Ran Hovav

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60u2009million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1–2 Myr ago, conferred about 30–36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum AtDt (in which ‘t’ indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

Partitioned expression of duplicated genes during development and evolution of a single cell in a polyploid plant

Polyploidy is an important driver of eukaryotic evolution, evident in many animals, fungi, and plants. One consequence of polyploidy is subfunctionalization, in which the ancestral expression profile becomes partitioned among duplicated genes (termed homoeologs). Subfunctionalization appears to be a common phenomenon insofar as it has been studied, at the scale of organs. Here, we use a high-resolution methodology to investigate the expression of thousands of pairs of homoeologs during the development of a single plant cell, using as a model the seed trichomes (“cotton fiber”) of allopolyploid (containing “A” and “D” genomes) cotton (Gossypium). We demonstrate that ≈30% of the homoeologs are significantly A- or D-biased at each of three time points studied during fiber development. Genes differentially biased toward the A or D genome belong to different biological processes, illustrating the functional partitioning of genomic contributions during cellular development. Interestingly, expression of the biased genes was shifted strongly toward the agronomically inferior D genome. Analyses of homoeologous gene expression during development of this cell showed that one-fifth of the genes exhibit changes in A/D ratios, indicating that significant alteration in duplicated gene expression is fairly frequent even at the level of development and maturation of a single cell. Comparing changes in homoeolog expression in cultivated versus wild cotton showed that most homoeolog expression bias reflects polyploidy rather than domestication. Evidence suggests, however, that domestication may increase expression bias in fibers toward the D genome, potentially implicating D-genome recruitment under human selection during domestication.

The Evolution of Spinnable Cotton Fiber Entailed Prolonged Development and a Novel Metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ∼22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.

A majority of cotton genes are expressed in single-celled fiber

Multicellular eukaryotes contain a diversity of cell types, presumably differing from one another in the suite of genes expressed during development. At present, little is known about the proportion of the genome transcribed in most cell types, nor the degree to which global patterns of expression change during cellular differentiation. To address these questions in a model plant system, we studied the unique and highly exaggerated single-celled, epidermal seed trichomes (“cotton”) of cultivated cotton (Gossypium hirsutum). By taking advantage of advances in expression profiling and microarray technology, we evaluated the transcriptome of cotton fibers across a developmental time-course, from a few days post-anthesis through primary and secondary wall synthesis stages. Comparisons of gene expression in populations of developing cotton fiber cells to genetically complex reference samples derived from 6 different cotton organs demonstrated that a remarkably high proportion of the cotton genome is transcribed, with 75–94% of the total genome transcribed at each stage. Compared to the reference samples, more than half of all genes were up-regulated during at least one stage of fiber development. These genes were clustered into seven groups of expression profiles that provided new insight into biological processes governing fiber development. Genes implicated in vesicle coating and trafficking were found to be overexpressed throughout all stages of fiber development studied, indicating their important role in maintaining rapid growth of this unique plant cell.

Gene expression in developing fibres of Upland cotton (Gossypium hirsutum L.) was massively altered by domestication

BackgroundUnderstanding the evolutionary genetics of modern crop phenotypes has a dual relevance to evolutionary biology and crop improvement. Modern upland cotton (Gossypium hirsutum L.) was developed following thousands of years of artificial selection from a wild form, G. hirsutum var. yucatanense, which bears a shorter, sparser, layer of single-celled, ovular trichomes (fibre). In order to gain an insight into the nature of the developmental genetic transformations that accompanied domestication and crop improvement, we studied the transcriptomes of cotton fibres from wild and domesticated accessions over a developmental time course.ResultsFibre cells were harvested between 2 and 25 days post-anthesis and encompassed the primary and secondary wall synthesis stages. Using amplified messenger RNA and a custom microarray platform designed to interrogate expression for 40,430 genes, we determined global patterns of expression during fibre development. The fibre transcriptome of domesticated cotton is far more dynamic than that of wild cotton, with over twice as many genes being differentially expressed during development (12,626 versus 5273). Remarkably, a total of 9465 genes were diagnosed as differentially expressed between wild and domesticated fibres when summed across five key developmental time points. Human selection during the initial domestication and subsequent crop improvement has resulted in a biased upregulation of components of the transcriptional network that are important for agronomically advanced fibre, especially in the early stages of development. About 15% of the differentially expressed genes in wild versus domesticated cotton fibre have no homology to the genes in databases.ConclusionsWe show that artificial selection during crop domestication can radically alter the transcriptional developmental network of even a single-celled structure, affecting nearly a quarter of the genes in the genome. Gene expression during fibre development within accessions and expression alteration arising from evolutionary change appears to be modular - complex genic networks have been simultaneously and similarly transformed, in a coordinated fashion, as a consequence of human-mediated selection. These results highlight the complex alteration of the global gene expression machinery that resulted from human selection for a longer, stronger and finer fibre, as well as other aspects of fibre physiology that were not consciously selected. We illustrate how the data can be mined for genes that were unwittingly targeted by aboriginal and/or modern domesticators during crop improvement and/or which potentially control the improved qualities of domesticated cotton fibre.See Commentary: http://www.biomedcentral.com/1741-7007/8/137

Global analysis of gene expression in cotton fibers from wild and domesticated Gossypium barbadense

SUMMARY Gossypium barbadense is widely cultivated because of its extra‐long staple cotton with superior luster, silkiness and high yield. These economically important traits were selected during initial domestication of an agronomically inferior wild ancestor, followed by millennia of human‐mediated selection. To reveal the effects of this history on the cotton fiber transcriptome, we conducted comparative expression profiling on mechanically isolated fiber cells at three different stages encompassing early, mid, and late fiber elongation in wild (K101) and domesticated (Pima S‐7) accessions, using a microarray platform that interrogates 42,429 unigenes. The distribution of differentially expressed genes across developmental stages was different in the two accessions, with a shift toward greater change earlier in cultivated than in wild G. barbadense. Approximately 4200 genes were differentially expressed between wild and domesticated accessions at one or more of the stages studied. Domestication appears to have led to enhanced modulation of cellular redox levels and the avoidance or delay of stress‐like processes. Prolonged fiber growth in cultivated relative to wild G. barbadense is associated with upregulation of signal transduction and hormone signaling genes and down‐regulation of cell wall maturation genes. Clues are provided into the processes and genes that may unwittingly have been selected by humans during domestication and development of modern elite lines. Several of the transcriptomic differences between wild and domesticated G. barbadense described here appear to have parallels in a second domesticated cotton species, Gossypium hirsutum, suggesting that replicated domestication of two different species has resulted in overlapping, parallel, metabolic transformations.

Parallel Domestication, Convergent Evolution and Duplicated Gene Recruitment in Allopolyploid Cotton

A putative advantage of allopolyploidy is the possibility of differential selection of duplicated (homeologous) genes originating from two different progenitor genomes. In this note we explore this hypothesis using a high throughput, SNP-specific microarray technology applied to seed trichomes (cotton) harvested from three developmental time points in wild and modern accessions of two independently domesticated cotton species, Gossypium hirsutum and G. barbadense. We show that homeolog expression ratios are dynamic both developmentally and over the several-thousand-year period encompassed by domestication and crop improvement, and that domestication increased the modulation of homeologous gene expression. In both species, D-genome expression was preferentially enhanced under human selection pressure, but for nonoverlapping sets of genes for the two independent domestication events. Our data suggest that human selection may have operated on different components of the fiber developmental genetic program in G. hirsutum and G. barbadense, leading to convergent rather than parallel genetic alterations and resulting morphology.

Parallel expression evolution of oxidative stress-related genes in fiber from wild and domesticated diploid and polyploid cotton (Gossypium)

BackgroundReactive oxygen species (ROS) play a prominent role in signal transduction and cellular homeostasis in plants. However, imbalances between generation and elimination of ROS can give rise to oxidative stress in growing cells. Because ROS are important to cell growth, ROS modulation could be responsive to natural or human-mediated selection pressure in plants. To study the evolution of oxidative stress related genes in a single plant cell, we conducted comparative expression profiling analyses of the elongated seed trichomes (fibers) of cotton (Gossypium), using a phylogenetic approach.ResultsWe measured expression changes during diploid progenitor species divergence, allopolyploid formation and parallel domestication of diploid and allopolyploid species, using a microarray platform that interrogates 42,429 unigenes. The distribution of differentially expressed genes in progenitor diploid species revealed significant up-regulation of ROS scavenging and potential signaling processes in domesticated G. arboreum. Similarly, in two independently domesticated allopolyploid species (G. barbadense and G. hirsutum) antioxidant genes were substantially up-regulated in comparison to antecedent wild forms. In contrast, analyses of three wild allopolyploid species indicate that genomic merger and ancient allopolyploid formation had no significant influences on regulation of ROS related genes. Remarkably, many of the ROS-related processes diagnosed as possible targets of selection were shared among diploid and allopolyploid cultigens, but involved different sets of antioxidant genes.ConclusionOur data suggests that parallel human selection for enhanced fiber growth in several geographically widely dispersed species of domesticated cotton resulted in similar and overlapping metabolic transformations of the manner in which cellular redox levels have become modulated.

Spotted cotton oligonucleotide microarrays for gene expression analysis

BackgroundMicroarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on three Gossypium species. Using these assemblies as templates, we describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species.ResultsSynthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included on the arrays, optimized to target the diversity of the transcriptome and previously studied cotton genes, transcription factors, and genes with homology to Arabidopsis. A small portion of the oligonucleotides target unidentified protein coding sequences, thereby providing an element of gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA libraries, the microarray has direct application for analysis of the fiber transcriptome. To illustrate the utility of the microarray, we hybridized labeled bud and leaf cDNAs from G. hirsutum and demonstrate technical consistency of results.ConclusionThe cotton oligonucleotide microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info.

A Transcriptome Profile for Developing Seed of Polyploid Cotton

Cotton ranks among the worlds important oilseed crops, yet relative to other oilseeds there are few studies of oil‐related biosynthetic and regulatory pathways. We present global transcriptome analyses of cotton seed development using RNA‐seq and four developmental time‐points. Because Upland cotton (Gossypium hirsutum L.) is an allopolyploid containing two genomes (A/D), we partitioned expression into the individual contributions of each homeologous gene copy. Data were explored with respect to genic and subgenomic patterns of expression, globally and with respect to seed pathways and networks. The most dynamic period of transcriptome change is from 20–30 d postanthesis (DPA), with about 20% of genes showing homeolog expression bias. Co‐expression analysis shows largely congruent homeolog networks, but also homeolog‐specific divergence. Functional enrichment tests show that flavonoid biosynthesis and lipid related genes were significantly represented early and later in seed development, respectively. An involvement of new features in oil biosynthesis was found, like the contribution of DGAT3 (diacylglycerol acyltransferase) to the total triglyceride expression pool. Also, catechin‐based and epicatechin‐based proanthocyanidin expression are reciprocally biased with respect to homeolog usage. This study provides the first temporal analysis of duplicated gene expression in cotton seed and a resource for understanding new aspects of oil and flavonoid biosynthetic processes.