Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 × 10⁴ in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex. This revised version was published online in August 2006 with corrections to the Cover Date.     

A full lengthTy3/gypsy-type retrotransposon in the fernAdiantum

Ty3/gypsy-type LTR-retrotransposons have been found only in lily and maize but not in cryptogam. In fernAdiantum, we recently found a full-lengthTy3/gypsy-type LTR-retrotransposon (ARET-1; 8284 bp). This retrotransposon has both 5′ and 3′ LTRs (1.2 kb), a primer binding site, a polypurine tract, and an RNA binding motif and its domain arrangement in thepol region is the same as that ofTy3/gypsy-type retrotransposon. These results suggest thatTy3/gypsy-type retrotransposons are widespread among vascular plants. The nucleotide sequence data reported will appear in the EMBL, DDBJ and GenBank Nucleotide Sequence Databases under the accession number AB003364.     

Retrotransposon populations of <Emphasis Type="Italic">Vicia</Emphasis> species with varying genome size

The (non-LTR) LINE and Ty3-gypsy-type LTR retrotransposon populations of three Vicia species that differ in genome size (Vicia faba, Vicia melanops and Vicia sativa) have been characterised. In each species the LINE retrotransposons comprise a complex, very heterogeneous set of sequences, while the Ty3-gypsy elements are much more homogeneous. Copy numbers of all three retrotransposon groups (Ty1-copia, Ty3-gypsy and LINE) in these species have been estimated by random genomic sequencing and Southern hybridisation analysis. The Ty3-gypsy elements are extremely numerous in all species, accounting for 18–35% of their genomes. The Ty1-copia group elements are somewhat less abundant and LINE elements are present in still lower amounts. Collectively, 20–45% of the genomes of these three Vicia species are comprised of retrotransposons. These data show that the three retrotransposon groups have proliferated to different extents in members of the Vicia genus and high proliferation has been associated with homogenisation of the retrotransposon population.Electronic Supplementary Material Supplementary material is available for this article at .     

Is Oryza malampuzhaensis Krish. et Chand. (Poaceae) a valid species? Evidence from morphological and molecular analyses

Phylogenetic relationship between O. malampuzhaensis Krish. et Chand. (2n = 4x = 48; Poaceae, Oryzeae), a South Indian endemic wild rice with a disputed taxonomic identity, and eight other species belonging to the O. officinalis complex of the genus Oryza was examined using 62 morphological characters and 445 random amplified polymorphic DNA (RAPD) markers. Multivariate and cluster analyses using both the data sets clearly separated all accessions of O. malampuzhaensis into a distinct group. Genetic distances between O. malampuzhaensis and other species in O. officinalis complex were comparable with the distance between any other two taxa with species rank in this complex. Case-by-case taxonomic treatment of O. malampuzhaensis in relation to other species examined is presented. A taxonomic key for the discrimination of O. malampuzhaensis from other species in the O. officinalis complex has been constructed. Based on the present results, we strongly argue to restore the species rank to O. malampuzhaensis, as originally proposed by Krishnaswamy and Chandrasekharan (1958).     

Specific endonuclease activity of integrase encoded by the mdg4 (gypsy) retrotransposon

To study the mechanism of precise excision ofgypsy from genomic sites, the integrase domain ofgypsy pol was cloned and expressed inEscherichia coli. The endonuclease activity of recombinant integrase was assayed with synthetic substrates corresponding to 3′-U5 ofgypsy LTR and to the known genomic insertion sites ofgypsy. Integrase nicked the 5′-A ⇓ YR-3′ triplet in the (+) strand of the double-stranded substrates; cleavage of a single-stranded substrate was nonspecific. Cleavage proved to be affected by the local conformation of the substrate: the (+) strand was cleaved more efficiently when the (−) strand had an unpaired base in the triplet and was not cleaved when the (−) strand was interrupted or branched. The triplet corresponded to the consensus region ofgypsy insertion (5′-YRYR ⇓ YR-3′), the site of cleavagein vitro coinciding with the site of insertionin vivo. The unique mechanism ofgypsy excision was assumed to depend to a great extent on the enzymic properties of its integrase.     

Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlue-Script II KS⁺ vector (pBSLTR), and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the lacZ gene; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACYCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 10^?4 to 10^?3, the frequency of spontaneously appearing blue colonies not exceeding 10^?9 to 10^?8. The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYCint) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the β-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them. gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).     

Polymorphism of canonical and noncanonical <Emphasis Type="Italic">gypsy</Emphasis> sequences in different species of <Emphasis Type="Italic">Drosophila </Emphasis><Emphasis Type="Italic">melanogaster</Emphasis> subgroup: possible evolutionary relations

Mobile genetic elements constitute a substantial part of eukaryotic genome and play an important role in its organization and functioning. Co-evolution of retrotransposons and their hosts resulted in the establishment of control systems employing mechanisms of RNA interference that seem to be impossible to evade. However, “active” copies of endogenous retrovirus gypsy escape cellular control in some cases, while its evolutionary elder “inactive” variants do not. To clarify the evolutionary relationship between “active” and “inactive” gypsy we combined two approaches: the analysis of gypsy sequences, isolated from G32 Drosophila melanogaster strain and from different Drosophila species of the melanogaster subgroup, as well as the study of databases, available on the Internet. No signs of “intermediate” (between “active” and “inactive”) gypsy form were found in GenBank, and four full-size G32 gypsy copies demonstrated a convergence that presumably involves gene conversion. No “active” gypsy were revealed among PCR generated gypsy ORF3 sequences from the various Drosophila species indicating that “active” gypsy appeared in some population of D. melanogaster and then started to spread out. Analysis of sequences flanking gypsy variants in G32 revealed their predominantly heterochromatic location. Discrepancy between the structure of actual gypsy sites in G32 and corresponding sequences in database might indicate significant inter-strain heterochromatin diversity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

A tourist element in the 5'-flanking region of the catalase gene CatA reveals evolutionary relationships among Oryza species with various genome types

Tourist-OsaCatA, a transposable element, was found in the 5′-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O. longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O. ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O. longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O. longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O. officinalis, O. ridleyi and O. meyeriana complexes then diverged from the ancestor of O. longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species. Received: 12 March 1999 / Accepted: 7 July 1999     

Molecular phylogeny and systematics of drosophila retrotransposons and retroviruses

Full classification of Drosophila melanogaster retrotransposons with long terminal repeats (LTR-retrotransposons) has been recomposed, and their evolutional analysis in sequenced genomes of different species of drosophila and other arthropods has been carried out. D. melanogaster LTR-retrotransposons are divided into three groups: gypsy (one, two, or three open reading frames (ORFs)), copia (one ORF), and BEL (one ORF). The gypsy group is divided into three subgroups. Subgroup I is underrepresented by retrotransposons-retroviruses with three ORFs and their derivatives, which have lost the env gene (ORF3). Subgroup II is underrepresented by retrotransposons with two ORFs, and subgroup III is underrepresented by retrotransposons with one ORF. A comparative analysis of homologs of gypsy group LTR-retrotransposons evidences that subgroups I and II are represented only in the genomes of Lepidoptera and Diptera. The gypsy group of LTR-retrotransposons with one and two ORFs is found in almost all genomes of arthropods. Most of the families of D. melanogaster gypsy group LTR-retrotransposons have close homologs in the genomes of other species of drosophila. A degree of identity of retrotransposons sequences is correlated with a degree of relation between species of drosophila, indicating vertical transmission of retrotransposons. Obvious cases of horizontal transfer of some mobile elements have been detected including retrotransposons without the env gene. Homologs of distinct ORFs of retrotransposons—genes gag and env—have been found. Gene-homolog of the gag gene—Grp (CG5680)—is under purifying selection, so it has an important function in drosophila genome.     

Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs

Summary Ninety-three accessions representing 21 species from the genus Oryza were examined for restriction fragment length polymorphism. The majority (78%) of the accessions, for which five individuals were tested, were found to be monomorphic. Most of the polymorphic accessions segregated for only one or two probes and appeared to be mixed pure lines. For most of the Oryza species tested, the majority of the genetic variation (83%) was found between accessions from different species with only 17% between accessions within species. Tetraploid species were found to have, on average, nearly 50% more alleles (unique fragments) per individual than diploid species reflecting the allopolyploid nature of their genomes.Classification of Oryza species based on RFLPs matches remarkably well previous classifications based on morphology, hybridization and isozymes. In the current study, four species complexes could be identified corresponding to those proposed by Vaughan (1989): the O. ridleyi complex, the O. meyeriana complex, the O. officinalis complex and the O. sativa complex. Within the O. sativa complex, accessions of O. rufipogon from Asia (including O. nivara) and perennial forms of O. rufipogon from Australia clustered together with accessions of cultivated rice O. sativa. Surprisingly, indica and japonica (the two major subspecies of cultivated rice) showed closer affinity with different accessions of wild O. Rufipogon than to each other, supporting a hypothesis of independent domestication events for these two types of rice. Australian annual wild rice O. meridionalis (previously classified as O. rufipogon) was clearly distinct from all other O. rufipogon accessions supporting its recent reclassification as O. meridionalis (Ng et al. 1981). Using genetic relatedness as a criterion, it was possible to identify the closest living diploid relatives of the currently known tetraploid rice species. Results from these analyses suggest that BBCC tetraploids (O. malampuzhaensis, O. punctata and O. minuta) are either of independent origins or have experienced introgression from sympatric C-genome diploid rice species. CCDD tetraploid species from America (O. latifolia, O. alta and O. grandiglumis) may be of ancient origin since they show a closer affinity to each other than to any known diploid species. Their closest living diploid relatives belong to C genome (O. eichingeri) and E genome (O. Australiensis) species. Comparisons among African, Australian and Asian rice species suggest that Oryza species in Africa and Australia are of polyphyletic origin and probably migrated to these regions at different times in the past.Finally, on a practical note, the majority of probes used in this study detected polymorphism between cultivated rice and its wild relatives. Hence, RFLP markers and maps based on such markers are likely to be very useful in monitoring and aiding introgression of genes from wild rice into modern cultivars.     

Ty1-copia group retrotransposons and the evolution of retroelements in the eukaryotes

Ty1-copia group retrotransposons are among the best studied transposable elements in the eukaryotes. This review discusses the extent of these transposons in the eukaryote kingdoms and compares models for the evolution of these genetic elements in the light of recent phylogenetic data. These data show that the Ty1-copia group is widespread among invertebrate eukaryotes, especially in the higher plant kingdom, where these genetic elements are unusually common and heterogeneous in their sequence. The phylogenetic data also suggest that the present day spectrum of Ty1-copia group retrotransposons has been influenced both by divergence during vertical transmission down evolving lineages and by horizontal transmission between distantly related species. Lastly, the factors affecting Ty1-copia group retrotransposon copy number and sequence heterogeneity in eukaryotic genomes and the effects of transpositional quiescence and defective retrotransposons upon evolution of Ty1-copia group retrotransposons are discussed.