Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae)

To study the consequences of hybridization and genome duplication on polyploid genome evolution and adaptation, we used independently formed hybrids (Spartina x townsendii and Spartina x neyrautii) that originated from natural crosses between Spartina alterniflora, an American introduced species, and the European native Spartina maritima. The hybrid from England, S. x townsendii, gave rise to the invasive allopolyploid, salt-marsh species, Spartina anglica. Recent studies indicated that allopolyploid speciation may be associated with rapid genetic and epigenetic changes. To assess this in Spartina, we performed AFLP (amplified fragment length polymorphism) and MSAP (methylation sensitive amplification polymorphism) on young hybrids and the allopolyploid. By comparing the subgenomes in the hybrids and the allopolyploid to the parental species, we inferred structural changes that arose repeatedly in the two independently formed hybrids. Surprisingly, 30% of the parental methylation patterns are altered in the hybrids and the allopolyploid. This high level of epigenetic regulation might explain the morphological plasticity of Spartina anglica and its larger ecological amplitude. Hybridization rather than genome doubling seems to have triggered most of the methylation changes observed in Spartina anglica.     

Molecular investigations in populations of Spartina anglica C.E. Hubbard (Poaceae) invading coastal Brittany (France)

Spartina anglica is a classical example of recent alloploid speciation. It arose during the end of the nineteenth century in England by hybridization between the indigenous Spartina maritima and the introduced East-American Spartina alterniflora. Duplication of the hybrid genome (Spartina x townsendii) gave rise to a vigorous allopolyploid involved in natural and artificial invasions on different continents. Spartina anglica was first recorded in France in 1906, and since then, it has spread all along the western French coast. Earlier studies revealed that native British populations display consistent morphological plasticity and lack of isozyme variation. In this paper, we use different molecular markers (randomly amplified polymorphic DNA, intersimple sequence repeats and restriction patterns from nuclear and chloroplast DNA sequences) to analyse the genetic patterns of the French populations of S. anglica. Our results show that French populations are mainly composed of one "major" multilocus genotype. This genotype is identical to the first-generation hybrid S. x townsendii from England. Losses of few markers from this genotype are observed but are restricted to a few populations from Brittany; it is likely that they appeared independently, subsequent to their introduction. In southern Brittany, no hybrids between S. anglica and S. maritima have been found where the two species co-occur. All French populations of S. anglica display the same chloroplast DNA sequences as S. alterniflora, the maternal genome donor. These findings are consistent with a severe genetic bottleneck at the time of the species formation, as a consequence of a unique origin of the species. Both parental nuclear sequences are present in the allopolyploid populations, revealing that for the markers investigated, no extensive changes have occurred in this young species.     

Spartina anglica C. E. Hubbard: a natural model system for analysing early evolutionary changes that affect allopolyploid genomes

Spartina anglica arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced East American Spartina alterniflora and following genome duplication of the hybrid ( S.  ×  townsendii ). This system allows investigations of the early evolutionary changes that accompany stabilization of a new allopolyploid species in natural populations. Various molecular data indicate that S. anglica has resulted from a unique parental genotype. This young species contains two distinctly divergent homoeologous genomes that have not undergone extensive change since their reunion. No burst of retroelements has been encountered in the F₁ hybrid or in the allopolyploid, suggesting a 'structural genomic stasis' rather than 'rapid genomic changes'. However, modifications of the methylation patterns in the genomes of S.  ×  townsendii and S. anglica indicate that in this system, epigenetic changes have followed both hybridization and polyploidization.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 475–484.     

Origin and genetic diversity of Spartina anglica (Poaceae) using nuclear DNA markers

Spartina alterniflora, introduced into the UK in the 1800s, was the seed parent in an interspecific hybridization with S. maritima. The sterile F1 hybrid S. ×townsendii gave rise to the fertile allopolyploid S. anglica by chromosomal doubling. Previous chromosome, isozyme, and cpDNA surveys did not reveal notable genetic variation within either the parental or the hybrid species. We used nuclear DNA markers (random amplified polymorphic DNA ([RAPD]) and inter-simple sequence repeats (ISSR) to further explore the origin, diversity, and parentage of S. anglica. We found DNA fragments in S. ×townsendii were the aggregate of diagnostic DNA fragments from S. maritima and S. alterniflora, thus confirming its hybrid origin. The S. ×townsendii genotype was identical to most of the S. anglica individuals analyzed, establishing the genetic concordance of these two taxa. We found widespread genetic variation within S. anglica. This could indicate that S. anglica arose several times, from different S. maritima sires. Alternatively, alleles could have been lost through recombination and/or through loss of entire chromosomes in S. anglica. Finally, all but one S. anglica individual had a S. alterniflora component that was indistinguishable from a S. alterniflora plant extant in Marchwood, UK, leaving open the possibility that this plant is the actual seed parent of S. anglica.     

The evolution of Spartina anglica C.E. Hubbard (Gramineae): origin and genetic variability

An extensive survey of isozyme phenotypes in British populations of the amphidiploid salt marsh grass Spartina anglica and its putative parents has confirmed that the species arose by chromosome doubling in S. × townsendii , a sterile hybrid between S. maritima and S. alterniflora. Isozyme phenotypes and seed protein profiles indicate that S. anglica is almost totally lacking in genetic variation. Isozyme evidence also indicates that the parental species are characterized by low levels of genetic variation. The lack of variation in S. anglica is proposed as being due to a narrow genetic base resulting from a single origin, or a multiple origin from uniform parents; the fact that many populations are derived from very small founder populations; and because preferential pairing between identical homologous chromosomes prevents recombination between the divergent component genomes of the species. The low levels of isozyme variation that occur appear to be due to chromosome loss.
The consequences for the future evolution of S. anglica , given its lack of genetic variation, are discussed.     

Molecular evidence for the maternal parentage in the hybrid origin of Spartina anglica C.E. Hubbard

Spartina anglica is a textbook example of a natural amphiploid, which originated from hybridization between S. alterniflora and S. maritima . Which of these species was the maternal parent has remained a mystery. Inheritance of chloroplast DNA in most angiosperms is strictly maternal and can thus be used to test the parentage of hybrid taxa. The DNA sequence of the chloroplast leucine tRNA gene intron was used to show that the introduced North American S. alterniflora is the female parent of the F ₁ hybrid S. x townsendii and the amphiploid S. anglica . A possible scenario for their origin is given.     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

The evolution of Spartina anglica G. E. Hubbard (Gramineae): genetic variation and status of the parental species in Britain

The perennial salt marsh grass Spartina anglica is one of the classic examples of allopolyploid speciation. It originated on the south coast of England at the end of the nineteenth century following chromosome doubling in S. × townsmdii , a hybrid between the native British S. marilima and a species introduced from the United States, S. alterniflora. The nature of the origin of S. anglica is beyond doubt; however, it is not known whether it had a single or multiple origin. In order to address this problem we undertook a survey of the genetic variation in the parental species of S. anglica using isozyme electrophoresis. The results show that S. alterniflora has no detectable variation and that S. maritima has extremely low levels of variation. These results, unfortunately, prevent the question of a single or multiple origin from being answered. Possible reasons for the low levels of variation and its influence on the future of the species are discussed. Another problem concerning the parental species is the rapid decline of S. maritima in Britain. It is often assumed that the major factor in this regression is the invasion of its habitats by S. anglica. We have examined the status of S. marilima throughout its range in Britain and have found that S. anglica rarely co-occurs with S. maritima. We propose that the decline of S. maritima is largely due to the physical erosion of its habitats and that this erosion may produce suitable habitats for colonization by S. anglica.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Genetic Diversity in Old Portuguese Durum Wheat Cultivars Assessed by Retrotransposon-Based Markers

Retrotransposon-based markers, such as the inter-retrotransposon-amplified polymorphism (IRAP) and the retrotransposon microsatellite-amplified polymorphism (REMAP) are highly informative, multilocus, and reveal insertional polymorphisms among plant individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, genetic mapping linkage, and for the detection of genetic rearrangements induced by polyploidization. In this study, we used IRAP and REMAP markers to assess the genetic diversity among 51 old Portuguese durum wheat cultivars belonging to 27 botanical varieties and to define their genetic relationships. Five IRAP and four REMAP primer combinations were used. IRAP markers revealed 66.3% of polymorphism and an average of 18.4 bands per primer combination which ranged in size from 450 to 3,100?bp. The REMAP technique allowed the detection of 86.36% of inter-cultivar polymorphism and an average of 11 bands per primer combination. The molecular weight of the REMAP bands ranged from 250 to 2,750?bp. The durum wheat cultivars analyzed here belong to 27 botanical varieties of the subspecies Triticum turgidum subsp. turgidum L. [syn. T. turgidum] and Triticum turgidum L. subsp. durum [syn. T. durum] (Desf.) Husn.. Our results showed that the genetic variability assessed by both the IRAP and REMAP markers did not allow the clustering of the durum wheat cultivars according to their taxonomical criteria (subspecies or botanical variety) or homonymy. Nonetheless, these markers were useful for the assessment of genetic diversity at the individual level, for the definition of genetic relationships among cultivars, and for estimation of the genetic structure of the Old collection under analysis. The achieved data could be valuable for future experiments of DNA fingerprinting, genetic improvement, and germplasm conservation in wheat.     

Retrotransposon based genetic status of North-West Himalayan Zingiber officinale revealed high heterogeneity

Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were successfully applied, for the first time, to analyze genetic diversity among 92 ginger landraces collected from north-western Himalayan region of India. Six IRAP primer/combinations generated 75 loci with an average of 12 loci/primer displaying an overall polymorphism of 95.95 %. On the other hand, twenty five REMAP primer combinations produced 414 loci with 96.5 % polymorphism. IRAP showed maximum Rp (5.39) and PIC (0.28) values, while the same in REMAP was observed to be 10.92 and 0.34. Cluster analysis using Jaccard’s similarity coefficient for IRAP and REMAP data ranged between 0.21 to 1.0 and 0.21 to 0.85, respectively distinguishing all the genotypes with diverse genetic makup. The results also confirmed the presence of sukkula retrotransposon (RT6) in the ginger genome which effectively acted as genetic marker revealing high regional genetic diversity in the ginger gene pool. The study will help in giving insight to the genetic constitution of vegetatively grown ginger crop and for its further utilization in improvement, conservation and management programmes.     

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.     

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.     

Genome merger: from sequence rearrangements in triticale to their elimination in wheat–rye addition lines

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat–rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat–rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale × wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat–rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat–rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.     

Retrotransposon Insertional Polymorphism in Iranian Bread Wheat Cultivars and Breeding Lines Revealed by IRAP and REMAP Markers

Inter-retrotransposon amplified polymorphisms (IRAPs) and retrotransposon-microsatellite amplified polymorphisms (REMAPs) were used to detect retrotransposon integration events and genetic diversity in 101 Iranian bread wheat (Triticum aestivum L.) cultivars and breeding lines. The 9 IRAP primers amplified 128 loci, and 20 REMAP primers amplified 263 loci. Percentage of polymorphic loci, average expected heterozygosity, number of effective alleles, and Shannon’s information index for the REMAP markers were slightly higher than those for the IRAP markers. The same estimated parameters calculated for native and nonnative retrotransposons were not considerably different. A Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation. Cluster analysis based on the Dice similarity coefficient and complete linkage algorithm using IRAP+REMAP loci identified five groups among the genotypes studied that could be applied as crossing parents in T. aestivum breeding programs.     

Uniformity of the nuclear and chloroplast genomes of Spartina maritima (Poaceae), a salt-marsh species in decline along the Western European Coast

Spartina maritima is a salt-marsh species from European and African Atlantic coasts. In the northern range of the species (including north-west France), a rapid decline of the populations has been observed during the 20th century. In this paper, the molecular diversity of 10 populations of S. maritima from France has been investigated using nuclear and chloroplast DNA markers: inter-simple sequence polymorphism (ISSR), randomly amplified polymorphic DNA (RAPD), inter-retrotransposon amplified polymorphism (IRAP), and PCR-RFLP of a 5 kb long portion of chloroplast DNA. The results reveal an extremely low level of genetic variation in this species: only one nuclear marker (out of 98) was polymorphic, with the presence of two genotypes randomly distributed among the populations. The lack of genetic diversity is interpreted in light of the almost exclusive vegetative propagation of the species in its northern range, the colonization history of the populations, and the origin of S. maritima (2n = 60) in the hexaploid lineage of the genus and in the context of the management of S. maritima populations.     

IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.