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.

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.

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.

Deleterious Mutations Accumulate Faster in Allopolyploid Than Diploid Cotton (Gossypium) and Unequally between Subgenomes.

Whole-genome duplication (polyploidization) is among the most dramatic mutational processes in nature, so understanding how natural selection differs in polyploids relative to diploids is an important goal. Population genetics theory predicts that recessive deleterious mutations accumulate faster in allopolyploids than diploids due to the masking effect of redundant gene copies, but this prediction is hitherto unconfirmed. Here, we use the cotton genus (Gossypium), which contains seven allopolyploids derived from a single polyploidization event 1-2 Million years ago, to investigate deleterious mutation accumulation. We use two methods of identifying deleterious mutations at the nucleotide and amino acid level, along with whole-genome resequencing of 43 individuals spanning six allopolyploid species and their two diploid progenitors, to demonstrate that deleterious mutations accumulate faster in allopolyploids than in their diploid progenitors. We find that, unlike what would be expected under models of demographic changes alone, strongly deleterious mutations show the biggest difference between ploidy levels, and this effect diminishes for moderately and mildly deleterious mutations. We further show that the proportion of nonsynonymous mutations that are deleterious differs between the two coresident subgenomes in the allopolyploids, suggesting that homoeologous masking acts unequally between subgenomes. Our results provide a genome-wide perspective on classic notions of the significance of gene duplication that likely are broadly applicable to allopolyploids, with implications for our understanding of the evolutionary fate of deleterious mutations. Finally, we note that some measures of selection (e.g., dN/dS, πN/πS) may be biased when species of different ploidy levels are compared.

Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number.

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication (WGD) events in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follows WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated the organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages.

Parallel and Intertwining Threads of Domestication in Allopolyploid Cotton.

The two cultivated allopolyploid cottons, Gossypium hirsutum and Gossypium barbadense, represent a remarkable example of parallel independent domestication, both involving dramatic morphological transformations under selection from wild perennial plants to annualized row crops. Deep resequencing of 643 newly sampled accessions spanning the wild-to-domesticated continuum of both species, and their allopolyploid relatives, are combined with existing data to resolve species relationships and elucidate multiple aspects of their parallel domestication. It is confirmed that wild G. hirsutum and G. barbadense were initially domesticated in the Yucatan Peninsula and NW South America, respectively, and subsequently spread under domestication over 4000-8000 years to encompass most of the American tropics. A robust phylogenomic analysis of infraspecific relationships in each species is presented, quantify genetic diversity in both, and describe genetic bottlenecks associated with domestication and subsequent diffusion. As these species became sympatric over the last several millennia, pervasive genome-wide bidirectional introgression occurred, often with striking asymmetries involving the two co-resident genomes of these allopolyploids. Diversity scans revealed genomic regions and genes unknowingly targeted during domestication and additional subgenomic asymmetries. These analyses provide a comprehensive depiction of the origin, divergence, and adaptation of cotton, and serve as a rich resource for cotton improvement.

pSONIC: Ploidy-aware Syntenic Orthologous Networks Identified via Collinearity.

With the rapid rise in availability of high-quality genomes for closely related species, methods for orthology inference that incorporate synteny are increasingly useful. Polyploidy perturbs the 1:1 expected frequencies of orthologs between two species, complicating the identification of orthologs. Here we present a method of ortholog inference, Ploidy-aware Syntenic Orthologous Networks Identified via Collinearity (pSONIC). We demonstrate the utility of pSONIC using four species in the cotton tribe (Gossypieae), including one allopolyploid, and place between 75% and 90% of genes from each species into nearly 32,000 orthologous groups, 97% of which consist of at most singletons or tandemly duplicated genes-58.8% more than comparable methods that do not incorporate synteny. We show that 99% of singleton gene groups follow the expected tree topology and that our ploidy-aware algorithm recovers 97.5% identical groups when compared to splitting the allopolyploid into its two respective subgenomes, treating each as separate "species."

Genomic diversifications of five Gossypium allopolyploid species and their impact on cotton improvement.

Polyploidy is an evolutionary innovation for many animals and all flowering plants, but its impact on selection and domestication remains elusive. Here we analyze genome evolution and diversification for all five allopolyploid cotton species, including economically important Upland and Pima cottons. Although these polyploid genomes are conserved in gene content and synteny, they have diversified by subgenomic transposon exchanges that equilibrate genome size, evolutionary rate heterogeneities and positive selection between homoeologs within and among lineages. These differential evolutionary trajectories are accompanied by gene-family diversification and homoeolog expression divergence among polyploid lineages. Selection and domestication drive parallel gene expression similarities in fibers of two cultivated cottons, involving coexpression networks and N6-methyladenosine RNA modifications. Furthermore, polyploidy induces recombination suppression, which correlates with altered epigenetic landscapes and can be overcome by wild introgression. These genomic insights will empower efforts to manipulate genetic recombination and modify epigenetic landscapes and target genes for crop improvement.

Genetic Analysis of the Transition from Wild to Domesticated Cotton (Gossypium hirsutum L.).

The evolution and domestication of cotton is of great interest from both economic and evolutionary standpoints. Although many genetic and genomic resources have been generated for cotton, the genetic underpinnings of the transition from wild to domesticated cotton remain poorly known. Here we generated an intraspecific QTL mapping population specifically targeting domesticated cotton phenotypes. We used 466 F2 individuals derived from an intraspecific cross between the wild Gossypium hirsutum var. yucatanense (TX2094) and the elite cultivar G. hirsutum cv. Acala Maxxa, in two environments, to identify 120 QTL associated with phenotypic changes under domestication. While the number of QTL recovered in each subpopulation was similar, only 22 QTL were considered coincident (i.e., shared) between the two locations, eight of which shared peak markers. Although approximately half of QTL were located in the A-subgenome, many key fiber QTL were detected in the D-subgenome, which was derived from a species with unspinnable fiber. We found that many QTL are environment-specific, with few shared between the two environments, indicating that QTL associated with G. hirsutum domestication are genomically clustered but environmentally labile. Possible candidate genes were recovered and are discussed in the context of the phenotype. We conclude that the evolutionary forces that shape intraspecific divergence and domestication in cotton are complex, and that phenotypic transformations likely involved multiple interacting and environmentally responsive factors.

The Genome Sequence of Gossypioides kirkii Illustrates a Descending Dysploidy in Plants.

One of the extraordinary aspects of plant genome evolution is variation in chromosome number, particularly that among closely related species. This is exemplified by the cotton genus (Gossypium) and its relatives, where most species and genera have a base chromosome number of 13. The two exceptions are sister genera that have n = 12 (the Hawaiian Kokia and the East African and Madagascan Gossypioides). We generated a high-quality genome sequence of Gossypioides kirkii (n = 12) using PacBio, Bionano, and Hi-C technologies, and compared this assembly to genome sequences of Kokia (n = 12) and Gossypium diploids (n = 13). Previous analysis demonstrated that the directionality of their reduced chromosome number was through large structural rearrangements. A series of structural rearrangements were identified comparing the de novo G. kirkii genome sequence to genome sequences of Gossypium, including chromosome fusions and inversions. Genome comparison between G. kirkii and Gossypium suggests that multiple steps are required to generate the extant structural differences.

De Novo Genome Sequence Assemblies of Gossypium raimondii and Gossypium turneri.

Cotton is an agriculturally important crop. Because of its importance, a genome sequence of a diploid cotton species (Gossypium raimondii, D-genome) was first assembled using Sanger sequencing data in 2012. Improvements to DNA sequencing technology have improved accuracy and correctness of assembled genome sequences. Here we report a new de novo genome assembly of G. raimondii and its close relative G. turneri The two genomes were assembled to a chromosome level using PacBio long-read technology, HiC, and Bionano optical mapping. This report corrects some minor assembly errors found in the Sanger assembly of G. raimondii We also compare the genome sequences of these two species for gene composition, repetitive element composition, and collinearity. Most of the identified structural rearrangements between these two species are due to intra-chromosomal inversions. More inversions were found in the G. turneri genome sequence than the G. raimondii genome sequence. These findings and updates to the D-genome sequence will improve accuracy and translation of genomics to cotton breeding and genetics.

Cytonuclear Coevolution following Homoploid Hybrid Speciation in Aegilops tauschii.

The diploid D-genome lineage of the Triticum/Aegilops complex has an evolutionary history involving genomic contributions from ancient A- and B/S-genome species. We explored here the possible cytonuclear evolutionary responses to this history of hybridization. Phylogenetic analysis of chloroplast DNAs indicates that the D-genome lineage has a maternal origin of the A-genome or some other closely allied lineage. Analyses of the nuclear genome in the D-genome species Aegilops tauschii indicate that accompanying and/or following this ancient hybridization, there has been biased maintenance of maternal A-genome ancestry in nuclear genes encoding cytonuclear enzyme complexes (CECs). Our study provides insights into mechanisms of cytonuclear coevolution accompanying the evolution and eventual stabilization of homoploid hybrid species. We suggest that this coevolutionary process includes likely rapid fixation of A-genome CEC orthologs as well as biased retention of A-genome nucleotides in CEC homologs following population level recombination during the initial generations.

Insights into the Evolution of the New World Diploid Cottons (Gossypium, Subgenus Houzingenia) Based on Genome Sequencing.

We employed phylogenomic methods to study molecular evolutionary processes and phylogeny in the geographically widely dispersed New World diploid cottons (Gossypium, subg. Houzingenia). Whole genome resequencing data (average of 33× genomic coverage) were generated to reassess the phylogenetic history of the subgenus and provide a temporal framework for its diversification. Phylogenetic analyses indicate that the subgenus likely originated following transoceanic dispersal from Africa about 6.6 Ma, but that nearly all of the biodiversity evolved following rapid diversification in the mid-Pleistocene (0.5-2.0 Ma), with multiple long-distance dispersals required to account for range expansion to Arizona, the Galapagos Islands, and Peru. Comparative analyses of cpDNAversus nuclear data indicate that this history was accompanied by several clear cases of interspecific introgression. Repetitive DNAs contribute roughly half of the total 880 Mb genome, but most transposable element families are relatively old and stable among species. In the genic fraction, pairwise synonymous mutation rates average 1% per Myr, with nonsynonymous changes being about seven times less frequent. Over 1.1 million indels were detected and phylogenetically polarized, revealing a 2-fold bias toward deletions over small insertions. We suggest that this genome down-sizing bias counteracts genome size growth by TE amplification and insertions, and helps explain the relatively small genomes that are restricted to this subgenus. Compared with the rate of nucleotide substitution, the rate of indel occurrence is much lower averaging about 17 nucleotide substitutions per indel event.

A Malvaceae mystery: A mallow maelstrom of genome multiplications and maybe misleading methods?

Previous research suggests that Gossypium has undergone a 5- to 6-fold multiplication following its divergence from Theobroma. However, the number of events, or where they occurred in the Malvaceae phylogeny remains unknown. We analyzed transcriptomic and genomic data from representatives of eight of the nine Malvaceae subfamilies. Phylogenetic analysis of nuclear data placed Dombeya (Dombeyoideae) as sister to the rest of Malvadendrina clade, but the plastid DNA tree strongly supported Durio (Helicteroideae) in this position. Intraspecific Ks plots indicated that all sampled taxa, except Theobroma (Byttnerioideae), Corchorus (Grewioideae), and Dombeya (Dombeyoideae), have experienced whole genome multiplications (WGMs). Quartet analysis suggested WGMs were shared by Malvoideae-Bombacoideae and Sterculioideae-Tilioideae, but did not resolve whether these are shared with each other or Helicteroideae (Durio). Gene tree reconciliation and Bayesian concordance analysis suggested a complex history. Alternative hypotheses are suggested, each involving two independent autotetraploid and one allopolyploid event. They differ in that one entails an allopolyploid origin for the Durio lineage, whereas the other invokes an allopolyploid origin for Malvoideae-Bombacoideae. We highlight the need for more genomic information in the Malvaceae and improved methods to resolve complex evolutionary histories that may include allopolyploidy, incomplete lineage sorting, and variable rates of gene and genome evolution.

Comparative Genomics of an Unusual Biogeographic Disjunction in the Cotton Tribe (Gossypieae) Yields Insights into Genome Downsizing.

Long-distance insular dispersal is associated with divergence and speciation because of founder effects and strong genetic drift. The cotton tribe (Gossypieae) has experienced multiple transoceanic dispersals, generating an aggregate geographic range that encompasses much of the tropics and subtropics worldwide. Two genera in the Gossypieae, Kokia and Gossypioides, exhibit a remarkable geographic disjunction, being restricted to the Hawaiian Islands and Madagascar/East Africa, respectively. We assembled and use de novo genome sequences to address questions regarding the divergence of these two genera from each other and from their sister-group, Gossypium. In addition, we explore processes underlying the genome downsizing that characterizes Kokia and Gossypioides relative to other genera in the tribe. Using 13,000 gene orthologs and synonymous substitution rates, we show that the two disjuncts last shared a common ancestor ∼5 Ma, or half as long ago as their divergence from Gossypium. We report relative stasis in the transposable element fraction. In comparison to Gossypium, there is loss of ∼30% of the gene content in the two disjunct genera and a history of genome-wide accumulation of deletions. In both genera, there is a genome-wide bias toward deletions over insertions, and the number of gene losses exceeds the number of gains by ∼2- to 4-fold. The genomic analyses presented here elucidate genomic consequences of the demographic and biogeographic history of these closest relatives of Gossypium, and enhance their value as phylogenetic outgroups.