Chromosome translocation affects multiple phenotypes, causes genome-wide dysregulation of gene expression, and remodels metabolome in hexaploid wheat.

Chromosomal rearrangements (CRs) may occur in newly formed polyploids due to compromised meiotic fidelity. Moreover, CRs can be more readily tolerated in polyploids allowing their longer-term retention and hence potential spreading/fixation within a lineage. The direct functional consequences of CRs in plant polyploids remain unexplored. Here, we identified a heterozygous individual from a synthetic allohexaploid wheat in which the terminal parts of the long-arms of chromosomes 2D (approximately 193 Mb) and 4A (approximately 167 Mb) were reciprocally translocated. Five homogeneous translocation lines including both unbalanced and balanced types were developed by selfing fertilization of the founder mutant (RT [2DL; 4AL]-ter/1, reciprocal translocation). We investigated impacts of these translocations on phenotype, genome-wide gene expression and metabolome. We find that, compared with sibling wild-type, CRs in the form of both unbalanced and balanced translocations induced substantial changes of gene expression primarily via trans-regulation in the nascent allopolyploid wheat. The CRs also manifested clear phenotypic and metabolic consequences. In particular, the genetically balanced, stable reciprocal translocations lines showed immediate enhanced reproductive fitness relative to wild type. Our results underscore the profound impact of CRs on gene expression in nascent allopolyploids with wide-ranging phenotypic and metabolic consequences, suggesting CRs are an important source of genetic variation that can be exploited for crop breeding.

Spatial and Temporal Transcriptomic Heredity and Asymmetry in an Artificially Constructed Allotetraploid Wheat (AADD).

Polyploidy, or whole-genome duplication (WGD), often induces dramatic changes in gene expression due to "transcriptome shock. " However, questions remain about how allopolyploidy (the merging of multiple nuclear genomes in the same nucleus) affects gene expression within and across multiple tissues and developmental stages during the initial foundation of allopolyploid plants. Here, we systematically investigated the immediate effect of allopolyploidy on gene expression variation in an artificial allopolyploidy system consisting of a constructed allotetraploid wheat (AADD genome, accession AT2) and its diploid progenitors Triticum urartu and Aegilops tauschii. We performed comprehensive RNA sequencing of 81 samples from different genotypes, tissues, and developmental stages. First, we found that intrinsic interspecific differences between the diploid parents played a major role in establishing the expression architecture of the allopolyploid. Nonetheless, allopolyploidy per se also induced dramatic and asymmetric patterns of differential gene expression between the subgenomes, and genes from the D subgenome exhibited a more drastic response. Second, analysis of homoeolog expression bias (HEB) revealed that the D subgenome exhibited significant expression bias and that de novo-generated HEB was attributed mainly to asymmetrical differential gene expression. Homoeolog-specific expression (HSE) analyses showed that the cis-only regulatory pattern was predominant in AT2, reflecting significant divergence between the parents. Co-expression network analysis revealed that homoeolog expression connectivity (HEC) was significantly correlated with sequence divergence in cis elements between subgenomes. Interestingly, allopolyploidy-induced reconstruction of network modules was also associated with different HSE patterns. Finally, a transcriptome atlas of spike development demonstrated that the phenotypic similarity of AT2 to T. urartu may be attributed to the combination of relatively stable expression of A-subgenome genes and drastic downregulation of their D-subgenome homoeologs. These findings provide a broad, multidimensional characterization of allopolyploidy-induced transcriptomic responses and suggest that allopolyploidy can have immediate and complex regulatory effects on the expression of nuclear genes.

Effects of homoeologous exchange on gene expression and alternative splicing in a newly formed allotetraploid wheat.

Homoeologous exchange (HE) is a major mechanism generating post-polyploidization genetic variation with important evolutionary consequences. However, the direct impacts of HE on gene expression and transcript diversity in allopolyploids without the intertwined evolutionary processes remain to be fully understood. Here, we analyzed high-throughput RNA-seq data of young leaves from plant groups of a synthetic allotetraploid wheat (AADD), which contained variable numbers of HEs. We aimed to investigate if and to which extent HE directly impacts gene expression and alternative splicing (AS). We found that HE impacts expression of genes located within HE regions primarily via a cis-acting dosage effect, which led to significant changes in the total expression level of homoeologous gene pairs, especially for homoeologs whose original expression was biased. In parallel, HE also influences expression of a large number of genes residing in non-HE regions by trans-regulation leading to convergent expression of homoeologs. Intriguingly, when taking the original relative homoeolog expression states into account, homoeolog pairs under trans-effect are more prone to manifesting a convergent response to the HEs whereas those under cis-regulation tended to show further exacerbated subgenome-biased expression. Moreover, HE-induced quantitative, largely individual-specific, changes of AS events were detected. Similar to homoeologous expression, homoeo-AS events under trans-effect were more responsive to HE. HE therefore exerts multifaceted immediate effects on gene expression and, to a less extent, on individualized transcript diversity in nascent allopolyploidy.

Balanced Genome Triplication in Wheat Causes Premature Growth Arrest and an Upheaval of Genome-Wide Gene Regulation.

Polyploidy, or whole genome duplication (WGD), is a driving evolutionary force across the tree of life and has played a pervasive role in the evolution of the plant kingdom. It is generally believed that a major genetic attribute contributing to the success of polyploidy is increased gene and genome dosage. The evolution of polyploid wheat has lent support to this scenario. Wheat has evolved at three ploidal levels: diploidy, tetraploidy, and hexaploidy. Ample evidence testifies that the evolutionary success, be it with respect to evolvability, natural adaptability, or domestication has dramatically increased with each elevation of the ploidal levels. A long-standing question is what would be the outcome if a further elevation of ploidy is superimposed on hexaploid wheat? Here, we characterized a spontaneously occurring nonaploid wheat individual in selfed progenies of synthetic hexaploid wheat and compared it with its isogenic hexaploid siblings at the phenotypic, cytological, and genome-wide gene-expression levels. The nonaploid manifested severe defects in growth and development, albeit with a balanced triplication of the three wheat subgenomes. Transcriptomic profiling of the second leaf of nonaploid, taken at a stage when phenotypic abnormality was not yet discernible, already revealed significant dysregulation in global-scale gene expression with ca. 25.2% of the 49,436 expressed genes being differentially expressed genes (DEGs) at a twofold change cutoff relative to the hexaploid counterpart. Both up- and downregulated DEGs were identified in the nonaploid vs. hexaploid, including 457 genes showing qualitative alteration, i.e., silencing or activation. Impaired functionality at both cellular and organismal levels was inferred from gene ontology analysis of the DEGs. Homoeologous expression analysis of 9,574 sets of syntenic triads indicated that, compared with hexaploid, the proportions showing various homeologous expression patterns were highly conserved in the nonaploid although gene identity showed moderate reshuffling among some of the patterns in the nonaploid. Together, our results suggest hexaploidy is likely the upper limit of ploidy level in wheat; crossing this threshold incurs severe ploidy syndrome that is preceded by disruptive dysregulation of global gene expression.

Homoeologous exchanges occur through intragenic recombination generating novel transcripts and proteins in wheat and other polyploids.

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexaploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous recombination hotspots, being biased toward low-copy, subtelomeric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high-resolution HE junctions was analyzed for allopolyploid Brassica, rice, Arabidopsis suecica, banana, and peanut. Intragenic recombination and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein sequences in nascent allopolyploids.

Transgenerationally Precipitated Meiotic Chromosome Instability Fuels Rapid Karyotypic Evolution and Phenotypic Diversity in an Artificially Constructed Allotetraploid Wheat (AADD).

Although a distinct karyotype with defined chromosome number and structure characterizes each biological species, it is intrinsically labile. Polyploidy or whole-genome duplication has played a pervasive and ongoing role in the evolution of all eukaryotes, and is the most dramatic force known to cause rapid karyotypic reconfiguration, especially at the initial stage. However, issues concerning transgenerational propagation of karyotypic heterogeneity and its translation to phenotypic diversity in nascent allopolyploidy, at the population level, have yet to be studied in detail. Here, we report a large-scale examination of transgenerationally propagated karyotypic heterogeneity and its phenotypic manifestation in an artificially constructed allotetraploid with a genome composition of AADD, that is, involving two of the three progenitor genomes of polyploid wheat. Specifically, we show that 1) massive organismal karyotypic heterogeneity is precipitated after 12 consecutive generations of selfing from a single euploid founder individual, 2) there exist dramatic differences in aptitudes between subgenomes and among chromosomes for whole-chromosome gain and/or loss and structural variations, 3) majority of the numerical and structural chromosomal variations are concurrent due to mutual contingency and possible functional constraint, 4) purposed and continuous selection and propagation for euploidy over generations did not result in enhanced karyotype stabilization, and 5) extent of karyotypic variation correlates with variability of phenotypic manifestation. Together, our results document that allopolyploidization catalyzes rampant and transgenerationally heritable organismal karyotypic heterogeneity that drives population-level phenotypic diversification, which lends fresh empirical support to the still contentious notion that whole-genome duplication enhances organismal evolvability.

Global Analysis of Gene Expression in Response to Whole-Chromosome Aneuploidy in Hexaploid Wheat.

Aneuploidy, a condition of unbalanced chromosome content, represents a large-effect mutation that bears significant relevance to human health and microbe adaptation. As such, extensive studies of aneuploidy have been conducted in unicellular model organisms and cancer cells. Aneuploidy also frequently is associated with plant polyploidization, but its impact on gene expression and its relevance to polyploid genome evolution/functional innovation remain largely unknown. Here, we used a panel of diverse types of whole-chromosome aneuploidy of hexaploid wheat (Triticum aestivum), all under the common genetic background of cv Chinese Spring, to systemically investigate the impact of aneuploidy on genome-, subgenome-, and chromosome-wide gene expression. Compared with prior findings in haploid or diploid aneuploid systems, we unravel additional and novel features of alteration in global gene expression resulting from the two major impacts of aneuploidy, cis- and trans-regulation, as well as dosage compensation. We show that the expression-altered genes map evenly along each chromosome, with no evidence for coregulating aggregated expression domains. However, chromosomes and subgenomes in hexaploid wheat are unequal in their responses to aneuploidy with respect to the number of genes being dysregulated. Strikingly, homeologous chromosomes do not differ from nonhomologous chromosomes in terms of aneuploidy-induced trans-acting effects, suggesting that the three constituent subgenomes of hexaploid wheat are largely uncoupled at the transcriptional level of gene regulation. Together, our findings shed new insights into the functional interplay between homeologous chromosomes and interactions between subgenomes in hexaploid wheat, which bear implications to further our understanding of allopolyploid genome evolution and efforts in breeding new allopolyploid crops.

Transcriptome shock invokes disruption of parental expression-conserved genes in tetraploid wheat.

Allopolyploidy often triggers phenotypic novelty and gene expression remolding in the resulting polyploids. In this study, we employed multiple phenotypic and genetic approaches to investigate the nature and consequences of allotetraploidization between A- and S-subgenome of tetraploid wheat. Results showed that karyotype of the nascent allopolyploid plants (AT2) is stable but they showed clear novelty in multiple morphological traits which might have positively contributed to the initial establishment of the tetraploids. Further microarray-based transcriptome profiling and gene-specific cDNA-pyrosequencing have documented that transcriptome shock was exceptionally strong in AT2, but a substantial proportion of the induced expression changes was rapidly stabilized in early generations. Meanwhile, both additive and nonadditive expression genes showed extensive homeolog expression remodeling and which have led to the subgenome expression dominance in leaf and young inflorescence of AT2. Through comparing the homeolog-expressing patterns between synthetic and natural tetraploid wheats, it appears that the shock-induced expression changes at both the total expression level and subgenome homeolog partitioning are evolutionarily persistent. Together, our study shed new light on how gene expression changes have rapidly occurred at the initial stage following allotetraploidization, as well as their evolutionary relevance, which may have implications for wheat improvements.

Evolution of the BBAA component of bread wheat during its history at the allohexaploid level.

Subgenome integrity in bread wheat (Triticum aestivum; BBAADD) makes possible the extraction of its BBAA component to restitute a novel plant type. The availability of such a ploidy-reversed wheat (extracted tetraploid wheat [ETW]) provides a unique opportunity to address whether and to what extent the BBAA component of bread wheat has been modified in phenotype, karyotype, and gene expression during its evolutionary history at the allohexaploid level. We report here that ETW was anomalous in multiple phenotypic traits but maintained a stable karyotype. Microarray-based transcriptome profiling identified a large number of differentially expressed genes between ETW and natural tetraploid wheat (Triticum turgidum), and the ETW-downregulated genes were enriched for distinct Gene Ontology categories. Quantitative RT-PCR analysis showed that gene expression differences between ETW and a set of diverse durum wheat (T. turgidum subsp durum) cultivars were distinct from those characterizing tetraploid cultivars per se. Pyrosequencing revealed that the expression alterations may occur to either only one or both of the B and A homoeolog transcripts in ETW. A majority of the genes showed additive expression in a resynthesized allohexaploid wheat. Analysis of a synthetic allohexaploid wheat and diverse bread wheat cultivars revealed the rapid occurrence of expression changes to the BBAA subgenomes subsequent to allohexaploidization and their evolutionary persistence.

Intrinsic karyotype stability and gene copy number variations may have laid the foundation for tetraploid wheat formation.

Polyploidy or whole-genome duplication is recurrent in plant evolution, yet only a small fraction of whole-genome duplications has led to successful speciation. A major challenge in the establishment of nascent polyploids is sustained karyotype instability, which compromises fitness. The three putative diploid progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes occurred sympatrically, and their cross-fertilization in different combinations may have resulted in fertile allotetraploids with various genomic constitutions. However, only SSAA or closely related genome combinations have led to the speciation of tetraploid wheats like Triticum turgidum and Triticum timopheevii. We analyzed early generations of four newly synthesized allotetraploid wheats with genome compositions S(sh)S(sh)A(m)A(m), S(l)S(l)AA, S(b)S(b)DD, and AADD by combined fluorescence and genomic in situ hybridization-based karyotyping. Results of karyotype analyses showed that although S(sh)S(sh)A(m)A(m) and S(l)S(l)AA are characterized by immediate and persistent karyotype stability, massive aneuploidy and extensive chromosome restructuring are associated with S(b)S(b)DD and AADD in which parental subgenomes showed markedly different propensities for chromosome gain/loss and rearrangements. Although compensating aneuploidy and reciprocal translocation between homeologs prevailed, reproductive fitness was substantially compromised due to chromosome instability. Strikingly, localized genomic changes in repetitive DNA and copy-number variations in gene homologs occurred in both chromosome stable lines, S(sh)S(sh)A(m)A(m) and S(l)S(l)AA. Our data demonstrated that immediate and persistent karyotype stability is intrinsic to newly formed allotetraploid wheat with genome combinations analogous to natural tetraploid wheats. This property, coupled with rapid gene copy-number variations, may have laid the foundation of tetraploid wheat establishment.

Persistent whole-chromosome aneuploidy is generally associated with nascent allohexaploid wheat.

Allopolyploidization has been a driving force in plant evolution. Formation of common wheat (Triticum aestivum L.) represents a classic example of successful speciation via allopolyploidy. Nevertheless, the immediate chromosomal consequences of allopolyploidization in wheat remain largely unexplored. We report here an in-depth investigation on transgenerational chromosomal variation in resynthesized allohexaploid wheats that are identical in genome constitution to common wheat. We deployed sequential FISH, genomic in situ hybridization (GISH), and homeolog-specific pyrosequencing, which enabled unequivocal identification of each of the 21 homologous chromosome pairs in each of >1,000 individual plants from 16 independent lines. We report that whole-chromosome aneuploidy occurred ubiquitously in early generations (from selfed generation S(1) to >S(20)) of wheat allohexaploidy although at highly variable frequencies (20-100%). In contrast, other types of gross structural variations were scant. Aneuploidy included an unexpected hidden type, which had a euploid chromosome number of 2n = 42 but with simultaneous loss and gain of nonhomeologous chromosomes. Of the three constituent subgenomes, B showed the most lability for aneuploidy, followed by A, but the recently added D subgenome was largely stable in most of the studied lines. Chromosome loss and gain were also unequal across the 21 homologous chromosome pairs. Pedigree analysis showed no evidence for progressive karyotype stabilization even with multigenerational selection for euploidy. Profiling of two traits directly related to reproductive fitness showed that although pollen viability was generally reduced by aneuploidy, the adverse effect of aneuploidy on seed-set is dependent on both aneuploidy type and synthetic line.