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.     

Evolution of duplicate gene expression in polyploid and hybrid plants

Allopolyploidy is a prominent mode of speciation in flowering plants. On allopolyploidy, genomic changes can take place, including chromosomal rearrangement and changes in gene expression; these processes continue over evolutionary time. Recent studies of gene expression in polyploid and hybrid plants, reviewed here, have examined expression in natural polyploids and synthetic neopolyploids as well as in diploid and F(1) hybrids. Considerable changes in gene expression have been observed in allopolyploids, including up- or downregulation of expression in the polyploids compared with their parents, unequal expression of duplicated genes, and silencing of one copy. Genes in a variety of functional categories show altered expression, and the patterns vary considerably by gene. Some changes seem to be stochastic, whereas others are repeatable. Gene expression changes can be organ specific. Reciprocal silencing of duplicates in different organs has been observed, suggesting subfunctionalization and long-term retention of duplicates. It has become clear that hybridization has a much greater effect than chromosome doubling on gene expression in allopolyploids. Diploid and triploid F(1) hybrids can show alterations of expression levels compared with their parents. Parent-of-origin effects on gene expression have been examined, and loss of gene imprinting has been shown. Some gene expression changes in polyploids and hybrids can be correlated with phenotypic effects. Demonstrated mechanisms of gene expression changes include DNA methylation, histone modifications, and antisense RNA. Several hypotheses have been proposed for why gene expression is altered in allopolyploids and hybrids.     

Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype

Many previous studies have provided evidence for genome changes in polyploids, but there are little data on the overall population dynamics of genome change and whether it causes phenotypic variability. We analyzed genetic, epigenetic, gene expression, and phenotypic changes in approximately 50 resynthesized Brassica napus lines independently derived by hybridizing double haploids of Brassica oleracea and Brassica rapa. A previous analysis of the first generation (S0) found that genetic changes were rare, and cytosine methylation changes were frequent. Our analysis of a later generation found that most S0 methylation changes remained fixed in their S5 progeny, although there were some reversions and new methylation changes. Genetic changes were much more frequent in the S5 generation, occurring in every line with lines normally distributed for number of changes. Genetic changes were detected on 36 of the 38 chromosomes of the S5 allopolyploids and were not random across the genome. DNA fragment losses within lines often occurred at linked marker loci, and most fragment losses co-occurred with intensification of signal from homoeologous markers, indicating that the changes were due to homoeologous nonreciprocal transpositions (HNRTs). HNRTs between chromosomes A1 and C1 initiated in early generations, occurred in successive generations, and segregated, consistent with a recombination mechanism. HNRTs and deletions were correlated with qualitative changes in the expression of specific homoeologous genes and anonymous cDNA amplified fragment length polymorphisms and with phenotypic variation among S5 polyploids. Our data indicate that exchanges among homoeologous chromosomes are a major mechanism creating novel allele combinations and phenotypic variation in newly formed B. napus polyploids.     

Polyploidy and DNA methylation: new tools available

Most plant species are recent or ancient polyploids (displaying at least one round of genome duplication in their history). Cultivated species (e.g. wheat, cotton, canola, sugarcane, coffee) and invasive species are often relatively recent polyploids, and frequently of hybrid origin (i.e. allopolyploids). Despite the genetic bottleneck occurring during the allopolyploid speciation process, the formation of such species from two divergent lineages leads to fixed heterozygosity decisive to their success. New phenotypes and new niche occupation are usually associated with this mode of speciation, as a result of both genomic rearrangements and gene expression changes of different magnitudes depending on the different polyploid species investigated. These gene expression changes affecting newly formed polyploid species may result from various, interconnected mechanisms, including (i) functional interactions between the homoeologous copies and between their products, that are reunited in the same nucleus and cell; (ii) the fate of duplicated copies, selective pressure on one of the parental copy being released which could lead to gene loss, pseudogenization, or alternatively, to subfunctionalization or neofunctionalization; and (iii) epigenetic landscape changes that in turn affect gene expression. As one of the interrelated processes leading to epigenetic regulation of gene expression, the DNA methylation status of newly formed species appears to be consistently affected following both hybridization and genome doubling. In this issue, Verhoeven et al. have investigated the fate of DNA methylation patterns that could affect naturally occurring new asexual triploid lineages of dandelions. As a result of such a ploidy level change, the authors demonstrate stably transmitted DNA methylation changes leading to unique DNA methylation patterns in each newly formed lineage. Most studies published to date on plant DNA methylation polymorphism were performed using restriction enzymes sensitive to methylation. Recently, new high‐throughput methods were made available, thanks to the development of ‘next‐generation sequencing’ techniques. The combination of these methods offers powerful and promising tools to investigate epigenetic variation in both model and non‐model systems.     

Sensitivity of 70-mer oligonucleotides and cDNAs for microarray analysis of gene expression in Arabidopsis and its related species

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60-70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance (anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from approximately 26,000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids.     

Epigenetic regulation of flowering time in polyploids

Polyploidy plays a significant role in the evolution of flowering plants. Understanding the effects of polyploidy on the epigenetic regulation of adaptive traits may resolve questions about the success of polyploids. One such trait, flowering time, has been the subject of several gene expression studies because it has one of the best characterized genetic networks and because polyploidy has a significant impact on generating variation in flowering time. Future research on the epigenetic consequences of polyploidy on flowering time should begin to examine natural variation in an ecological context, while continuing to make use of resynthesized polyploids.     

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.     

Polyploid organisms

Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants and animals, and is an important mechanism of speciation. Genome sequencing and related molecular systematics and bioinformatics studies on plants and animals in recent years support the view that species have been shaped by whole genome duplication during evolution. The stability of polyploids depends on rapid genome recombination and changes in gene expression after formation. The formation of polyploids and subsequent diploidization are important aspects in long-term evolution. Polyploids can be formed in various ways. Among them, hybrid organisms formed by distant hybridization could produce unreduced gametes and thus generate offspring with doubled chromosomes, which is a fast, efficient method of polyploidization. The formation of fertile polyploids not only promoted the interflow of genetic materials among species and enriched the species diversity, but also laid the foundation for polyploidy breeding. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits.     

Big roles for small RNAs in polyploidy, hybrid vigor, and hybrid incompatibility

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (ta-siRNAs), mediate gene expression and epigenetic regulation. While siRNAs are highly diverged, miRNAs and ta-siRNAs are generally conserved but many are differentially expressed between related species and in interspecific hybrids and allopolyploids. On one hand, combination of diverged maternal and paternal siRNAs in the same nucleus may exert cis-acting and trans-acting effects on transposable elements (TEs) and TE-associated genes, leading to genomic instability and endosperm and embryo failures, constituting a bottleneck for the evolution of hybrids and polyploids. On the other hand, cis and trans-acting small RNAs induce quantitative and qualitative changes in epigenetic regulation, leading to morphological variation and hybrid vigor in F1 hybrids and stable allopolyploids as well as transgressive phenotypes in the progeny, increasing a potential for adaptive evolution.     

Polyploidy in Dalea formosa (Fabaceae) on the Chihuahuan Desert

Dalea formosa consists of diploids (n = 7), tetraploids (n = 14), and hexaploids (n = 21), the polyploids restricted to the Chihuahuan Desert region or its immediate borders. There is very little morphological differentiation between the three chromosome races and, therefore, the polyploids are assumed to be primarily autoploid. Tetraploids discovered were few and were very similar to hexaploids; the two ploidy levels were combined as “polyploids” for analyses of geographically and cytologically correlated morphological variation. Pollen length generally was found to be greater in known polyploids than in known diploids. Through the use of pollen length and geographic origin, chromosomally unknown specimens were estimated as to ploidy level. This produced four groups, known diploids and polyploids, and putative diploids and polyploids, which were then subjected to stepwise discriminant analysis (SDA) to search for other morphological characters that might indicate ploidy level, to evaluate the assignments to putative ploidy level in unknown plants, and to assess correlation of these plants of putative ploidy level to geographic regions. SDA also indicated that pollen length, among ten morphological features, is the primary discriminator between ploidy levels, and that putative polyploids are confined primarily to the Chihuahuan Desert. Chromosomally unknown specimens that were originally assigned to one ploidy level, but were classified by SDA as another, are viewed as indicative of areas where further cytological sampling is particularly needed. These areas are southeastern Arizona, where pollen among known diploids is comparatively large, northeastern New Mexico, where polyploids might occur off the Chihuahuan Desert, east edge of the Chihuahuan Desert in Texas, a cytologically poorly sampled contact zone between diploids and polyploids, and central Coahuila, where no cytological sampling has been done. Canonical variate analysis is used to aid in the visualization of the general morphological relationship between diploids and polyploids.     

Genome redundancy and plasticity within ancient and recent Brassica crop species

The crop species within the genus Brassica have highly replicated genomes. Three base 'diploid' species, Brassica oleracea , B. nigra and B. rapa , are likely ancient polyploids, and three derived allopolyploid species, B. carinata , B. juncea and B. napus , are created from the interspecific hybridization of these base genomes. The base Brassica genome is thought to have hexaploid ancestry, and both recent and ancient polyploidization events have been proposed to generate a large number of genome rearrangements and novel genetic variation for important traits. Here, we revisit and refine these hypotheses. We have examined the B. oleracea linkage map using the Arabidopsis thaliana genome sequence as a template and suggest that there is strong evidence for genome replication and rearrangement within the base Brassicas, but less evidence for genome triplication. We show that novel phenotypic variation within the base Brassicas can be achieved by replication of a single gene, BrFLC , that acts additively to influence flowering time. Within the derived allopolyploids, intergenomic heterozygosity is associated with higher seed yields. Some studies have reported that de novo genomic variation occurs within derived polyploid genomes, whereas other studies have not detected these changes. We discuss reasons for these different findings. Large translocations and tetrasomic inheritance can explain some but not all genomic changes within the polyploids. Transpositions and other small-scale sequence changes probably also have contributed to genomic novelty. Our results have shown that the Brassica genomes are remarkably plastic, and that polyploidy generates novel genetic variation through gene duplication, intergenomic heterozygosity and perhaps epigenetic change.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 665–674.     

The development of an Arabidopsis model system for genome-wide analysis of polyploidy effects

Arabidopsis is a model system not only for studying numerous aspects of plant biology, but also for understanding mechanisms of the rapid evolutionary process associated with genome duplication and polyploidization. Although in animals interspecific hybrids are often sterile and aneuploids are related to disease syndromes, both Arabidopsis autopolyploids and allopolyploids occur in nature and can be readily formed in the laboratory, providing an attractive system for comparing changes in gene expression and genome structure among relatively 'young' and 'established' or 'ancient' polyploids. Powerful reverse and forward genetics in Arabidopsis offer an exceptional means by which regulatory mechanisms of gene and genome duplication may be revealed. Moreover, the Arabidopsis genome is completely sequenced; both coding and non-coding sequences are available. We have developed spotted oligo-gene and chromosome microarrays using the complete Arabidopsis genome sequence. The oligo-gene microarray consists of ∼26 000 70-mer oligonucleotides that are designed from all annotated genes in Arabidopsis , and the chromosome microarray contains 1 kb genomic tiling fragments amplified from a chromosomal region or the complete sequence of chromosome 4. We have demonstrated the utility of microarrays for genome-wide analysis of changes in gene expression, genome organization and chromatin structure in Arabidopsis polyploids and related species.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 689–700.     

Other tetraploid species and conspecific diploids as sources of genetic variation for an autotetraploid

? Premise of the study: Most plants are polyploid and have more than two copies of the genome. The evolutionary success of polyploids is often attributed to their potential to harbor increased genetic variation, but it is poorly understood how polyploids can attain such variation. Because of their formation bottleneck, newly formed tetraploids start out with little variation. Tetraploids may attain genetic variation through a combination of new mutations, recurrent formation, and gene exchange with diploid ancestors or related tetraploid species. We explore the role of gene exchange and introgression in autotetraploid Rorippa amphibia, a species that harbors more genetic variation than its diploid ancestors. ? Methods: We crossed autotetraploid R. amphibia to diploid conspecifics and tetraploid R. sylvestris and backcrossed resulting F(1) hybrids. We used flow cytometry to determine the ploidy of all progeny. ? Key results: Tetraploids of R. amphibia and R. sylvestris were interfertile; F(1) hybrids were fertile and could backcross. Crosses between diploids and tetraploids yielded a small number of viable, often tetraploid progeny. This indicates that unreduced gametes can facilitate gene flow from diploids to tetraploids. We detected a frequency of unreduced gametes of around 2.7 per 1000, which was comparable between diploids and tetraploids. ? Conclusions: Introgression from tetraploid R. sylvestris provides a realistic source of variation in autotetraploid R. amphibia. Only in a scenario where other compatible partners are absent, for example immediately after tetraploidization, gene flow through unreduced gametes from diploids could be an important source of genetic variation for tetraploids.     

MicroRNA expression changes following synthesis of three full-sib <Emphasis Type="Italic">Populus</Emphasis> triploid populations with different heterozygosities

Key message

Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage.

Abstract

Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

     

FLAVONOID RACES OF CLAYTONIA VIRGINICA (PORTULACACEAE)

Four flavonoid races have been found in the Claytonia virginica aneuploid complex, differing from one another in the accumulation of 12 kaempferol and quercetin 3-glycosides. Each of these races has a distinctive geographical distribution within the overall range of the species. The three major diploid cytotypes of C. virginica each belong to different races, two of which also include polyploids; a fourth race consists entirely of polyploids. An examination of biosynthetic pathways indicates that a small number of genetic changes are responsible for the observed variation; polyploidy per se does not appear to have contributed significantly to the production of novel compounds. Flavonoid data also suggest strongly that polyploidy within rather than between modern chemical races has been responsible for the bulk of chromosome number variation in the species.     

Exploring the genomic mysteries of polyploidy in cotton

For several years allopolyploid cottons have been the subject of evolutionary investigations into the genomic mysteries of polyploidy. An array of genomic interactions have been documented, including interlocus concerted evolution, differential rates of genomic evolution and intergenomic sequence transfer. Substantial alterations in gene expression have occurred in response to allopolyploidization, including gene silencing and expression changes that vary by organ. Some of the molecular phenomena occurring in polyploids appear to be non-Mendelian. Many of the genomic and expression alterations have occurred on an evolutionary timescale, whereas others reflect more immediate consequences of genomic merger.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 573–581.     

Life history divergence of sympatric diploid and polyploid populations of brine shrimp Artemia parthenogenetica

In order to study how polyploidy affects life history patterns in animals, we have examined sympatric diploid and polyploid brine shrimp (Artemia parthenogenetica) from China, Italy and Spain under laboratory conditions. At optimal temperature and salinity (25°C and 90 ppt), diploids from the three populations had much higher intrinsic rates of increase, higher fecundity, faster developmental rates, and larger brood sizes than their sympatric polyploids. The Chinese and Italian populations were selected for further analysis to determine the life history responses of diploids and polyploids to temperature and salinity changes. Under intermediate and high salinities, Chinese and Italian polyploids produced most of their offspring as dormant cysts while their sympatric diploids produced most of their offspring as nauplii. This relationship is reversed in the Spanish diploid-polyploid complex. For the Chinese population at 25° C, pentaploid clones had higher developmental rates than diploid clones at 35 ppt; at 90 ppt, diploid clones had higher developmental rates than the pentaploids. Italian diploids and tetraploids had different responses to variation in both temperature (25° C and 31° C) and salinity (30 ppt and 180 ppt). Our results demonstrate that relative fitness of the two cytotypes is a function of environmental conditions and that sympatric diploids and polyploids respond differently to environmental changes. Chinese and Italian polyploids are expected to have lower fitness than their sympatric diploids when the physical environment is not stressful and when intraspecific competition is important. However, polyploids may have advantages over sympatric diploids in stressful habitats or when they encounter short-term lethal temperatures. These results suggest that polyploid Artemia have evolved a suite of life-history characteristics adapting them to environments that contrast to those of their sympatric diploids.     

Polyploidy induces centromere association

Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes.