Evolutionary dynamics of chromatin structure and duplicate gene expression in diploid and allopolyploid cotton.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n=4X=52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this inter-genomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.

iJAZ-based approach to engineer lepidopteran pest resistance in multiple crop species.

The fall armyworm (FAW) poses a significant threat to global crop production. Here we showed that overexpression of jasmonate ZIM-domain (JAZ) protein GhJAZ24 confers resistance to cotton bollworm and FAW, while also causing sterility in transgenic cotton by recruiting TOPLESS and histone deacetylase 6. We identified the NGR motif of GhJAZ24 that recognizes and binds the aminopeptidase N receptor, enabling GhJAZ24 to enter cells and disrupt histone deacetylase 3, leading to cell death. To overcome plant sterility associated with GhJAZ24 overexpression, we developed iJAZ (i, induced), an approach involving damage-induced expression and a switch from intracellular to extracellular localization of GhJAZ24. iJAZ transgenic cotton maintained fertility and showed insecticidal activity against cotton bollworm and FAW. In addition, iJAZ transgenic rice, maize and tobacco plants showed insecticidal activity against their lepidopteran pests, resulting in an iJAZ-based approach for generating alternative insecticidal proteins with distinctive mechanisms of action, thus holding immense potential for future crop engineering.

Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex.

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Polyploid plants take cytonuclear perturbations in stride.

Hybridization in plants is often accompanied by nuclear genome doubling (allopolyploidy), which has been hypothesized to perturb interactions between nuclear and organellar (mitochondrial and plastid) genomes by creating imbalances in the relative copy number of these genomes and producing genetic incompatibilities between maternally derived organellar genomes and the half of the allopolyploid nuclear genome from the paternal progenitor. Several evolutionary responses have been predicted to ameliorate these effects, including selection for changes in protein sequences that restore cytonuclear interactions; biased gene retention/expression/conversion favoring maternal nuclear gene copies; and fine-tuning of relative cytonuclear genome copy numbers and expression levels. Numerous recent studies, however, have found that evolutionary responses are inconsistent and rarely scale to genome-wide generalities. The apparent robustness of plant cytonuclear interactions to allopolyploidy may reflect features that are general to allopolyploids such as the lack of F2 hybrid breakdown under disomic inheritance, and others that are more plant-specific, including slow sequence divergence in organellar genomes and preexisting regulatory responses to changes in cell size and endopolyploidy during development. Thus, cytonuclear interactions may only rarely act as the main barrier to establishment of allopolyploid lineages, perhaps helping to explain why allopolyploidy is so pervasive in plant evolution.

Evolutionary insights into the organization of chromatin structure and landscape of transcriptional regulation in plants.

Development of complex traits necessitates the functioning and coordination of intricate regulatory networks involving multiple genes. Understanding 3D chromatin structure can facilitate insight into the regulation of gene expression by regulatory elements. This potential, of visualizing the role of chromatin organization in the evolution and function of regulatory elements, remains largely unexplored. Here, we describe new perspectives that arise from the dual considerations of sequence variation of regulatory elements and chromatin structure, with a special focus on whole-genome doubling or polyploidy. We underscore the significance of hierarchical chromatin organization in gene regulation during evolution. In addition, we describe strategies for exploring chromatin organization in future investigations of regulatory evolution in plants, enabling insights into the evolutionary influence of regulatory elements on gene expression and, hence, phenotypes.

Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants.

Cell type-specific cytonuclear coevolution in three allopolyploid plant species.

Cytonuclear disruption may accompany allopolyploid evolution as a consequence of the merger of different nuclear genomes in a cellular environment having only one set of progenitor organellar genomes. One path to reconcile potential cytonuclear mismatch is biased expression for maternal gene duplicates (homoeologs) encoding proteins that target to plastids and/or mitochondria. Assessment of this transcriptional form of cytonuclear coevolution at the level of individual cells or cell types remains unexplored. Using single-cell (sc-) and single-nucleus (sn-) RNAseq data from eight tissues in three allopolyploid species, we characterized cell type-specific variations of cytonuclear coevolutionary homoeologous expression and demonstrated the temporal dynamics of expression patterns across development stages during cotton fiber development. Our results provide unique insights into transcriptional cytonuclear coevolution in plant allopolyploids at the single-cell level.

Domestication over Speciation in Allopolyploid Cotton Species: A Stronger Transcriptomic Pull.

Cotton has been domesticated independently four times for its fiber, but the genomic targets of selection during each domestication event are mostly unknown. Comparative analysis of the transcriptome during cotton fiber development in wild and cultivated materials holds promise for revealing how independent domestications led to the superficially similar modern cotton fiber phenotype in upland (G. hirsutum) and Pima (G. barbadense) cotton cultivars. Here we examined the fiber transcriptomes of both wild and domesticated G. hirsutum and G. barbadense to compare the effects of speciation versus domestication, performing differential gene expression analysis and coexpression network analysis at four developmental timepoints (5, 10, 15, or 20 days after flowering) spanning primary and secondary wall synthesis. These analyses revealed extensive differential expression between species, timepoints, domestication states, and particularly the intersection of domestication and species. Differential expression was higher when comparing domesticated accessions of the two species than between the wild, indicating that domestication had a greater impact on the transcriptome than speciation. Network analysis showed significant interspecific differences in coexpression network topology, module membership, and connectivity. Despite these differences, some modules or module functions were subject to parallel domestication in both species. Taken together, these results indicate that independent domestication led G. hirsutum and G. barbadense down unique pathways but that it also leveraged similar modules of coexpression to arrive at similar domesticated phenotypes.

Meiotic pairing irregularity and homoeologous chromosome compensation cause rapid karyotype variation in synthetic allotetraploid wheat.

Allopolyploidization may initiate rapid evolution due to heritable karyotypic changes. The types and extents of these changes, the underlying causes, and their effects on phenotype remain to be fully understood. Here, we designed experimental populations suitable to address these issues using a synthetic allotetraploid wheat. We show that extensive variation in both chromosome number (NCV) and structure (SCV) accumulated in a selfed population of a synthetic allotetraploid wheat (genome Sb Sb DD). The combination of NCVs and SCVs generated massive organismal karyotypic heterogeneity. NCVs and SCVs were intrinsically correlated and highly variable across the seven sets of homoeologous chromosomes. Both NCVs and SCVs stemmed from meiotic pairing irregularity (presumably homoeologous pairing) but were also constrained by homoeologous chromosome compensation. We further show that homoeologous meiotic pairing was positively correlated with sequence synteny at the subtelomeric regions of both chromosome arms, but not with genic nucleotide similarity per se. Both NCVs and SCVs impacted phenotypic traits but only NCVs caused significant reduction in reproductive fitness. Our results implicate factors influencing meiotic homoeologous chromosome pairing and reveal the type and extent of karyotypic variation and its immediate phenotypic manifestation in synthetic allotetraploid wheat. This has relevance for our understanding of allopolyploid evolution.

Cotton fiber as a model for understanding shifts in cell development under domestication.

Cotton fiber provides the predominant plant textile in the world, and it is also a model for plant cell wall biosynthesis. The development of the single-celled cotton fiber takes place across several overlapping but discrete stages, including fiber initiation, elongation, the transition from elongation to secondary cell wall formation, cell wall thickening, and maturation and cell death. During each stage, the developing fiber undergoes a complex restructuring of genome-wide gene expression change and physiological/biosynthetic processes, which ultimately generate a strikingly elongated and nearly pure cellulose product that forms the basis of the global cotton industry. Here, we provide an overview of this developmental process focusing both on its temporal as well as evolutionary dimensions. We suggest potential avenues for further improvement of cotton as a crop plant.

Genomic rearrangements and evolutionary changes in 3D chromatin topologies in the cotton tribe (Gossypieae).

BACKGROUND: Analysis of the relationship between chromosomal structural variation (synteny breaks) and 3D-chromatin architectural changes among closely related species has the potential to reveal causes and correlates between chromosomal change and chromatin remodeling. Of note, contrary to extensive studies in animal species, the pace and pattern of chromatin architectural changes following the speciation of plants remain unexplored; moreover, there is little exploration of the occurrence of synteny breaks in the context of multiple genome topological hierarchies within the same model species. RESULTS: Here we used Hi-C and epigenomic analyses to characterize and compare the profiles of hierarchical chromatin architectural features in representative species of the cotton tribe (Gossypieae), including Gossypium arboreum, Gossypium raimondii, and Gossypioides kirkii, which differ with respect to chromosome rearrangements. We found that (i) overall chromatin architectural territories were preserved in Gossypioides and Gossypium, which was reflected in their similar intra-chromosomal contact patterns and spatial chromosomal distributions; (ii) the non-random preferential occurrence of synteny breaks in A compartment significantly associate with the B-to-A compartment switch in syntenic blocks flanking synteny breaks; (iii) synteny changes co-localize with open-chromatin boundaries of topologically associating domains, while TAD stabilization has a greater influence on regulating orthologous expression divergence than do rearrangements; and (iv) rearranged chromosome segments largely maintain ancestral in-cis interactions. CONCLUSIONS: Our findings provide insights into the non-random occurrence of epigenomic remodeling relative to the genomic landscape and its evolutionary and functional connections to alterations of hierarchical chromatin architecture, on a known evolutionary timescale.

The Gossypium herbaceum L. Wagad genome as a resource for understanding cotton domestication.

Gossypium herbaceum is a species of cotton native to Africa and Asia that is one of the 2 domesticated diploids. Together with its sister-species G. arboreum, these A-genome taxa represent models of the extinct A-genome donor of modern polyploid cotton, which provide about 95% of cotton grown worldwide. As part of a larger effort to characterize variation and improve resources among diverse diploid and polyploid cotton genomes, we sequenced and assembled the genome of G. herbaceum cultivar (cv.) Wagad, representing the first domesticated accession for this species. This chromosome-level genome was generated using a combination of PacBio long-read technology, HiC, and Bionano optical mapping and compared to existing genome sequences in cotton. We compare the genome of this cultivar to the existing genome of wild G. herbaceum subspecies africanum to elucidate changes in the G. herbaceum genome concomitant with domestication and extend these analyses to gene expression using available RNA-seq. Our results demonstrate the utility of the G. herbaceum cv. Wagad genome in understanding domestication in the diploid species, which could inform modern breeding programs.

The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum).

Zanthoxylum armatum and Zanthoxylum bungeanum, known as 'Chinese pepper', are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.

Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

Dual Domestication, Diversity, and Differential Introgression in Old World Cotton Diploids.

Domestication in the cotton genus is remarkable in that it has occurred independently four different times at two different ploidy levels. Relatively little is known about genome evolution and domestication in the cultivated diploid species Gossypium herbaceum and Gossypium arboreum, due to the absence of wild representatives for the latter species, their ancient domestication, and their joint history of human-mediated dispersal and interspecific gene flow. Using in-depth resequencing of a broad sampling from both species, we provide support for their independent domestication, as opposed to a progenitor-derivative relationship, showing that diversity (mean π = 6 × 10-3) within species is similar, and that divergence between species is modest (FST = 0.413). Individual accessions were homozygous for ancestral single-nucleotide polymorphisms at over half of variable sites, while fixed, derived sites were at modest frequencies. Notably, two chromosomes with a paucity of fixed, derived sites (i.e., chromosomes 7 and 10) were also strongly implicated as having experienced high levels of introgression. Collectively, these data demonstrate variable permeability to introgression among chromosomes, which we propose is due to divergent selection under domestication and/or the phenomenon of F2 breakdown in interspecific crosses. Our analyses provide insight into the evolutionary forces that shape diversity and divergence in the diploid cultivated species and establish a foundation for understanding the contribution of introgression and/or strong parallel selection to the extensive morphological similarities shared between species.

Compensatory Genetic and Transcriptional Cytonuclear Coordination in Allopolyploid Lager Yeast (Saccharomyces pastorianus).

Cytonuclear coordination between biparental-nuclear genomes and uniparental-cytoplasmic organellar genomes in plants is often resolved by genetic and transcriptional cytonuclear responses. Whether this mechanism also acts in allopolyploid members of other kingdoms is not clear. Additionally, cytonuclear coordination of interleaved allopolyploid cells/individuals within the same population is underexplored. The yeast Saccharomyces pastorianus provides the opportunity to explore cytonuclear coevolution during different growth stages and from novel dimensions. Using S. pastorianus cells from multiple growth stages in the same environment, we show that nuclear mitochondria-targeted genes have undergone both asymmetric gene conversion and growth stage-specific biased expression favoring genes from the mitochondrial genome donor (Saccharomyces eubayanus). Our results suggest that cytonuclear coordination in allopolyploid lager yeast species entails an orchestrated and compensatory genetic and transcriptional evolutionary regulatory shift. The common as well as unique properties of cytonuclear coordination underlying allopolyploidy between unicellular yeasts and higher plants offers novel insights into mechanisms of cytonuclear evolution associated with allopolyploid speciation.

Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons.

Allotetraploid cotton (Gossypium) species represents a model system for the study of plant polyploidy, molecular evolution, and domestication. Here, chromosome-scale genome sequences were obtained and assembled for two recently described wild species of tetraploid cotton, Gossypium ekmanianum [(AD)6, Ge] and Gossypium stephensii [(AD)7, Gs], and one early form of domesticated Gossypium hirsutum, race punctatum [(AD)1, Ghp]. Based on phylogenomic analysis, we provide a dated whole-genome level perspective for the evolution of the tetraploid Gossypium clade and resolved the evolutionary relationships of Gs, Ge, and domesticated G. hirsutum. We describe genomic structural variation that arose during Gossypium evolution and describe its correlates-including phenotypic differentiation, genetic isolation, and genetic convergence-that contributed to cotton biodiversity and cotton domestication. Presence/absence variation is prominent in causing cotton genomic structural variations. A presence/absence variation-derived gene encoding a phosphopeptide-binding protein is implicated in increasing fiber length during cotton domestication. The relatively unimproved Ghp offers the potential for gene discovery related to adaptation to environmental challenges. Expanded gene families enoyl-CoA δ isomerase 3 and RAP2-7 may have contributed to abiotic stress tolerance, possibly by targeting plant hormone-associated biochemical pathways. Our results generate a genomic context for a better understanding of cotton evolution and for agriculture.

Variation in cytonuclear expression accommodation among allopolyploid plants.

Cytonuclear coevolution is a common feature among plants, which coordinates gene expression and protein products between the nucleus and organelles. Consequently, lineage-specific differences may result in incompatibilities between the nucleus and cytoplasm in hybrid taxa. Allopolyploidy is also a common phenomenon in plant evolution. The hybrid nature of allopolyploids may result in cytonuclear incompatibilities, but the massive nuclear redundancy created during polyploidy affords additional avenues for resolving cytonuclear conflict (i.e. cytonuclear accommodation). Here we evaluate expression changes in organelle-targeted nuclear genes for 6 allopolyploid lineages that represent 4 genera (i.e. Arabidopsis, Arachis, Chenopodium, and Gossypium) and encompass a range in polyploid ages. Because incompatibilities between the nucleus and cytoplasm could potentially result in biases toward the maternal homoeolog and/or maternal expression level, we evaluate patterns of homoeolog usage, expression bias, and expression-level dominance in cytonuclear genes relative to the background of noncytonuclear expression changes and to the diploid parents. Although we find subsets of cytonuclear genes in most lineages that match our expectations of maternal preference, these observations are not consistent among either allopolyploids or categories of organelle-targeted genes. Our results indicate that cytonuclear expression evolution may be subtle and variable among genera and genes, likely reflecting a diversity of mechanisms to resolve nuclear-cytoplasmic incompatibilities in allopolyploid species.

A temporal gradient of cytonuclear coordination of chaperonins and chaperones during RuBisCo biogenesis in allopolyploid plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) has long been studied from many perspectives. As a multisubunit (large subunits [LSUs] and small subunits[SSUs]) protein encoded by genes residing in the chloroplast (rbcL) and nuclear (rbcS) genomes, RuBisCo also is a model for cytonuclear coevolution following allopolyploid speciation in plants. Here, we studied the genomic and transcriptional cytonuclear coordination of auxiliary chaperonin and chaperones that facilitate RuBisCo biogenesis across multiple natural and artificially synthesized plant allopolyploids. We found similar genomic and transcriptional cytonuclear responses, including respective paternal-to-maternal conversions and maternal homeologous biased expression, in chaperonin/chaperon-assisted folding and assembly of RuBisCo in different allopolyploids. One observation is about the temporally attenuated genomic and transcriptional cytonuclear evolutionary responses during early folding and later assembly process of RuBisCo biogenesis, which were established by long-term evolution and immediate onset of allopolyploidy, respectively. Our study not only points to the potential widespread and hitherto unrecognized features of cytonuclear evolution but also bears implications for the structural interaction interface between LSU and Cpn60 chaperonin and the functioning stage of the Raf2 chaperone.

Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels.

Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA sequencing (RNA-seq) to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9 to 82.3%) of messenger RNA (mRNA) transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3 to 3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than 10-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.