3.23%
0 0 0
2.0 0.2 1.8 0.2 1.9 0.2
19.0 0.2 19.1 0.2 19.1 0.2
70 63
8 (11.4) 6 (9.5) 10.5%
FES-1 FES-2 Average
45 60
3 (6.7) 5 (8.3) 7.5%
1 (2.2) 0 1.1%
2.2 0.2 2.1 0.1 2.2 0.1
18.7 0.2 18.8 0.1 18.8 0.1
206 134
15 (6.8) 10 (7.5) 7.2%
CS-1 CS-2 Average
40 43
2 (5.0) 2 (4.7) 4.85%
0 1 (2.2) 1.1%
2.1 0.2 2.1 0.2 2.1 0.1
18.9 0.2 19.0 0.2 18.9 0.1
149 185
15 (10.1) 12 (6.5) 8.3%
Plant
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 individuals did not show compromised phenotypes related to organismal 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 chromosome 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; Jackson & 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; Feldman & Levy, 2012; Li et al., 2014). In light of this, it is conceivable 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 phenotype. However, given the prior observation (Zhang et al., 2013), listed as (2) earlier, an apparent conundrum is what would New Phytologist (2018) www.newphytologist.com
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 treatments, operating in separation, have enabled the selection of cohorts from an early selfed generation (F7) of a synthetic allohexaploid wheat 960 (Zhang et al., 2013), which showed heritable tolerance to the respective stresses. Unexpectedly, these selected plants also manifested significantly increased euploid frequencies 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 tolerance 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 hypermorphic 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 segregating in the selfed generations. The variants were cryptic under normal conditions but became phenotypically revealing under Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
New Phytologist the stresses. We consider the variants were unlikely standing polymorphisms 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 meiosis 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 tolerance (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 indirectly selected and maintained. This scenario is consistent with findings in other plant neopolyploids. For example, in newly synthesized 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 populations 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 organismal fitness, thus implying stable meiosis was not immediately under selection. Recently, the importance of environmental factors 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 meiosis 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 meiosisrelated 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 (homologues of PDS5 and SMC6b) and artificially downregulated their expression via VIGS in CS. Intriguingly, we observed compromised meiosis stability in the VIGS-downregulated plants of CS Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust
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for each of the two genes studied and which, remarkably, phenocopies features of the synthetic wheat, including both cytological manifestations of meiosis instability and subgenome and chromosome biases. It is thus tempting to speculate that an allohexaploidization-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 possibility 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 modifications 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 dedicate 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.
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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 different comparisons from leaf and various stages of anther
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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 synthetic hexaploid wheat (960) at metaphase I and telophase I.
Notes S4 Meiosis analysis of VIGS-manipulated common wheat (cv CS) plants at metaphase I and telophase I. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
Notes S3 Expression levels and classification of 442 wheat homologs of meiosis-related genes identified in Arabidopsis thaliana.
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