Synthetic Allopolyploids Research Articles

Subgenome Dominance in an Interspecific Hybrid, Synthetic Allopolyploid, and a 140-Year-Old Naturally Established Neo-Allopolyploid Monkeyflower.

Recent studies have shown that one of the parental subgenomes in ancient polyploids is generally more dominant, having retained more genes and being more highly expressed, a phenomenon termed subgenome dominance. The genomic features that determine how quickly and which subgenome dominates within a newly formed polyploid remain poorly understood. To investigate the rate of emergence of subgenome dominance, we examined gene expression, gene methylation, and transposable element (TE) methylation in a natural, <140-year-old allopolyploid (Mimulus peregrinus), a resynthesized interspecies triploid hybrid (M. robertsii), a resynthesized allopolyploid (M. peregrinus), and progenitor species (M. guttatus and M. luteus). We show that subgenome expression dominance occurs instantly following the hybridization of divergent genomes and significantly increases over generations. Additionally, CHH methylation levels are reduced in regions near genes and within TEs in the first-generation hybrid, intermediate in the resynthesized allopolyploid, and are repatterned differently between the dominant and recessive subgenomes in the natural allopolyploid. Subgenome differences in levels of TE methylation mirror the increase in expression bias observed over the generations following hybridization. These findings provide important insights into genomic and epigenomic shock that occurs following hybridization and polyploid events and may also contribute to uncovering the mechanistic basis of heterosis and subgenome dominance.

Cytological and morphological analysis of hybrids between Brassicoraphanus, and Brassica napus for introgression of clubroot resistant trait into Brassica napus L.

Interspecific hybridization is a powerful tool for improvement of crop species, it has the potential to broaden the genetic base and create new plant forms for breeding programs. Synthetic allopolyploid is a widely-used model for the study of genetic recombination and fixed heterosis in Brassica. In Brassica napus breeding, identification and introgression of new sources of clubroot resistance trait from wild or related species into it by hybridization is a long-term crop management strategy for clubroot disease. Radish (Raphanus sativus L.) is a close relative of the Brassica and most radish accessions are immune to the clubroot disease. A synthesized allotetraploid Brassicoraphanus (RRCC, 2n = 36) between R. sativus cv. HQ-04 (2n = 18, RR) and Brassica oleracea var. alboglabra (L.H Bailey) (2n = 18, CC) proved resistant of multiple clubroot disease pathogen P. brassicae. To predict the possibility to transfer the clubroot resistance trait from the RR subgenome of allotetraploid Brassicoraphanus (RRCC, 2n = 36) into Brassica napus (AACC, 2n = 38), we analyzed the frequency of chromosome pairings in the F1 hybrids produced from a cross between B. napus cv. HS5 and the allotetraploid, characterize the genomic composition of some backcrossed progeny (BC1) using GISH, BAC-FISH and AFLP techniques. The level of intergenomic pairing between A and R genomes in the F1 hybrid was high, allosyndetic bivalents formed in 73.53% PMCs indicative of significant level of homeologous recombination between two genomes and high probability of incorporating chromosomal segments/genes from R-genome into A/C-genomes. The BC1 plants inherited variant extra R chromosomes or fragments from allotetraploid as revealed by GISH and AFLP analysis. 13.51% BC2 individuals were resistant to clubroot disease, and several resistance lines had high pollen fertility, Overall, the genetic material presented in this work represents a potential new genetic resource for practical use in breeding B. napus clubroot resistant cultivars.

Altered expression of TaRSL4 gene by genome interplay shapes root hair length in allopolyploid wheat.

Polyploidy is a major driving force in plant evolution and speciation. Phenotypic changes often arise with the formation, natural selection and domestication of polyploid plants. However, little is known about the consequence of hybridization and polyploidization on root hair development. Here, we report that root hair length of synthetic and natural allopolyploid wheats is significantly longer than those of their diploid progenitors, whereas no difference is observed between allohexaploid and allotetraploid wheats. The expression of wheat gene TaRSL4, an orthologue of AtRSL4 controlling the root hair development in Arabidopsis, was positively correlated with the root hair length in diploid and allotetraploid wheats. Moreover, transcript abundance of TaRSL4 homoeologue from A genome (TaRSL4-A) was much higher than those of other genomes in natural allopolyploid wheat. Notably, increased root hair length by overexpression of the TaRSL4-A in wheat led to enhanced shoot fresh biomass under nutrient-poor conditions. Our observations indicate that increased root hair length in allohexaploid wheat originated in the allotetraploid progenitors and altered expression of TaRSL4 gene by genome interplay shapes root hair length in allopolyploid wheat.

Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

An efficient method of developing synthetic allopolyploid rice (Oryza spp.)

Allopolyploid rice is an important genetic resource for biology research and rice breeding. However, it is difficult to obtain allopolyploid rice in both natural and artificial conditions. We have established an efficient technology, development of synthetic allopolyploid rice, for obtaining allopolyploid rice based on a wild cross, embryo rescue and in vitro colchicine treatment. Fourteen typological allopolyploid rice lines among 6 interspecies and 7 different genomes have been developed using this technology. It will enable the development of new rice germplasms with the potential to double yields and enrich varieties.

Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis.

Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.

The BOY NAMED SUE Quantitative Trait Locus Confers Increased Meiotic Stability to an Adapted Natural Allopolyploid of Arabidopsis

Whole-genome duplication resulting from polyploidy is ubiquitous in the evolutionary history of plant species. Yet, polyploids must overcome the meiotic challenge of pairing, recombining, and segregating more than two sets of chromosomes. Using genomic sequencing of synthetic and natural allopolyploids of Arabidopsis thaliana and Arabidopsis arenosa, we determined that dosage variation and chromosomal translocations consistent with homoeologous pairing were more frequent in the synthetic allopolyploids. To test the role of structural chromosomal differentiation versus genetic regulation of meiotic pairing, we performed sequenced-based, high-density genetic mapping in F2 hybrids between synthetic and natural lines. This F2 population displayed frequent dosage variation and deleterious homoeologous recombination. The genetic map derived from this population provided no indication of structural evolution of the genome of the natural allopolyploid Arabidopsis suecica, compared with its predicted parents. The F2 population displayed variation in meiotic regularity and pollen viability that correlated with a single quantitative trait locus, which we named BOY NAMED SUE, and whose beneficial allele was contributed by A. suecica. This demonstrates that an additive, gain-of-function allele contributes to meiotic stability and fertility in a recently established allopolyploid and provides an Arabidopsis system to decipher evolutionary and molecular mechanisms of meiotic regularity in polyploids.

Allopolyploidy and the evolution of plant virus resistance.

BackgroundThe relationship between allopolyploidy and plant virus resistance is poorly understood. To determine the relationship of plant evolutionary history and basal virus resistance, a panel of Nicotiana species from diverse geographic regions and ploidy levels was assessed for resistance to non-coevolved viruses from the genus Nepovirus, family Secoviridae. The heritability of resistance was tested in a panel of synthetic allopolyploids. Leaves of different positions on each inoculated plant were tested for virus presence and a subset of plants was re-inoculated and assessed for systemic recovery.ResultsDepending on the host-virus combination, plants displayed immunity, susceptibility or intermediate levels of resistance. Synthetic allopolyploids showed an incompletely dominant resistance phenotype and manifested systemic recovery. Plant ploidy was weakly negatively correlated with virus resistance in Nicotiana species, but this trend did not hold when synthetic allopolyploids were taken into account. Furthermore, a relationship between resistance and geographical origin was observed.ConclusionThe gradients of resistance and virulence corresponded to a modified matching allele model of resistance. Intermediate resistance responses of allopolyploids corresponded with a model of multi-allelic additive resistance. The variable virus resistance of extant allopolyploids suggested that selection-based mechanisms surpass ploidy with respect to evolution of basal resistance to viruses.

Cytomorphological Characterization of the Backcross Progeny of Synthetic Amphiploid Rice (AABB) and Tetraploid Oryza sativa (AAAA)

DCW008, a synthetic allopolyploid rice (2n = 4X = 48, AABB), was obtained from chromosome doubling of interspecific hybrids between Oryza sativa (2n = 24, AA) and O. punctata (2n = 24, BB). The F1 and backcross (BC1) hybrids were produced by crossing DCW008 as a female parent with a high seed set tetraploid rice Sg99012 - 4X (2n = 4X = 48, AAAA). BC1F1 and BC1F2 overcame many of the wild-type traits of DCW008; they had yellow-hulled grains, seed set ranged from 0% to 71.31% and the grain morphology was similar to that of cultivated rice. Variable numbers of chromosomes were observed in pollen mother cells (PMCs) from the BC1 plant. Genomic in situ hybridization (GISH) revealed that the majority of somatic cells and PMCs contained six chromosomes of O. punctata with fragment recombination observed in two of them. The backcross selection method employed in this study to generate allopolyploid progeny provides a reliable way of transferring useful genes from wild species into cultivated rice.

The reorganization of plant genomes during allopolyploidization

Molecular mechanisms governing plant genome reorganization in the course of allopolyploidization are reviewed. The special role of early genetic changes, which were studied using a unique model of synthetic allopolyploids, is emphasized. The data on various genomic changes at early stages of allopolyploidization, including the activation of mobile elements, chromosomal rearrangements, epigenetic and transcriptomic changes, etc., are summarized. We note that these changes provide for the evolutionary success of allopolyploids and their prevalence among higher plants because of the formation of a stable organization of the allopolyploid genome.

Stochastic changes affect Solanum wild species following autopolyploidization.

Polyploidy is very common within angiosperms, and several studies are in progress to ascertain the effects of early polyploidization at the molecular, physiological, and phenotypic level. Extensive studies are available only in synthetic allopolyploids. By contrast, less is known about the consequences of autopolyploidization. The current study aimed to assess the occurrence and extent of genetic, epigenetic, and anatomical changes occurring after oryzaline-induced polyploidization of Solanum commersonii Dunal and Solanum bulbocastanum Dunal, two diploid (2n=2×=24) potato species widely used in breeding programmes. Microsatellite analysis showed no polymorphisms between synthetic tetraploids and diploid progenitors. By contrast, analysis of DNA methylation levels indicated that subtle alterations at CG and CHG sites were present in tetraploids of both species. However, no change occurred concurrently in all tetraploids analysed with respect to their diploid parent, revealing a stochastic trend in the changes observed. The morpho-anatomical consequences of polyploidization were studied in leaf main veins and stomata. With only a few exceptions, analyses showed no clear superiority of tetraploids in terms of leaf thickness and area, vessel number, lumen size and vessel wall thickness, stomata pore length and width, guard cell width, and stomatal density compared with their diploid progenitors. These results are consistent with the hypothesis that there are no traits systematically associated with autopolyploidy.

Prevalence of gene expression additivity in genetically stable wheat allohexaploids

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34,820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.

Homoeolog expression bias and expression level dominance in allopolyploid cotton

Allopolyploidy is an evolutionary and mechanistically intriguing process, in that it entails the reconciliation of two or more sets of diverged genomes and regulatory interactions. In this study, we explored gene expression patterns in interspecific hybrid F(1), and synthetic and natural allopolyploid cotton using RNA-Seq reads from leaf transcriptomes. We determined how the extent and direction of expression level dominance (total level of expression for both homoeologs) and homoeolog expression bias (relative contribution of homoeologs to the transcriptome) changed from hybridization through evolution at the polyploid level and following cotton domestication. Genome-wide expression level dominance was biased toward the A-genome in the diploid hybrid and natural allopolyploids, whereas the direction was reversed in the synthetic allopolyploid. This biased expression level dominance was mainly caused by up- or downregulation of the homoeolog from the 'non-dominant' parent. Extensive alterations in homoeolog expression bias and expression level dominance accompany the initial merger of two diverged diploid genomes, suggesting a combination of regulatory (cis or trans) and epigenetic interactions that may arise and propagate through the transcriptome network. The extent of homoeolog expression bias and expression level dominance increases over time, from genome merger through evolution at the polyploid level. Higher rates of transgressive and novel gene expression patterns as well as homoeolog silencing were observed in natural allopolyploids than in F(1) hybrid and synthetic allopolyploid cottons. These observations suggest that natural selection reconciles the regulatory mismatches caused by initial genomic merger, while new gene expression conditions are generated for evaluation by selection.

Genome Evolution Due to Allopolyploidization in Wheat

The wheat group has evolved through allopolyploidization, namely, through hybridization among species from the plant genera Aegilops and Triticum followed by genome doubling. This speciation process has been associated with ecogeographical expansion and with domestication. In the past few decades, we have searched for explanations for this impressive success. Our studies attempted to probe the bases for the wide genetic variation characterizing these species, which accounts for their great adaptability and colonizing ability. Central to our work was the investigation of how allopolyploidization alters genome structure and expression. We found in wheat that allopolyploidy accelerated genome evolution in two ways: (1) it triggered rapid genome alterations through the instantaneous generation of a variety of cardinal genetic and epigenetic changes (which we termed "revolutionary" changes), and (2) it facilitated sporadic genomic changes throughout the species' evolution (i.e., evolutionary changes), which are not attainable at the diploid level. Our major findings in natural and synthetic allopolyploid wheat indicate that these alterations have led to the cytological and genetic diploidization of the allopolyploids. These genetic and epigenetic changes reflect the dynamic structural and functional plasticity of the allopolyploid wheat genome. The significance of this plasticity for the successful establishment of wheat allopolyploids, in nature and under domestication, is discussed.

Development and characterization of synthetic amphiploid (AABB) between Oryza sativa and Oryza punctata

AbstractOryza punctata, a wild relative of cultivated rice, belongs to the BB genome of Oryza. Interspecific hybrids (CW008, AB) between Oryza sativa (2n = 24 AA) and O. punctata (2n = 24 BB) were obtained using embryo rescue technique. Synthetic allopolyploid (DCW008, AABB) were produced through chromosome doubling by colchicine. Hybrids overcame many wild traits except the shattering and awn. The synthetic amphiploid plants showed obvious superiority in growth and production. Interspecific hybrids CW008 were completely infertile but DCW008 had better seed set after selfing. Genomic in situ hybridization investigations were performed on DCW008. The result indicated that the A genome was closed to the B genome and translocations occurred between some chromosomes of cultivated and wild rice. Meiosis was nearly normal in the amphiploid hybrid but was disrupted in the diploid hybrid, which resulted in different fertility of them.

Independent, Rapid and Targeted Loss of Highly Repetitive DNA in Natural and Synthetic Allopolyploids of Nicotiana tabacum

Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6–1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation.

Intertribal hybrid plants produced from crossing Arabidopsis thaliana with apomictic Boechera

Arabidopsis thaliana and Boechera belong to different tribes of the Brassicaceae and last shared a common ancestor 13-35 million years ago. A. thaliana reproduces sexually but some Boechera accessions reproduce by apomixis (asexual reproduction by seed). The two species are reproductively isolated, preventing introgression of the trait(s) controlling apomixis from Boechera into A. thaliana and their molecular characterisation. To identify if "escapers" from such hybridisation barriers exist, we crossed diploid or tetraploid A. thaliana mothers carrying a conditional male sterile mutation with a triploid Boechera apomict. These cross-pollinations generated zygotes and embryos. Most aborted or suffered multiple developmental defects at all stages of growth, but some seed matured and germinated. Seedlings grew slowly but eventually some developed into mature plants that were novel synthetic allopolyploid hybrids. With one exception, intertribal hybrids contained three Boechera plus either one or two A. thaliana genomes (depending on maternal ploidy) and were male and female sterile. The exception was a semi-fertile, sexual partial hybrid with one Boechera plus two A. thaliana genomes. The synthesis of "escapers" that survive rigorous early developmental challenges in crosses between A. thaliana and Boechera demonstrates that the inviability form of postzygotic reproductive isolation separating these distantly related species is not impenetrable. The recovery of a single semi-fertile partial hybrid also demonstrates that hybrid sterility, another form of postzygotic reproductive isolation, can be overcome between these species.

Methylation, transcription, and rearrangements of transposable elements in synthetic allopolyploids.

Transposable elements (TEs) constitute over 90% of the wheat genome. It was suggested that “genomic stress” such as hybridity or polyploidy might activate transposons. Intensive investigations of various polyploid systems revealed that allopolyploidization event is associated with widespread changes in genome structure, methylation, and expression involving low- and high-copy, coding and noncoding sequences. Massive demethylation and transcriptional activation of TEs were also observed in newly formed allopolyploids. Massive proliferation, however, was reported for very limited number of TE families in various polyploidy systems. The aim of this review is to summarize the accumulated data on genetic and epigenetic dynamics of TEs, particularly in synthetic allotetraploid and allohexaploid wheat species. In addition, the underlying mechanisms and the potential biological significance of TE dynamics following allopolyploidization are discussed.

Differential contributions to the transcriptome of duplicated genes in response to abiotic stresses in natural and synthetic polyploids

Polyploidy has occurred throughout plant evolution and can result in considerable changes to gene expression when it takes place and over evolutionary time. Little is known about the effects of abiotic stress conditions on duplicate gene expression patterns in polyploid plants. We examined the expression patterns of 60 duplicated genes in leaves, roots and cotyledons of allotetraploid Gossypium hirsutum in response to five abiotic stress treatments (heat, cold, drought, high salt and water submersion) using single-strand conformation polymorphism assays, and 20 genes in a synthetic allotetraploid. Over 70% of the genes showed stress-induced changes in the relative expression levels of the duplicates under one or more stress treatments with frequent variability among treatments. Twelve pairs showed opposite changes in expression levels in response to different abiotic stress treatments. Stress-induced expression changes occurred in the synthetic allopolyploid, but there was little correspondence in patterns between the natural and synthetic polyploids. Our results indicate that abiotic stress conditions can have considerable effects on duplicate gene expression in a polyploid, with the effects varying by gene, stress and organ type. Differential expression in response to environmental stresses may be a factor in the preservation of some duplicated genes in polyploids.

Nuclear DNA content and chromosome number in somatic hybrid allopolyploids of Solanum

An attempt was made to change the proportion of the parental genomes in interspecific hybrids Solanum nigrum + S. tuberosum (ngr + tbr) by means of repeated protoplast fusion. In order to enlarge the potato input into the hybrid genome, the protoplasts of two ngr + tbr hybrids of different ploidy (7x and 8x) were fused with the protoplasts of two different diploid potato clones in three combinations. Protoclonal variability was studied in three populations of new ngr + tbr allopolyploids maintained in vitro. The absolute nuclear DNA content (2C) was measured using flow cytometry to estimate the ploidy of the hybrids. The ploidy level of the selected clones was verified by chromosome counts in root meristems. The newly synthesized allopolyploids (75 clones) showed only a small gain in nuclear DNA content above the mean value determined for the parents, instead of the expected addition of an entire diploid potato genome to the combined parental ngr + tbr genome. An increase in nuclear DNA was observed mostly in the clones having the 7x hybrid as a parent (75% of allopolyploids from two combinations). When the 8x hybrid was used as a parent, only two allopolyploids (5%) exhibited a significantly increased nuclear DNA content. The 8x level of ngr + tbr allopolyploids was shown to be stable and was only occasionally exceeded. Somatic hybrids ngr + tbr offer a model system for studying the molecular mechanism(s) and processes involved in stabilization and establishment of the synthetic Solanum allopolyploids.