Polyploid formation in cotton is not accompanied by rapid genomic changes.

Recent work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis. To address the question of whether rapid genomic changes also occur in allopolyploid cotton (Gossypium) species, amplified fragment length polymorphism (AFLP) analysis was performed to evaluate nine sets of newly synthesized allotetraploid and allohexaploid plants, their parents, and the selfed progeny from colchicine-doubled synthetics. Using both methylation-sensitive and methylation-insensitive enzymes, the extent of fragment additivity in newly combined genomes was ascertained for a total of approximately 22,000 genomic loci. Fragment additivity was observed in nearly all cases, with the few exceptions most likely reflecting parental heterozygosity or experimental error. In addition, genomic Southern analysis on six sets of synthetic allopolyploids probed with five retrotransposons also revealed complete additivity. Because no alterations were observed using methylation-sensitive isoschizomers, epigenetic changes following polyploid synthesis were also minimal. These indications of genomic additivity and epigenetic stasis during allopolyploid formation provide a contrast to recent evidence from several model plant allopolyploids, most notably wheat and Brassica, where rapid and unexplained genomic changes have been reported. In addition, the data contrast with evidence from repetitive DNAs in Gossypium, some of which are subject to non-Mendelian molecular evolutionary phenomena in extant polyploids. These contrasts indicate polyploid speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa.     

The contribution of polyploidy to variation in Brassica species

The genus Brassica includes species with two levels of polyploidy: diploids that have replicated genomes and appear to be ancient polyploids, and allopolyploids that were recently derived from hybridization of the diploid species. Research on these species has provided evidence that polyploidy contributes to phenotypic variation through several mechanisms. Polyploidy increases the potential variation of dosage-regulated gene expression, and this mechanism appears to affect flowering time variation through the effects of replicated copies of the flowering time gene FLC . Homoeologous chromosome transpositions occur in allopolyploids that alter allele composition, and this has created novel flowering time variation in newly formed Brassica allopolyploids. New allopolyploids also may have epigenetic changes or altered regulatory interactions that affect gene expression and phenotypic variation. Continued research on Brassica and other species should provide insight into the relative importance of these mechanisms for generating novel variation in polyploids.     

Genetic and epigenetic interactions in allopolyploid plants

Allopolyploid plants are hybrids that contain two copies of the genome from each parent. Whereas wild and cultivated allopolyploids are well adapted, man-made allopolyploids are typically unstable, displaying homeotic transformation and lethality as well as chromosomal rearrangements and changes in the number and distribution of repeated DNA sequences within heterochromatin. Large increases in the length of some chromosomes has been documented in allopolyploid hybrids and could be caused by the activation of dormant retrotransposons, as shown to be the case in marsupial hybrids. Synthetic (man-made) allotetraploids of Arabidopsis exhibit rapid changes in gene regulation, including gene silencing. These regulatory abnormalities could derive from ploidy changes and/or incompatible interactions between parental genomes, although comparison of auto- and allopolyploids suggests that intergenomic incompatibilities play the major role. Models to explain intergenomic incompatibilities incorporate both genetic and epigenetic mechanisms. In one model, the activation of heterochromatic transposons (McClintock's genomic shock) may lead to widespread perturbation of gene expression, perhaps by a silencing interaction between activated transposons and euchromatic genes. Qualitatively similar responses, of lesser intensity, may occur in intraspecific hybrids. Therefore, insight into genome function gained from the study of allopolyploidy may be applicable to hybrids of any type and may even elucidate positive interactions, such as those responsible for hybrid vigor.     

Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops–Triticum) Group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.     

Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops-Triticum) group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and TRITICUM: In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.     

Nonadditive changes in genome size during allopolyploidization in the wheat (aegilops-triticum) group

Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes. Despite these studies, it is not yet clear whether the C value of an allopolyploid is the sum of its diploid parents. To address this question, six newly synthesized wheat allopolyploids and their parental plants were investigated. It was found that allopolyploids have a genome size significantly smaller than the expected value. The reduction of the nuclear genome size in the synthetic allotetraploids and allohexaploids was 2 pg DNA at 2C. It was also found that changes in the genome size already existed in the first generation amphiploids, indicating that the change was a rapid event. There was no difference in the reduction of nuclear genome size between the allotetraploid and the allohexaploid. These data clearly show that genome differentiation in allopolyploids was not related to the ploidy level. The data obtained clearly suggested that the nonadditive change in genome size that occurred during allopolyploidization may represent a preprogrammed adaptive response to genomic stress caused by hybridization and allopolyploidy, which serves to stabilize polyploid genomes.     

Nuclear DNA amount and genome downsizing in natural and synthetic allopolyploids of the genera Aegilops and Triticum

Recent molecular studies in the genera Aegilops and Triticum showed that allopolyploidization (interspecific or intergeneric hybridization followed by chromosome doubling) generated rapid elimination of low-copy or high-copy, non-coding and coding DNA sequences. The aims of this work were to determine the amount of nuclear DNA in allopolyploid species of the group and to see to what extent elimination of DNA sequences affected genome size. Nuclear DNA amount was determined by the flow cytometry method in 27 natural allopolyploid species (most of which were represented by several lines and each line by several plants) as well as 14 newly synthesized allopolyploids (each represented by several plants) and their parental plants. Very small intraspecific variation in DNA amount was found between lines of allopolyploid species collected from different habitats or between wild and domesticated forms of allopolyploid wheat. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various allopolyploid species, at both the tetraploid and hexaploid levels. In most allopolyploids nuclear DNA amount was significantly less than the sum of DNA amounts of the parental species. Newly synthesized allopolyploids exhibited a similar decrease in nuclear DNA amount in the first generation, indicating that genome downsizing occurs during and (or) immediately after the formation of the allopolyploids and that there are no further changes in genome size during the life of the allopolyploids. Phylogenetic considerations of the origin of the B genome of allopolyploid wheat, based on nuclear DNA amount, are discussed.     

Genome evolution in allopolyploid wheat-a revolutionary reprogramming followed by gradual changes

Allopolyploidy accelerates genome evolution in wheat in two ways： 1） allopolyploidization triggers rapid genome alterations （revolutionary changes） through the instantaneous generation of a variety of cardinal genetic and epigenetic changes, and 2） the allopolyploid condition facilitates sporadic genomic changes during the life of the species （evolutionary changes） that are not attainable at the diploid level. The revolutionary alterations, occurring during the formation of the allopolyploid and leading to rapid cytological and genetic diploidization, facilitate the successful establishment of the newly formed allopolyploid in nature. On the other hand, the evolutionary changes, occurring during the life of the allopolyploids, increase the intra-specific genetic diversity, and consequently, increased fimess, adaptability and competitiveness. These phenomena, emphasizing the dynamic plasticity of the allopolyploid wheat genome with regards to both structure and function, are described and discussed in this review.     

The legacy of diploid progenitors in allopolyploid gene expression patterns

Allopolyploidization (hybridization and whole-genome duplication) is a common phenomenon in plant evolution with immediate saltational effects on genome structure and gene expression. New technologies have allowed rapid progress over the past decade in our understanding of the consequences of allopolyploidy. A major question, raised by early pioneer of this field Leslie Gottlieb, concerned the extent to which gene expression differences among duplicate genes present in an allopolyploid are a legacy of expression differences that were already present in the progenitor diploid species. Addressing this question necessitates phylogenetically well-understood natural study systems, appropriate technology, availability of genomic resources and a suitable analytical framework, including a sufficiently detailed and generally accepted terminology. Here, we review these requirements and illustrate their application to a natural study system that Gottlieb worked on and recommended for this purpose: recent allopolyploids of Tragopogon (Asteraceae). We reanalyse recent data from this system within the conceptual framework of parental legacies on duplicate gene expression in allopolyploids. On a broader level, we highlight the intellectual connection between Gottlieb''s phrasing of this issue and the more contemporary framework of cis- versus trans-regulation of duplicate gene expression in allopolyploid plants.     

Nonadditive changes to cytosine methylation as a consequence of hybridization and genome duplication in Senecio (Asteraceae)

The merger of two or more divergent genomes within an allopolyploid nucleus can facilitate speciation and adaptive evolution in flowering plants. Widespread changes to gene expression have been shown to result from interspecific hybridisation and polyploidy in a number of plant species, and attention has now shifted to determining the epigenetic processes that drive these changes. We present here an analysis of cytosine methylation patterns in triploid F(1) Senecio (ragwort) hybrids and their allohexaploid derivatives. We observe that, in common with similar studies in Arabidopsis, Spartina and Triticum, a small but significant proportion of loci display nonadditive methylation in the hybrids, largely resulting from interspecific hybridisation. Despite this, genome duplication results in a secondary effect on methylation, with reversion to additivity at some loci and novel methylation status at others. We also observe differences in methylation state between different allopolyploid generations, predominantly in cases of additive methylation with regard to which parental methylation state is dominant. These changes to methylation state in both F(1) triploids and their allohexaploid derivatives largely mirror the overall patterns of nonadditive gene expression observed in our previous microarray analyses and may play a causative role in generating those expression changes. These similar global changes to DNA methylation resulting from hybridisation and genome duplication may serve as a source of epigenetic variation in natural populations, facilitating adaptive evolution. Our observations that methylation state can also vary between different generations of polyploid hybrids suggests that newly formed allopolyploid species may display a high degree of epigenetic diversity upon which natural selection can act.     

Rapid genomic changes in interspecific and intergeneric hybrids and allopolyploids of Triticeae.

Allopolyploidy is preponderant in plants, which often leads to speciation. Some recent studies indicate that the process of wide hybridization and (or) genome doubling may induce rapid and extensive genetic and epigenetic changes in some plant species and genomic stasis in others. To further study this phenomenon, we analyzed three sets of synthetic allopolyploids in the Triticeae by restriction fragment length polymorphism (RFLP) using a set of expressed sequence tags (ESTs) and retrotransposons as probes. It was found that 40-64.7% of the ESTs detected genomic changes in the three sets of allopolyploids. Changes included disappearance of parental hybridization fragment(s), simultaneous appearance of novel fragment(s) and loss of parental fragment(s), and appearance of novel fragment(s). Some of the changes occurred as early as in the F1 hybrid, whereas others occurred only after allopolyploid formation. Probing with retrotransposons revealed numerous examples of disappearance of sequences. No gross chromosome structural changes or physical elimination of sequences were found. It is suggested that DNA methylation and localized recombination at the DNA level were probably the main causes for the genomic changes. Possible implications of the genomic changes for allopolyploid genome evolution are discussed.     

A genetic appraisal of a new synthetic Nicotiana tabacum (Solanaceae) and the Kostoff synthetic tobacco

Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973) and Kostoff (1938) generated synthetic tobacco (N. tabacum) using the parents ♀N. sylvestris × ♂N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations. Two plants of TR1 (TR1-A, TR1-B) have the expected number, structure, and chromosomal distribution of rDNA families, in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopolyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy.     

Alterations in subtelomeric tandem repeats during early stages of allopolyploidy in wheat.

The genomic content of the subtelomeric repeated sequences Spelt1 and Spelt52 was studied by dot, Southern, and in situ hybridization in 11 newly synthesized amphiploids of Aegilops and Triticum, and data were compared with the parental plants. Spelt1 had reduced copy numbers in the first generation of three synthetic amphiploids, but two others did not change; Spelt52 was amplified in nine amphiploids and did not change in two. In the second allopolyploid generation, Spelt1 copy number did not change, whereas there was amplification of Spelt52 in some allopolyploids and decreases in others. Neither allopolyploidy level nor the direction of the cross affected the patterns of change in the newly synthesized amphiploids. Changes did not result from intergenomic recombination because similar alterations were noticed in allopolyploids with and without Ph1, a gene that suppresses homoeologous pairing. No differences in Spelt1 and Spelt52 tandem organization were found by Southern hybridization. The significance of these data are discussed in relation to the establishment of newly formed allopolyploids.     

Next-generation sequencing and genome evolution in allopolyploids

?Premise of the study: Hybridization and polyploidization (allopolyploidy) are ubiquitous in the evolution of plants, but tracing the origins and subsequent evolution of the constituent genomes of allopolyploids has been challenging. Genome doubling greatly complicates genetic analyses, and this has long hindered investigation in that most allopolyploid species are "nonmodel" organisms. However, recent advances in sequencing and genomics technologies now provide unprecedented opportunities to analyze numerous genetic markers in multiple individuals in any organism. ?Methods: Here we review the application of next-generation sequencing technologies to the study of three aspects of allopolyploid genome evolution: duplicated gene loss and expression in two recently formed Tragopogon allopolyploids, intergenomic interactions and chromosomal evolution in Tragopogon miscellus, and repetitive DNA evolution in Nicotiana allopolyploids. ?Key results: For the first time, we can explore on a genomic scale the evolutionary processes that are ongoing in natural allopolyploids and not be restricted to well-studied crops and genetic models. ?Conclusions: These approaches can be easily and inexpensively applied to many other plant species-making any evolutionarily provocative system a new "model" system.     

Genome evolution of allopolyploids: a process of cytological and genetic diploidization

Allopolyploidy is a prominent mode of speciation in higher plants. Due to the coexistence of closely related genomes, a successful allopolyploid must have the ability to invoke and maintain diploid-like behavior, both cytologically and genetically. Recent studies on natural and synthetic allopolyploids have raised many discrepancies. Most species have displayed non-Mendelian behavior in the allopolyploids, but others have not. Some species have demonstrated rapid genome changes following allopolyploid formation, while others have conserved progenitor genomes. Some have displayed directed, non-random genome changes, whereas others have shown random changes. Some of the genomic changes have appeared in the F1 hybrids, which have been attributed to the union of gametes from different progenitors, while other changes have occurred during or after genome doubling. Although these observations provide significant novel insights into the evolution of allopolyploids, the overall mechanisms of the event are still elusive. It appears that both genetic and epigenetic operations are involved in the diploidization process of allopolyploids. Overall, genetic and epigenetic variations are often associated with the activities of repetitive sequences and transposon elements. Specifically, genomic sequence elimination and chromosome rearrangement are probably the major forces guiding cytological diploidization. Gene non-functionalization, sub-functionalization, neo-functionalization, as well as other kinds of epigenetic modifications, are likely the leading factors promoting genetic diploidization.