Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops

By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization.     

Rapid changes of microsatellite flanking sequence in the allopolyploidization of new synthesized hexaploid wheat

Polyploidy has been found to be very common inplants. Comparative genome studies have revealed thateven species that were considered as typical diploidsincluding maize[1], soybean[2], Arabidopsis[3] have un-dergone polyploidization during their evolution. Ge-nome polyploidization is a major force of evolutionthat affects genome size and gene copy number[4,5]. Polyploids can be formed via the duplication ofgenomes, either of the same genomes (autopolyploid)or of diverged genomes with homoe…     

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.     

Rapid changes of microsatellite flanking sequence in the allopolyploidization of new synthesized hexaploid wheat

It was suggested that the rapid changes of DNA sequence and gene expression occurred at the early stages of allopolyploid formation. In this study, we revealed the microsatellite (SSR) differences between newly formed allopolyploids and their donor parents by using 21 primer sets specific for D genome of wheat. It was indicated that rapid changes had occurred in the “shock” process of the allopolyploid formation between tetraploid wheat and Aegilops tauschii. The changes of SSR flanking sequence resulted in appearance of novel bands or disappearance of parental bands. The disappearance of the parental bands showed much higher frequencies in comparison with that of appearance of novel bands. Disappearance of the parental bands was not random. The frequency of disappearance in tetraploid wheat was much higher than in Ae. tauschii, i. e. the disappearance frequency in AABB genome was much higher than in D genome. Changes of SSR flanking sequence occurred at the early stage of F₁ hybrid or just after chromosome doubling. From the above results, it can be inferred that SSR flanking sequence region was very active and was amenable to change in the process of polyploidization. This suggested that SSR flanking sequence probably had special biological function at the early stage of ployploidization. The rapid and directional changes at the early stage of polyploidization might contribute to the rapid evolution of the newly formed allopolyploid and allow the divergent genomes to act in harmony.     

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.     

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.     

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.     

Analysis of 5S rDNA changes in synthetic allopolyploids <Emphasis Type="Italic">Triticum</Emphasis> × <Emphasis Type="Italic">Aegilops</Emphasis>

Changes of 5S rDNA at the early stage of allopolyploidization were investigated in three synthetic allopolyploids: Aegilops sharonensis × Ae. umbellulata (2n = 28), Triticum urartu × Ae. tauschii (2n = 28), and T. dicoccoides × Ae. tauschii (2n = 42). Fluorescent in situ hybridization (FISH) revealed quantitative changes affecting separate loci of one of the parental genomes in S₃ plants of each hybrid combination. Southern hybridization with genomic DNA of the allopolyploid T. urartu × Ae. tauschii (TMU38 × TQ27) revealed a lower intensity of signals from Ae. tauschii fragments compared with those derived from T. urartu. This confirmed the signal reduction revealed for chromosome 1D of this hybrid by FISH. Neither Southern hybridization nor PCR testing of 5–15 plants of the S₂-S₃ generations revealed an appearance of new 5S rDNA fragments or a complete disappearance of parental fragments from the allopolyploids under study. No changes were found by aligning nine 5S rDNA sequences of the allopolyploid TMU38 × TQ27 with corresponding sequences of the parental species. The similarity between one of the synthetic allopolyploids examined and a natural allopolyploid with the same genome composition points to an early formation of the 5S rDNA organization unique for each allopolyploid.     

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.     

Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae)

Spartina x townsendii arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced Spartina alterniflora, native to the eastern seaboard of North America. Duplication of the hybrid genome gave rise to Spartina anglica, a vigorous allopolyploid involved in natural and artificial invasions on several continents. This system allows investigation of the early evolutionary changes that accompany stabilization of new allopolyploid species. Because allopolyploidy may be a genomic shock, eliciting retroelement insertional activity, we examined whether retrotransposons present in the parental species have been activated in the genome of S. anglica. For this purpose we used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposons-microsatellite amplified polymorphism (REMAP) markers, which are multilocus PCR-based methods detecting retrotransposon integration events in the genome. IRAP and REMAP allowed the screening of insertional polymorphisms in populations of S. anglica. The populations are composed mainly of one major multilocus genotype, identical to the first-generation hybrid S. x townsendii. Few new integration sites were encountered in the young allopolyploid genome. We also found strict additivity of the parental subgenomes in the allopolyploid. Both these findings indicate that the genome of S. anglica has not undergone extensive changes since its formation. This contrasts with previous results from the literature, which report rapid structural changes in experimentally resynthesized allopolyploids.     

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.     

Sequence of events leading to near-complete genome turnover in allopolyploid Nicotiana within five million years

Analyses of selected bacterial artificial chromosomes (BACs) clones suggest that the retrotransposon component of angiosperm genomes can be amplified or deleted, leading to genome turnover. Here, Nicotiana allopolyploids were used to characterize the nature of sequence turnover across the whole genome in allopolyploids known to be of different ages. Using molecular-clock analyses, the likely age of Nicotiana allopolyploids was estimated. Genomic in situ hybridization (GISH) and tandem repeat characterization were used to determine how the parental genomic compartments of these allopolyploids have diverged over time. Paternal genome sequence losses, retroelement activity and intergenomic translocation have been reported in early Nicotiana tabacum evolution (up to 200,000 yr divergence). Here it is shown that within 1 million years of allopolyploid divergence there is considerable exchange of repeats between parental chromosome sets. After c. 5 million years of divergence GISH fails. This GISH failure may represent near-complete genome turnover, probably involving the replacement of nongenic sequences with new, or previously rare sequence types, all occurring within a conserved karyotype structure. This mode of evolution may influence or be influenced by long-term diploidization processes that characterize angiosperm polyploidy-diploid evolutionary cycles.     

Genomic changes at early stages of formation of allopolyploid Aegilops longissima x Triticum urartu

Using the model of synthetic allopolyploid Aegilops longissima TL05 x Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing of PCR spectra obtained while amplifying genome DNA of allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for subtelomere DNA sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some PCR fragments typical for parental genome in RAPD specters of allopolyploid TL05 x TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomere primers such changes in PSR specters were shown only for one of 66 pays of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in specter of synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids

Allopolyploid formation requires the adaptation of two nuclear genomes within a single cytoplasm, which may involve programmed genetic and epigenetic changes during the initial generations following genome fusion. To study the dynamics of genome change, we synthesized 49 isogenic Brassica napus allopolyploids and surveyed them with 76 restriction fragment length polymorphism (RFLP) probes and 30 simple sequence repeat (SSR) primer pairs. Here, we report on the types and distribution of genetic and epigenetic changes within the S(1) genotypes. We found that insertion/deletion (indel) events were rare, but not random. Of the 57,710 (54,383 RFLP and 3,327 SSR) parental fragments expected among the amphidiploids, we observed 56,676 or 99.9%. Three loci derived from Brassica rapa had indels, and one indel occurred repeatedly across 29% (14/49) of the lines. Loss of one parental fragment was due to the 400-bp reduction of a guanine-adenine dinucleotide repeat-rich sequence. In contrast to the 4% (3/76) RFLP probes that detected indels, 48% (35/73) detected changes in the CpG methylation status between parental genomes and the S1 lines. Some loci were far more likely than others to undergo epigenetic change, but the number of methylation changes within each synthetic polyploid was remarkably similar to others. Clear de novo methylation occurred at a much higher frequency than de novo demethylation within allopolyploid sequences derived from B. rapa. Our results suggest that there is little genetic change in the S(0) generation of resynthesized B. napus polyploids. In contrast, DNA methylation was altered extensively in a pattern that indicates tight regulation of epigenetic changes.     

Size matters in Triticeae polyploids: larger genomes have higher remodeling

Polyploidization is one of the major driving forces in plant evolution and is extremely relevant to speciation and diversity creation. Polyploidization leads to a myriad of genetic and epigenetic alterations that ultimately generate plants and species with increased genome plasticity. Polyploids are the result of the fusion of two or more genomes into the same nucleus and can be classified as allopolyploids (different genomes) or autopolyploids (same genome). Triticeae synthetic allopolyploid species are excellent models to study polyploids evolution, particularly the wheat-rye hybrid triticale, which includes various ploidy levels and genome combinations. In this review, we reanalyze data concerning genomic analysis of octoploid and hexaploid triticale and different synthetic wheat hybrids, in comparison with other polyploid species. This analysis reveals high levels of genomic restructuring events in triticale and wheat hybrids, namely major parental band disappearance and the appearance of novel bands. Furthermore, the data shows that restructuring depends on parental genomes, ploidy level, and sequence type (repetitive, low copy, and (or) coding); is markedly different after wide hybridization or genome doubling; and affects preferentially the larger parental genome. The shared role of genetic and epigenetic modifications in parental genome size homogenization, diploidization establishment, and stabilization of polyploid species is discussed.     

Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploid formation between <Emphasis Type="Italic">Raphanus sativus</Emphasis> L. and <Emphasis Type="Italic">Brassica alboglabra</Emphasis>Bailey

Background  

Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations.     

Genomic changes at early stages of formation of allopolyploid <Emphasis Type="Italic">Aegilops longissima × Triticum urartu</Emphasis>

Using the model of synthetic allopolyploid Aegilops longissima TL05 × Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing PCR fragments of the allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for these chromosomal regions sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some RAPD fragments typical for parental genome in allopolyploid TL05 × TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomeric primers the changes in PCR fragments were shown only for one of 66 pairs of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in the synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat

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, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between AE: sharonensis x AE: umbellulata, 14% of the loci from AE: sharonensis were eliminated compared with only 0.5% from AE: umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between AE: longissima x T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in approximately 13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.