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.

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.

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.

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.

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.

A high-quality chromosome-level genome assembly of rohu carp, Labeo rohita, and its utilization in SNP-based exploration of gene flow and sex determination.

Labeo rohita (rohu) is a carp important to aquaculture in South Asia, with a production volume close to Atlantic salmon. While genetic improvements to rohu are ongoing, the genomic methods commonly used in other aquaculture improvement programs have historically been precluded in rohu, partially due to the lack of a high-quality reference genome. Here we present a high-quality de novo genome produced using a combination of next-generation sequencing technologies, resulting in a 946 Mb genome consisting of 25 chromosomes and 2,844 unplaced scaffolds. Notably, while approximately half the size of the existing genome sequence, our genome represents 97.9% of the genome size newly estimated here using flow cytometry. Sequencing from 120 individuals was used in conjunction with this genome to predict the population structure, diversity, and divergence in three major rivers (Jamuna, Padma, and Halda), in addition to infer a likely sex determination mechism in rohu. These results demonstrate the utility of the new rohu genome in modernizing some aspects of rohu genetic improvement programs.

Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification.

We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

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.

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.

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.

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.

Parental legacy versus regulatory innovation in salt stress responsiveness of allopolyploid cotton (Gossypium) species.

Polyploidy provides an opportunity for evolutionary innovation and species diversification, especially under stressful conditions. In allopolyploids, the conditional dynamics of homoeologous gene expression can be either inherited from ancestral states pre-existing in the parental diploids or novel upon polyploidization, the latter potentially permitting a wider range of phenotypic responses to stresses. To gain insight into regulatory mechanisms underlying the diversity of salt resistance in Gossypium species, we compared global transcriptomic responses to modest salinity stress in two allotetraploid (AD-genome) cotton species, Gossypium hirsutum and G. mustelinum, relative to their model diploid progenitors (A-genome and D-genome). Multivariate and pairwise analyses of salt-responsive changes revealed a profound alteration of gene expression for about one third of the transcriptome. Transcriptional responses and associated functional implications of salt acclimation varied across species, as did species-specific coexpression modules among species and ploidy levels. Salt responsiveness in both allopolyploids was strongly biased toward the D-genome progenitor. A much lower level of transgressive downregulation was observed in the more salt-tolerant G. mustelinum than in the less tolerant G. hirsutum. By disentangling inherited effects from evolved responses, we show that expression biases that are not conditional upon salt stress approximately equally reflect parental legacy and regulatory novelty upon allopolyploidization, whereas stress-responsive biases are predominantly novel, or evolved, in allopolyploids. Overall, our work suggests that allopolyploid cottons acquired a wide range of stress response flexibility relative to their diploid ancestors, most likely mediated by complex suites of duplicated genes and regulatory factors.

Global Patterns of Subgenome Evolution in Organelle-Targeted Genes of Six Allotetraploid Angiosperms.

Whole-genome duplications (WGDs) are a prominent process of diversification in eukaryotes. The genetic and evolutionary forces that WGD imposes on cytoplasmic genomes are not well understood, despite the central role that cytonuclear interactions play in eukaryotic function and fitness. Cellular respiration and photosynthesis depend on successful interaction between the 3,000+ nuclear-encoded proteins destined for the mitochondria or plastids and the gene products of cytoplasmic genomes in multi-subunit complexes such as OXPHOS, organellar ribosomes, Photosystems I and II, and Rubisco. Allopolyploids are thus faced with the critical task of coordinating interactions between the nuclear and cytoplasmic genes that were inherited from different species. Because the cytoplasmic genomes share a more recent history of common descent with the maternal nuclear subgenome than the paternal subgenome, evolutionary "mismatches" between the paternal subgenome and the cytoplasmic genomes in allopolyploids might lead to the accelerated rates of evolution in the paternal homoeologs of allopolyploids, either through relaxed purifying selection or strong directional selection to rectify these mismatches. We report evidence from six independently formed allotetraploids that the subgenomes exhibit unequal rates of protein-sequence evolution, but we found no evidence that cytonuclear incompatibilities result in altered evolutionary trajectories of the paternal homoeologs of organelle-targeted genes. The analyses of gene content revealed mixed evidence for whether the organelle-targeted genes are lost more rapidly than the non-organelle-targeted genes. Together, these global analyses provide insights into the complex evolutionary dynamics of allopolyploids, showing that the allopolyploid subgenomes have separate evolutionary trajectories despite sharing the same nucleus, generation time, and ecological context.

Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wilt resistance.

Sea Island cotton (Gossypium barbadense) is the source of the world's finest fibre quality cotton, yet relatively little is understood about genetic variations among diverse germplasms, genes underlying important traits and the effects of pedigree selection. Here, we resequenced 336 G. barbadense accessions and identified 16 million SNPs. Phylogenetic and population structure analyses revealed two major gene pools and a third admixed subgroup derived from geographical dissemination and interbreeding. We conducted a genome-wide association study (GWAS) of 15 traits including fibre quality, yield, disease resistance, maturity and plant architecture. The highest number of associated loci was for fibre quality, followed by disease resistance and yield. Using gene expression analyses and VIGS transgenic experiments, we confirmed the roles of five candidate genes regulating four key traits, that is disease resistance, fibre length, fibre strength and lint percentage. Geographical and temporal considerations demonstrated selection for the superior fibre quality (fibre length and fibre strength), and high lint percentage in improving G. barbadense in China. Pedigree selection breeding increased Fusarium wilt disease resistance and separately improved fibre quality and yield. Our work provides a foundation for understanding genomic variation and selective breeding of Sea Island cotton.

Embryogenic Calli Induction and Salt Stress Response Revealed by RNA-Seq in Diploid Wild Species Gossypium sturtianum and Gossypium raimondii.

Wild cotton species can contribute to a valuable gene pool for genetic improvement, such as genes related to salt tolerance. However, reproductive isolation of different species poses an obstacle to produce hybrids through conventional breeding. Protoplast fusion technology for somatic cell hybridization provides an opportunity for genetic manipulation and targeting of agronomic traits. Transcriptome sequencing analysis of callus under salt stress is conducive to study salt tolerance genes. In this study, calli were induced to provide materials for extracting protoplasts and also for screening salt tolerance genes. Calli were successfully induced from leaves of Gossypium sturtianum (C1 genome) and hypocotyls of G. raimondii (D5 genome), and embryogenic calli of G. sturtianum and G. raimondii were induced on a differentiation medium with different concentrations of 2, 4-D, KT, and IBA, respectively. In addition, embryogenic calli were also induced successfully from G. raimondii through suspension cultivation. Transcriptome sequencing analysis was performed on the calli of G. raimondii and G. sturtianum, which were treated with 200 mM NaCl at 0, 6, 12, 24, and 48 h, and a total of 12,524 genes were detected with different expression patterns under salt stress. Functional analysis showed that 3,482 genes, which were differentially expressed in calli of G. raimondii and G. sturtianum, were associated with biological processes of nucleic acid binding, plant hormone (such as ABA) biosynthesis, and signal transduction. We demonstrated that DEGs or TFs which related to ABA metabolism were involved in the response to salt stress, including xanthoxin dehydrogenase genes (ABA2), sucrose non-fermenting 1-related protein kinases (SnRK2), NAM, ATAT1/2, and CUC2 transcription factors (NAC), and WRKY class of zinc-finger proteins (WRKY). This research has successfully induced calli from two diploid cotton species and revealed new genes responding to salt stress in callus tissue, which will lay the foundation for protoplast fusion for further understanding of salt stress responses in cotton.

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility.

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here, we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (Gossypium davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.