Making the Bread: Insights from Newly Synthesized Allohexaploid Wheat

Please cite this article in press as: Li et al., Making the Bread: Insights from Newly Synthesized Allohexaploid Wheat, Molecular Plant (2015), http:// dx.doi.org/10.1016/j.molp.2015.02.016

Molecular Plant Review Article

Making the Bread: Insights from Newly Synthesized Allohexaploid Wheat Ai-li Li1,3, Shuai-feng Geng1,3, Lian-quan Zhang2, Deng-cai Liu2 and Long Mao1,* 1

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

2

Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China

3These

authors contributed equally to this article.

*Correspondence: Long Mao ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.02.016

ABSTRACT Bread wheat (or common wheat, Triticum aestivum) is an allohexaploid (AABBDD, 2n = 6x = 42) that arose by hybridization between a cultivated tetraploid wheat T. turgidum (AABB, 2n = 4x = 28) and the wild goatgrass Aegilops tauschii (DD, 2n = 2x = 14). Polyploidization provided niches for rigorous genome modification at cytogenetic, genetic, and epigenetic levels, rendering a broader spread than its progenitors. This review summarizes the latest advances in understanding gene regulation mechanisms in newly synthesized allohexaploid wheat and possible correlation with polyploid growth vigor and adaptation. Cytogenetic studies reveal persistent association of whole-chromosome aneuploidy with nascent allopolyploids, in contrast to the genetic stability in common wheat. Transcriptome analysis of the euploid wheat shows that small RNAs are driving forces for homoeo-allele expression regulation via genetic and epigenetic mechanisms. The ensuing non-additively expressed genes and those with expression level dominance to the respective progenitor may play distinct functions in growth vigor and adaptation in nascent allohexaploid wheat. Further genetic diploidization of allohexaploid wheat is not random. Regional asymmetrical gene distribution, rather than subgenome dominance, is observed in both synthetic and natural allohexaploid wheats. The combinatorial effects of diverged genomes, subsequent selection of specific gene categories, and subgenome-specific traits are essential for the successful establishment of common wheat. Key words: allopolyploidy, synthetic wheat, heterosis, adaptation, expression level dominance Li A.-l., Geng S.-f., Zhang L.-q., Liu D.-c., and Mao L. (2015). Making the Bread: Insights from Newly Synthesized Allohexaploid Wheat. Mol. Plant. --, 1–13.

INTRODUCTION Polyploidy plays a significant role in the evolutionary history of eukaryotes including fungi, metazoans, and green plants (Wolfe, 2001) and is a major force in shaping plant biodiversity (Meyers and Levin, 2006; Rieseberg and Willis, 2007; Hegarty and Hiscock, 2008; Leitch and Leitch, 2008; Wood et al., 2009; Buggs et al., 2011; Matsushita et al., 2012). Many agricultural crops, such as cotton, canola, and bread wheat, are polyploids. Bread wheat (common wheat, Triticum aestivum) is a major staple food that feeds more than 35% of the world’s population (Paux et al., 2008; Singh et al., 2011). Common wheat is an allohexaploid (2n = 6x = 42; AABBDD) that arose about 8000 years ago in the Fertile Crescent (Kihara, 1944; McFadden and Sears, 1944, 1946) by hybridizations between the cultivated tetraploid wheat (T. turgidum 2n = 4x = 28; AABB) and the diploid wild goatgrass Aegilops tauschii (2n = 14; DD). Like most allopolyploid plants, the heterogeneity among subgenomes is considered fundamental in conferring hexaploid

wheat with enhanced adaptability to a wide range of climates (Dubcovsky and Dvorak, 2007). Luckily, new allohexaploid wheat can still be synthesized in the laboratory, although only tetraploid species can be used as maternal parents (Mestiri et al., 2010). Nascent hexaploid wheat exhibits certain hybrid vigor and adaptive traits, such as robust seedling growth, larger spikes, and salt tolerance when compared with the parents (He et al., 2003; Colmer et al., 2006; Yang et al., 2014). Morphologically, nascent hexaploid wheat is more similar to that of T. turgidum in plant height and spike shape, while the longer rachis internodes seem to be inherited from the paternal progenitor Ae. tauschii. Nascent hexaploid wheat also resembled T. turgidum in their spring-type flowering behavior, presumably because of the dominant VERNALIZATION

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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Molecular Plant (Vrn) genes in the tetraploid wheat (He et al., 2003; Li et al., 2014). Polyploidization also suppresses the shattering habit in Ae. tauschii and further domestication renders non-free-threshing grains as free-threshing ones. The robust performance in adaptation and the favorable agronomic traits make common wheat one of the most popular crops on Earth (Peng et al., 2011). For decades, the phenomenon of the widespread success of polyploid plants has encouraged scientists to make tremendous efforts to decipher the underlying molecular mechanisms. The advent and subsequent wide utilization of microarray and next-generation sequencing technologies led to genome-wide exploration of gene expression patterns in a variety of polyploid plants, such as Arabidopsis (Wang et al., 2006; Shi et al., 2012), cotton (Flagel and Wendel, 2010), Tragopogon miscellus (Buggs et al., 2011), and wheat (Pumphrey et al., 2009; Chague et al., 2010; Qi et al., 2012; Chelaifa et al., 2013; Li et al., 2014). Here, we review the most recent progress in the efforts to understand the mechanisms that may underlie polyploid growth vigor and adaptation from cytological, genetic, and epigenetic perspectives and mostly focus on analyses of gene and small RNA expression patterns in newly synthesized allohexaploid wheat.

CHROMOSOME NUMBER INSTABILITY IN NEWLY SYNTHESIZED ALLOHEXAPLOID WHEAT Chromosome-level perturbation is considered the first manifestation of nascent allopolyploidization (Xiong et al., 2011). In early generations of resynthesized allotetraploid Brassica, extensive aneuploidy is observed in successive generations and could reach up to 95% even after 10 selfed generations (Xiong et al., 2011). A similar phenomenon is also reported in allotetraploid Tragopogon (Chester et al., 2012). The common features in chromosomal variations between these two phylogenetically divergent plant taxa implicate gene dosage balance as a major restrictive mechanism for aneuploid retention (Xiong et al., 2011; Chester et al., 2012). Similarly, studies in nascent allohexaploid wheats show that whole-chromosome aneuploidy is also a common phenomenon (Zhang et al., 2013a). Whole-chromosome aneuploids often involve only one homolog (i.e., hemizygous condition), and are potentially revertible to euploid and hence the survivable allohexaploids (Zhang et al., 2013a). The persistent aneuploidy in nascent allohexaploid wheat echoes the notion that aneuploids may serve as a novel source of adaptive variations (Chester et al., 2012). A similar scenario is demonstrated experimentally in yeast, in which aneuploidy alone represents a large-effect mutation that may confer fitness gains under specific conditions (Rancati et al., 2008). The aneuploidy might be alleviated by further cytological diploidization resulting from preferred DNA elimination from one genome versus the other, thus increasing the divergence between homoeologous chromosomes (Feldman et al., 2012b). On the other hand, aneuploidy displays progenitor dependency, i.e., some hybrids show higher frequency of aneuploids and others have a much lower ratio (Mestiri et al., 2010; Zhang et al., 2010, 2013a). In fact, not all synthetic hexaploid wheat plants generate aneuploids; many of them are 2

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Insights from Nascent Allohexaploid Wheat euploids (Zhang et al., 2013a) and may establish quicker than the aneuploids. It is plausible that the actual parental genotypes contributing to the formation of early allohexaploid individuals that evolved into the modern common wheat did not generate persistent aneuploids. Nevertheless, rapid and extensive structural instabilities such as DNA elimination are found as major genetic changes in newly synthesized wheat allotetraploids (Ozkan and Feldman, 2001; Shaked et al., 2001; Kashkush et al., 2002, 2003). Proper genome combinations, such as AABB (or AASS), rather than AADD, are crucial for karyotype stabilization, which, together with variation in copy number of coding genes and localized changes in genomic repeats, may contribute to successful speciation (Zhang et al., 2013b). Studies in nascent allohexaploid wheat demonstrated similar rapid and extensive DNA elimination (Ozkan et al., 2003; Han et al., 2005; Feldman and Levy, 2009; Bento et al., 2011; Qi et al., 2012; Guo and Han, 2014). As reported, natural wheat allopolyploids contain 2%–10% less DNA than the sum of their two progenitors, while synthetic allopolyploids exhibit a similar loss, indicating that DNA elimination occurs soon after allopolyploidization (Eilam et al., 2008, 2010). Moreover, experiments in triticale (an allopolyploid between wheat and rye) reveal that different genomes are not affected equally: wheat genomic sequences are preferentially preserved, whereas rye genomic sequences are more prone to be changed or eliminated (Ma et al., 2004; Ma and Gustafson, 2005, 2006, 2008). Similar bias of DNA elimination is also found for hexaploid wheat where genome D appears to undergo considerable reduction in the amount of DNA while A and B genomes do not (Eilam et al., 2008, 2010). These studies also showed that elimination of non-coding and coding DNA sequences occurred from one genome only and not from the other, thus increasing the divergence between homoeologous chromosomes. Such biased DNA elimination may contribute to cytological diploidization, which provides the physical basis for the diploid-like meiotic behavior in newly formed allohexaploid wheat, i.e., prevention of inter-genomic pairing and recombination, a process that is crucial for the establishment and maintenance of the allopolyploid nature of allohexaploid wheat (Feldman et al., 2012b). However, some recent studies did not find obvious DNA rearrangements in nascent hexaploid wheats (Mestiri et al., 2010; Zhao et al., 2011a; Luo et al., 2012), suggesting that DNA changes may be parental genotype dependent. The genetic stability in such wheat genotypes makes them desirable materials to analyze transcriptional changes and epigenetic regulation during wheat polyploidization.

POLYPLOIDY HETEROSIS AND NONADDITIVE GENE EXPRESSION IN NEWLY SYNTHESIZED ALLOHEXAPLOID WHEAT Hybridization of two divergent species may incur heterosis in the allopolyploid progeny, which can be permanently fixed by subsequent polyploidization (Comai, 2005; Chen, 2007, 2013). The popular hypothesis to explain ‘‘hybrid heterosis’’ is that complementation in the hybrids of different slightly deleterious recessive homozygous alleles from the two parents may account for increased biomass and fertility in the hybrid

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Insights from Nascent Allohexaploid Wheat progeny when compared with both parents (Bruce, 1910; Jones, 1917; Shull, 1948; Washburn and Birchler, 2014). However, such a model cannot properly explain some of the observations in polyploids, such as the so-called ‘‘progressive heterosis,’’ in which allopolyploids appear to have higher heterosis than autopolyploids and tetraploids higher than diploids (Groose et al., 1989; Bingham et al., 1994; Riddle et al., 2010; Yao et al., 2013). The effects in polyploid hybrids are deduced to be caused by increased genome dosage, allelic heterozygosity, and/or epigenetic changes (Chen, 2007, 2010; Riddle et al., 2010; Yao et al., 2013). In other words, polyploidy should be a new factor to consider in plant heterosis studies. One major mechanism for heterosis is modulation by dosage-sensitive factors that involve allelic diversity across parental genomes (Yao et al., 2013). Polyploids allow experiments to be conducted that are not possible in diploids and the insights gained can be incorporated into modified models to explain heterosis at all levels (Washburn and Birchler, 2014). Newly synthesized wheat allohexaploids indeed exhibits certain hybrid vigor and adaptive traits as mentioned earlier (He et al., 2003; Colmer et al., 2006; Yang et al., 2014). Moreover, the tractable genetic pedigree and high ploidy of synthetic wheat should provide special values in studying the molecular basis of polyploid heterosis. Heterotic traits outperform, or at minimum are equal to, the parent with the best value for the trait (best-parent heterosis) or the average of its two parents (so-called mid-parent heterosis). Accordingly, reprogramming of gene expression in polyploids should deviate from that of the best parent or the mid-parent. Non-additive expression is used to describe the expression patterns of such genes with the expression level not equal to the sum of two parental levels or mid-parent value (MPV), leading to expression activation or repression. In wheat, before the availability of genomes of any kind, diploid or hexaploid, non-additive genes were detected using microarray technology. Such analyses, however, estimated MPVs by using a mixture of RNA from the two progenitors in a ratio of 1:1 (AABB/DD), or 2:1 (Akhunova et al., 2010; Chague et al., 2010; Qi et al., 2012). These studies detected 2.8%–19% of nonadditively expressed genes in the synthetic allohexaploids studied. In contrast, a near-complete additivity is found in hexaploid progeny when a tetraploid progenitor (AABB) ‘‘extracted’’ from the natural common wheat is used as a putative parent for comparison, indicating that the parental genotype is critical in non-additive expression evaluation (Chelaifa et al., 2013). Alternatively, selection of only genes exhibiting differences between progenitors may increase the proportion of nonadditively expressed genes (Akhunova et al., 2010). Recently, the draft genome sequences of the two putative genome donors of common wheat, T. urartu (AA) and Ae. tauschii (DD) (Ling et al., 2013; Jia et al., 2013) became available and have been applied in studying this scenario (Li et al., 2014). With RNA-seq technology, gene expression can be counted by read numbers (in RPKM, or reads per kilobase per million reads) and therefore MPVs can be calculated from the progenitor expression levels. Consistent with microarray analysis, only a handful non-additive expression genes are found in young spikes (0.9% of 20 204 expressed genes), which display more prominent growth vigor than other organs (Li et al., 2014). There are even less in seedlings (0.1%) and none can be detected in immature seeds (Li et al., 2014).

Molecular Plant On the other hand, global analyses using various tissues and research platforms reveal different functions of non-additive expressing genes. For instance, two studies reported that Gene Ontology (GO) photosynthesis and reproduction functions are enriched for non-additively activated genes, and defense function is enriched among down-regulation genes using shoot tissues from different synthetic wheat allohexaploids (Chague et al., 2010; Chelaifa et al., 2013). A third study showed the enrichment of vesicle function in leaves (Qi et al., 2012). RNA-seq analysis showed that in young spikes, non-additively up-regulated genes are enriched for cell growth function while genes non-additively down-regulated are enriched for carbohydrate metabolic processes (Li et al., 2014). Despite this, some correlation between non-additively expressed genes and phenotypes may be worthy of attention. For instance, genes involved in cell size regulation (such as expansins) and auxin homoeostasis (such as GH3.1) are non-additively activated in young spikes of newly synthesized wheat, which are much larger than those of their parental lines. These data indicate that non-additive expression genes may partly contribute to the formation of polyploid heterosis. Moreover, non-additive expression genes are moderately stable in intergenerational inheritance. Chague et al. (2010) reported that up to 47% of non-additively expressed genes were transmittable between first and second selfed generations, while the rates reported by Qi et al. (2012) and Li et al. (2014) were
20%. Direct comparison of overall gene expression levels revealed high similarity between synthetics and between synthetic and natural wheat, indicating that, upon wheat allohexaploidization, gene expression is established in early generations and is largely maintained over generations (Chague et al., 2010). Such observations support the hypothesis that a mechanism for rapid and stochastic establishment of non-additively expressed genes exists in allohexaploids, which may lead to natural variation for adaptive selection and domestication (Chen, 2007; Jackson and Chen, 2010). Analysis of gene expression in synthetic allohexaploid Senecio cambrensis clearly shows that interspecific hybridization is indeed the primary cause of changes in gene expression. The ‘‘transcriptome shock’’ observed in the triploid F1 S. x baxteri is ‘‘ameliorated’’ after genome doubling in the first generation of synthetic S. cambrensis and this altered expression pattern is maintained in subsequent generations. Importantly, the sudden alterations that different generations of the synthetic allohexaploids display in gene expression may represent sources of genetic novelty for environmental adaptation (Hegarty et al., 2006).

FUNCTIONAL DIFFERENTIATION OF PARENTAL EXPRESSION LEVEL DOMINANCE GENES In addition to non-additive expression genes, genes with ‘‘expression level dominance’’ have been widely investigated in allopolyploids (Rapp et al., 2009; Flagel and Wendel, 2010; Grover et al., 2012). These genes are inferred in the allopolyploids when their direct or putative progenitors are known. The Senecio analysis shows that polyploidization itself results in a secondary change in gene expression to a state resembling that in one of the original parent taxa (Hegarty et al., 2006). Expression level Molecular Plant --, 1–13, -- 2015 ª The Author 2015.

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Molecular Plant dominance describes the expression condition in an allopolyploid where, for a given gene, the total expression of homoeologs is statistically similar to only one of the parents. This concept is therefore different from homoeolog expression bias. It was originally described in cotton allopolyploids by Rapp et al. (2009), extended by Flagel and Wendel (2010), and subsequently confirmed in both Spartina (Chelaifa et al., 2013) and Coffea (Bardil et al., 2011). In cotton and coffee, for example, significant portions of the genes are expressed at a level equal to that of one progenitor while different from that of the other, irrespective of the MPV and expression additivity (Rapp et al., 2009; Flagel and Wendel, 2010; Grover et al., 2012). In nascent allohexaploid wheat, biased expression dominance toward the AB genome progenitor is observed using microarray analyses (Akhunova et al., 2010; Chague et al., 2010; Qi et al., 2012). RNA-seq analysis not only finds a significant number of genes displaying parental expression level dominance but also biased expression toward the AB genome parent (Li et al., 2014). In seedlings of allohexaploid progeny, genes with expression levels statistically similar to those in T. turgidum (socalled AB parent expression level dominance, or ELD-ab) are enriched for GO molecular function ‘‘catalytic activity’’, while those D parent expression level dominance (or ELD-d) genes are enriched for the biological process term ‘‘response to stimulus’’. In young spikes, significant enrichment of ‘‘cytoplasm’’ and ‘‘cell wall and membrane’’ functions are found for ELD-ab and ELD-d genes, respectively, demonstrating clear functional distinctions and developmental stage dependency between the two groups of genes in nascent allohexaploid progeny. The potential biological roles of ELD genes are further substantiated by the presence of key transcription factors such as homologs of MADS-box genes PISTILLATA (PI), APETALA3 (AP3), and AGAMOUS (AG), which are essential for flower development among the spike ELD-ab genes, indicating that ELD-ab genes may actively participate in allohexaploid wheat development. On the other hand, ELD-d genes encode heat shock proteins, chitinases, and NBS-LRRs, as well as homologs of CONSTANS (CO) and Late Elongated Hypocotyl 1 (LHY1), and may be involved in biotic and abiotic stress tolerance and flowering condition flexibility. Such patterns are largely heritable among early generations of selfpollinated progeny, and even in the common wheat Chinese Spring (CS), suggesting that functional divergence between parental specific ELD genes is to maintain advantageous gene regulation modes from each parent and should have a positive role in allohexaploid wheat evolution and domestication (Li et al., 2014).

DYNAMIC REGULATION OF HOMOEOALLELE EXPRESSIONS DURING WHEAT HEXAPLOIDIZATION The merging of multiple genomes in polyploid plants may cause interactions between diverged regulatory networks, stoichiometric disruption of genes and pathways due to differential dosage sensitivity, and possible de novo genetic and epigenetic alterations (Jackson and Chen, 2010; Madlung, 2013). Homoeologous genes may follow one of the many possible evolutionary fates: non-functionalization (deleted or pseudogenized), neofunctionalization (taking on novel functions), 4

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Insights from Nascent Allohexaploid Wheat and subfunctionalization (partitioning of ancestral functions among alleles or homoeoalleles) (Lynch and Conery, 2000; Lynch and Force, 2000; Prince and Pickett, 2002; Chaudhary et al., 2009). In allohexaploid wheat, early attempts to analyze homoeolog contribution to gene expression levels mostly used low throughput techniques such as expressed sequence tags and single strand confirmation polymorphism analyses (Mochida et al., 2004; Bottley et al., 2006; Bottley and Koebner, 2008). No significant bias toward selective silencing of a particular subgenome was found (Mochida et al., 2004; Bottley et al., 2006). Later, microarray-based homoeoallele-specific analysis using parental specific fragments showed the dominance of the tetraploid transcripts (designated as AB transcripts) in the transcriptome of resynthesized allopolyploid wheat (Akhunova et al., 2010). Nevertheless, microarray approaches cannot efficiently distinguish homoeologs and often cross hybridize with each other, which may cause false positives (Arnaud et al., 2013). In this regard, RNA-seq technology measures homoeolog expression using reads tagged with parental sequence single nucleotide polymorphisms, which detected more AB homoeologs in higher expression categories than D homoeologs in nascent allohexaploid wheat (Li et al., 2014). For a significant portion of genes, such a bias toward higher expression of AB homoeologs is mostly achieved by dynamic regulation of both AB and D homoeologs during allopolyploidization, instead of being simply inherited from their progenitors (Li et al., 2014; Zhang et al., 2014). A good example was recently reported in the literature for the interaction among homoeoalleles, i.e., the acquisition of enhanced salt tolerance in hexaploid wheat compared with its tetraploid progenitor. Such an advantageous trait is considered to be caused by the change of expression patterns of the D subgenome HKT1;5 homoeoallele. The HKT1;5 gene is responsible for Na+ removal from the xylem vessels and is reprogrammed in transcription immediately after allohexaploidization, i.e., from constitutively high basal expression in Ae. tauschii (the D genome donor) to saltinducible expression in nascent hexaploid wheat (Yang et al., 2014). Changes in gene expression modes may provide the plasticity required to improve the fitness and adaptation of the newly formed allopolyploid and to increase its competitive efficiency for successful establishment in nature. Nevertheless, except for Yang et al. (2014), nearly all the above studies did not distinguish A homoeolog expression level from that of the B homoeologs and considered the A and B expression as the total. The recent availability of the hexaploid genome sequence provides the opportunity to dissect expression patterns of each homoeoallele during wheat allopolyploidization in detail and in a genome-wide manner with much improved accuracy (International Wheat Genome Sequencing Consortium (IWGSC), 2014).

MICRORNAS IN NEWLY SYNTHESIZED HEXAPLOID WHEAT AND THEIR REGULATION OF HOMOEOLOG EXPRESSION MicroRNA (miRNA) genes occupy distinct genetic loci in the plant genomes. They perform post-transcriptional gene silencing by

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Insights from Nascent Allohexaploid Wheat degrading target mRNAs or by affecting their translational efficiency through the effector RNA-induced silencing complex (Chapman and Carrington, 2007; Voinnet, 2009). In Arabidopsis, miRNAs appear to be sensitive to polyploidization and may play important roles during polyploidization (Ha et al., 2009; Ng et al., 2012). For instance, more than half of miRNAs (35,
51%) are differentially expressed in leaves between A. thaliana and A. arenosa or between an allotetraploid (F7 or A. suecica) and MPV (Ha et al., 2009). In newly synthesized wheat, the percentage of miRNAs with parental differential expression is also high, up to 40%, in young spikes. More than 20% miRNAs are also non-additively expressed in spikes of early generation plants (Li et al., 2014). This is in contrast to the scarcity of non-additively expressed protein-coding genes (0.9%). The trend of differential accumulation of many miRNAs between two progenitors or between progeny and MPV demonstrates their fundamental roles in allopolyploid formation and evolution. Interestingly, miRNA non-additive expression also exhibits species and tissue specificity. In Arabidopsis, miR172 and miR163 in leaves and miR398 in flowers are non-additively activated (Ha et al., 2009), while in wheat tiller tissues, miR156, miR160, miR159, miR396, and miR168 are significantly over represented relative to MPV (Kenan-Eichler et al., 2011). Among these five miRNAs, miR159 and miR396 are also non-additively activated in wheat young spikes (Li et al., 2014). On the other hand, in both Arabidopsis leaves and flowers, miR156, miR157, miR159, miR161, miR164, miR169, miR171, miR173, and miR390 are non-additively repressed (Ha et al., 2009), while miR158 and miR393 are repressed only in leaves and miR163, miR164, miR167, miR168, miR170, miR172, miR162, miR319, and miR399 only in flowers. In wheat tiller tissues, miR156, miR165, and miR1135 are significantly under represented (Kenan-Eichler et al., 2011). In wheat young spikes, functionally wellcharacterized miRNAs, such as miR169, miR172, miR319, and miR827, which are conserved between monocots and eudicots, as well as grass-specific miRNAs, such as miR5200, miR9672, and miR9863, are significantly under represented relative to MPV (Li et al., 2014). Despite the phylogenetic distance between Arabidopsis and wheat, non-additive expression of miR156 (in leaves or tiller tissues) and miR169, miR172, and miR319 (in inflorescence or spikes) are conserved in the two species. Since most of these miRNAs are involved in flowering and stress (Sunkar et al., 2012; Spanudakis and Jackson, 2014), miRNAs and their target genes may be intrinsic regulatory components during plant polyploidization and are preserved by natural and human selection. Detailed work on Arabidopsis shows that both cis and trans regulation may occur in miRNA genes (Ng et al., 2011). The differential expression of miRNAs may provide a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses. For example, the newly evolved miR163 is highly expressed in A. thaliana but not in A. arenosa. It is repressed in resynthesized allotetraploids, causing up-regulation of its target genes, which encode a family of small molecule methyltransferases implicated in secondary metabolite biosynthetic pathways and are inducible by the fungal elicitor alamethicin. Thus, change in miR163 expression causes differential accumulation

Molecular Plant of secondary metabolites that are involved in defense response (Ng et al., 2011). Similarly, wheat miR9863 was previously predicted to target R gene analogs (Wei et al., 2009) and was recently confirmed in barley to regulate distinct Mla alleles (Liu et al., 2014). We hypothesize that higher expression of miR9863 in Ae. tauschii relative to T. turgidum and its non-additive repression (hence, up-regulation of R genes) in allohexaploid progeny may underlie the enhanced powdery mildew resistance of newly synthesized wheat (Li et al., 2014). Differential expression of divergent miRNAs may cause differential regulation of homoeologous transcripts. In cotton (Gossypium hirsutum, AADD), two miRNAs, miR828 and miR858, generate trans-acting siRNAs (ta-siRNAs) in the TAS4 family and respectively target the two MYB2 homoeologs, GhMYB2A and GhMYB2D that are involved in promoting fiber development, demonstrating specific roles of miRNAs in expression divergence of target homoeologs (Guan et al., 2014a, 2014b). In wheat, despite the detection of a negative correlation between the changes in miRNA and target gene expression levels, not much is known about the mode of action of miRNAs on homoeologous gene expression regulation, mostly because of the limitation of current reference genome quality and the complexity of wheat as a hexaploid (Li et al., 2014).

ROLES OF SIRNAS IN GENOME STABILITY AND HOMOEOLOG EXPRESSION Small interfering RNAs (siRNAs) may serve as a genetic buffer for balancing genome shock (genetic chaos) in newly formed allopolyploids (Ha et al., 2009; Renny-Byfield and Wendel, 2014). Most siRNAs are derived from transposable elements (TEs) or repetitive sequences that are characteristic of a given species. siRNAs, especially those with 24 nucleotides, exert their functions through the RNA-directed DNA methylation (RdDM) pathway by changing the methylation status at regulatory sites (Groszmann et al., 2013). Therefore, changes in siRNA density along the genomes of hybrids or allopolyploids may cause differential modification of TEs and hence the differential expression of neighboring genes. For related species, the genetic loci of siRNA clusters are conserved between species and are often stably inherited in the progeny. For example, about 52%–57% of clustered siRNA loci in A. thaliana can be found in A. suecica and resynthesized F1 and F7 allotetraploids (Ha et al., 2009). In allohexaploid wheat, up to 95% of siRNA loci detected in the progenitors are conserved in the hexaploid progeny and transmittable across early generations (Li et al., 2014). The steady siRNA locations suggest their roles in maintaining the stability of the wheat genome. Wheat is notorious for its high repetitive sequence content (Jia et al., 2013; Ling et al., 2013). Epigenetic silencing by cytosine methylation of DNA sequences (Kashkush et al., 2002) and the activity of small RNA molecules (Kenan-Eichler et al., 2011) have been reported for limited genomic regions or sequences. With the assistance of recently sequenced A and D draft genomes, Li et al. (2014) reported that up to 70% of gene models are co-localized with TEs (henceforth, TE-associated genes, or TAGs), much higher than in Arabidopsis (46%) (Lu Molecular Plant --, 1–13, -- 2015 ª The Author 2015.

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Molecular Plant et al., 2012). Thus, wheat genes are more likely to be regulated by epigenetic mechanisms such as RdDM. In Arabidopsis, interspecific hybrids and allotetraploids can only be produced using A. thaliana as a maternal parent (Comai et al., 2000; Wang et al., 2004). A. thaliana loci are specifically repressed (Wang et al., 2006), which is consistent with the accumulation of A. thaliana siRNAs (Chen et al., 2008). Such biased repression of maternal homoeologs is probably achieved via a mechanism similar to transposon repression by maternally transmitted siRNAs as shown in Drosophila (Brennecke et al., 2008). For wheat, allohexaploids can only be produced using the tetraploid T. turgidum as a maternal parent (Zhang et al., 2010). During allohexaploidization, increased siRNA density is detected for D subgenome TAGs, while no such increase of siRNA density on the A subgenome is observed (Li et al., 2014). Such differential modification of the A and D subgenome TAGs may partly explain biased suppression of D homoeologs in nascent allohexaploid wheat. Intriguingly, TAGs with altered siRNA expression during hexaploidization are enriched for stress response functions, potentially offering expanded adaptability to allohexaploid wheat. Epigenetically regulated loci are probably intrinsic targets of natural selection and domestication.

TRANSPOSABLE ELEMENTS AND DNA METHYLATION IN NEWLY SYNTHESIZED ALLOHEXAPLOID WHEAT The high repeat content (mostly from TEs) may provide more complicated yet versatile gene regulation modes via epigenetic modifications such as DNA and histone methylation. Following allopolyploidization, activation of the basic expression level of LTR retrotransposons can be easily detected in wheat (Kashkush et al., 2002, 2003; Kraitshtein et al., 2010; Yaakov and Kashkush, 2011a, 2011b; Zhao et al., 2011b). A similar genomic reaction is also observed in synthetic Arabidopsis allopolyploid hybrids (Madlung et al., 2005) and in many cases is associated with changes in siRNA levels at TEs (Lu et al., 2012). Studies of methylation status around an LTR retrotransposon Veju found in the first four generations of a newly formed wheat allopolyploid that epigenetic read-out activities occur early during polyploidization (Kraitshtein et al., 2010). A similar pattern of hypomethylation of Veju elements is also observed in the first three generations of a synthetic allotetraploid (Yaakov and Kashkush, 2011b), indicating consistent epigenetic modification on constituent genomes during wheat polyploidization. rRNA genes are long tandem repeats and are targets of gene loss, structural rearrangement, and expression modification during plant evolution (Schubert and Kunzel, 1990; Wendel et al., 1995; Mishima et al., 2002; Pontes et al., 2004; Ksiazczyk et al., 2011). In common wheat, rDNA loci from the A and D genomes are largely lost during the evolutionary process. Such biased DNA elimination is related to asymmetric transcription and epigenetic modifications on divergent rRNA loci, particularly associated TEs (Guo and Han, 2014). It may also contribute to the stabilization of allopolyploid wheat with increased differentiation and diversity. As a matter of fact, nucleolar dominance has been extensively studied in several 6

Molecular Plant --, 1–13, -- 2015 ª The Author 2015.

Insights from Nascent Allohexaploid Wheat genetic systems. In triticale, suppression of rye origin rRNA genes is observed (Heslop-Harrison, 1990; Vieira et al., 1990; Neves et al., 1995). Treating the root tip cells with 5-aza-20 deoxycytidine (5AC), an inhibitor of DNA methyltransferase, induces changes in the inter-rRNA gene spacer methylation patterns of rye origin rDNA (Sardana et al., 1993; Neves et al., 1995). However, the inheritance of 5AC effects on rRNA gene expression observed in the following generation is developmental stage dependent (Amado et al., 1997). In light of the complexity of epigenetic modification, which involves a variety of modifications on DNA and histones, the impact of polyploidization on homoeologous gene by epigenetic mechanisms can be more complicated than expected.

REGIONAL ASYMMETRICAL GENE DISTRIBUTION BUT GENOME DOMINANCE IN SYNTHETIC AND NATURAL ALLOHEXAPLOID WHEAT As in diploids, divergence between the parental species may be the major determinants for heterosis in newly synthesized polyploids (Schnable and Springer, 2013). Divergence between the subgenomes may continue after polyploidization and probably be expedited with the presence of duplicated genomes. Similar to those observed in paleopolyploids (e.g., Arabidopsis and maize), the duplicated genomes often experience nonequivalent gene losses (or genome fractionation), with one genome or genomic region retaining more genes (dominant) than the other (more fractionated). Together with dominant expression of genes on one genome or genomic region, they exhibit the so-called genome dominance phenomenon (Wang et al., 2006; Schnable et al., 2011; Cheng et al., 2012; Garsmeur et al., 2014). Such phenomenon has also been reported in canola, an ancient tetraploid of about 4 mya (Cheng et al., 2012). Common wheat has a much shorter history of
10 000 years. Despite this, accelerated accumulation of gene structure changes, such as alternative splicing, non-synonymous and premature termination codon mutations, have been observed (Akhunov et al., 2013). Such changes resulted in rapid divergence of wheat genomes by generating thousands of pseudogenes, mostly via TEmediated pseudogenization, many of which were not even conserved between wheat subgenomes (Wicker et al., 2011). It is estimated that between 10 000 and 16 000 genes have been lost since the presence of common wheat (Brenchley et al., 2012). A study of wheat syntenome and conserved orthologous gene sets proposed that for hexaploid wheat, the first neotetraploidization event resulted in subgenome dominance with the A subgenome dominant over the B subgenome, while the second neohexaploidization event led to a supra-dominance, with the D subgenome dominant over the tetraploid (subgenomes A and B) (Pont et al., 2013). But, among newly released CS genomes obtained by single chromosome sequencing, no extensively biased gene content in any of the subgenomes was detected; local, regional, or cell type and developmental stage dependent genome dominance was found (International Wheat Genome Sequencing Consortium (IWGSC), 2014; Pfeifer et al., 2014). Such regional genome dominance seems to

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Molecular Plant

Insights from Nascent Allohexaploid Wheat A

Chr 1 TaGH3.8

Chr 2

Chr 3 TaGH3.8 TaGH3.8 TaGH3.2 TaGH3.11

TaGH3.5 B

Chr 4

TaGH3.8 TaGH3.11 TaGH3.2 TaGH3.11

TaGH3.7 TaGH3.7

TaGH3.3

TaGH3.1 TaGH3.1 TaGH3.3

Chr 7

TaGH3.7

TaGH3.8

TaGH3.3 TaGH3.2 TaGH3.2

TaGH3.5 D

TaGH3.7 TaGH3.11

TaGH3.7

TaGH3.5

Figure 1. Asymmetrical Distribution of the GH3 Family Genes in the Chinese Spring Genome. A, B, and D represent the three subgenomes in allohexaploid wheat. Red lines indicate triplet homoeologs that are present on all three homoeologous chromosomes. Purple lines indicate duplet homoeologs that are present on two homoeologous chromosomes. Black lines indicate singletons that are present only on one homoeologous chromosomes. Genes are positioned according to their chromosome arm locations and are not in their exact positions and order on the chromosomes. See Supplemental Table 1 for annotation information.

be correlated with the asymmetrical distribution of important agronomic traits among wheat subgenomes (Feldman et al., 2012a). Previous work estimated that about 10% of the loci in common wheat is composed of genes of which only one genome is active, while the homoeoalleles on the other genome(s) are either eliminated or partially or completely suppressed by genetic or epigenetic means (Feldman et al., 2012a). It seems that genetic diploidization is not a random process. Specific gene categories and their corresponding traits are distinctly affected with a clear-cut division of tasks between the constituent genomes of allopolyploid wheat. For example, the A genome preferentially controls morphological traits, while the B genome in allotetraploid wheat and the B and D genomes in allohexaploid wheat preferentially control the reaction to biotic and abiotic factors (Feldman et al., 2012a, 2012b). The ability of one genome to suppress the activity of genes of another genome renders full control of a set of traits in allopolyploids, preventing conflict in gene expression that is detrimental to plant development and may reduce the fitness of the progeny, and hence is especially important for the evolutionary success of hexaploid wheat (Feldman et al., 2012a, 2012b).

Characteristic gene expression in allopolyploids may also be caused by subgenome divergence existing before and after polyploidization. The plant GH3 (Gretchen Hagen3) genes participate in auxin homeostasis by catalyzing auxin conjugation and converting free indoleacetic acid to amino acids (Domingo et al., 2009; Zhao et al., 2013; Lin et al., 2014). In newly synthesized allohexaploid wheat, one homolog TaGH3.1 is non-additively expressed in young spikes of newly synthesized wheat, while a second homolog TaGH3.2 showed tetraploid progenitor expression level dominance, or ELD-ab (Li et al., 2014). Mapping of the GH3 gene family members to the IWGS CS gene sets found that there were 31 OsGH3 homologs or homoeologs in modern common wheat (Figure 1). Among them, 27 are located on the A and B subgenomes and four on the D subgenome. For GH3.1, two members are located on chromosome 3A and none on B or D chromosomes. Meanwhile, GH3.2 has one copy each on chromosomes 2A and 2B, respectively, and two additional members on 3B but none on D chromosomes (Supplemental Table 1). Thus, the GH3 family genes appear to distribute asymmetrically on three subgenomes in CS. Because there are 12 GH3 genes in Ae. tauschii and 10 in T. urartu (Supplemental Table 1), it is possible that the elimination of GH3 genes on the D chromosomes may occur during or after allopolyploidization. Molecular Plant --, 1–13, -- 2015 ª The Author 2015.

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Molecular Plant

Insights from Nascent Allohexaploid Wheat AABB ǀ

DD ǁ

X A

B

Genome stability and neighboring gene regulaon

Target gene expression regulaon

Allohexaploid (AABBDD)

a AAAAA

b c d

siRNA

miRNA

AAAAA AAAAA

RdDM

mRNA cleavage

?

Homoeolog expression regulaon

C Expression level dominance

Nonaddive expression

Expression level dominance

ELD-ab

ELD-d acvaon AABB

DD

DD ELD-ab AABB

AABBDD

• Development

AABBDD repression

• Reproducon • Cell growth etc.

AABB

ELD-d AABBDD

DD

• Stress • Flowering

Keys: AABB, T. turgidum; DD, Ae. tauschii; AABBDD, nascent allohexaploid progeny; RdDM, RNA-directed DNA methylaon; ELD-ab, genes with expression level similar to that in T. turgidum; ELD-d, genes with expression level similar to that in Ae. tauschii; , putave methylated sites; , transposable elements (TEs); , siRNA; and , miRNAs; , transcripon; , indicates gene silencing; ?, means other regulators.

Figure 2. A Model to Summarize the Molecular Mechanisms that May Be Involved in Homoeolog Expressions Leading to Nonadditive Gene Expression and Parental Expression Level Dominance Genes that May Be Relevant to the Growth Vigor and Enhanced Adaptation in Nascent Allohexaploid Wheat. (A) siRNA regulation module. Genome stabilization by siRNA mediated chromatin modification (a), gene expression (b, c), and TAG silencing (d). (B) miRNA regulation module. Homoeolog-specific cleavage by diverged miRNAs and their differential expressions. (C) Regulation of protein-coding gene expressions and their distinct functions (development, growth vigor, and adaptation) in nascent allohexaploid wheat. The two red bars represent gene expression levels in the allohexaploid progeny. The question mark in the middle indicates unknown cis and trans mechanisms for protein-coding gene regulation. Modified from Li et al. (2014).

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Molecular Plant

Insights from Nascent Allohexaploid Wheat In light of their potential functions in auxin homeostasis, which is essential for plant development and defense response (Domingo et al., 2009; Zhao et al., 2013; Lin et al., 2014), the relevance of these genes with wheat spike growth vigor or heterosis is worthy of further investigation.

CONCLUSIONS The Triticeae family comprises a rich pool of species that can cross with each other with minimal human assistance (Zhang et al., 2010). The existence of various polyploid plants at various ploidy levels provides excellent materials to study important genetics questions, such as progressive heterosis (Chen, 2013). Allohexaploid wheat provides a good example of successful ‘‘human-made’’ polyploid species. Besides some instantly acquired traits, such as enhanced salt tolerance (Yang et al., 2014), it is also a good model to study structural, functional, and epigenetic changes in polyploid plants. In fact, synthetic wheat has been employed in breeding to introduce unique agronomic traits that are lacking among current genetic breeding pools by direct crossing with wheat cultivars to produce more superior breeding materials (Yang et al., 2009). Comparative studies should shed light on the molecular mechanisms underlying the successful evolution and domestication of allohexaploid wheat into modern common wheat, which should provide useful lessons for wheat breeding. The work on nascent allohexaploid wheat should provide new insights into the mechanisms for the success of common wheat. The current model proposes that growth vigor and adaptation may be underpinned by both protein-coding genes and small non-coding RNAs, in the manner of non-additive expression and/or probably expression level dominance. Figure 2 describes three major regulatory modules. In the siRNA module, increased siRNA density in hexaploid progeny may result in biased down-regulation of D homoeologs through RdDM, which involves dynamic regulation of both D and AB homoeologs. In the miRNA module, non-additive expression of important miRNAs (both conserved and grass specific) may cause expression changes in their targets involving plant growth, stress, and flowering. In the protein-coding gene module, genes with expression levels similar to those of Ae. tauschii (ELD-d) may contribute to flowering-related adaptations (speculatively based on LHY and CO), disease resistance (based on R genes), and salt tolerance (based on AKT1). Meanwhile, genes with expression levels similar to those of T. turgidum (PI, AP3, and AG) and the non-additively activated genes (GH3.1 and expansins) may contribute to spike development and growth vigor. Overall, the molecular underpinnings established during the early allopolyploidization events should have laid the ground work for the successful advent of common wheat. Despite the genome complexity of the Triticeae species, the recent availability of the diploid and hexaploid wheat genomes provides new tools to examine this important process. Unlike tetraploid Arabidopsis (Wang et al., 2006; Chen, 2010), it seems that both nonadditive expression genes and ELD genes may contribute to growth vigor and adaptability of nascent allohexaploid wheat. Reductionist approaches using proteomics, metabolomics, and epigenetics approaches may be needed to dissect the specific contributions of corresponding regulatory networks to individual

vigor and adaptation traits and to facilitate the identification of key regulators in the pathways that can be experimentally tested using transgenic approaches. A better understanding of the molecular basis for allopolyploid heterosis and adaptation may ultimately help us to improve current wheat cultivars and, hence, to make better bread for mankind.

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

FUNDING This work is supported by National Science Foundation of China (Nos. 91331117 and 31271716) and the CAAS Innovation Project.

ACKNOWLEDGMENTS We thank Drs. Michael Freeling, James Birchler, Johnathan Wendel, Xiaowu Wang, Bao Liu, and the two anonymous reviewers for constructive communications and comments. No conflict of interest declared. Received: November 5, 2014 Revised: February 13, 2015 Accepted: February 25, 2015 Published: March 5, 2015

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