Ty1-copia Group Research Articles

Structural Diversity of a Novel LTR Retrotransposon, RTPOSON, in the Genus Oryza.

Retrotransposons with long terminal repeats (LTRs) are the most abundant transposable elements in plant genomes. A novel LTR retrotransposon named RTPOSON primarily occurs in the genus Oryza and in several species of the Poaceae family. RTPOSON has been identified in the Ty1-copia group of retrotransposons because two of its open reading frames encode an uncharacterized protein and UBN2_2 and zinc knuckle, respectively. More than 700 RTPOSONs were identified in Oryza genomes; 127 RTPOSONs with LTRs and gag-pol elements were classified into three subgroups. The subgroup RTPOSON_sub3 had the smallest DNA size and 97% (32/33) of RTPOSON elements from Oryza punctata are classified in this group. The insertion time of these RTPOSONs varied and their proliferation occurred within the last 8 Mya, with two bursting periods within the last 1.5–5.0 Mya. A total of 37 different orthologous insertions of RTPOSONs, with different nested transposable elements and gene fragments, were identified by comparing the genomes of ssp. japonica cv. Nipponbare and ssp. indica cv. 93–11. A part of intact RTPOSON elements was evolved independently after the divergence of indica and japonica. In addition, intact RTPOSONs and homologous fragments were preferentially retained or integrated in genic regions. This novel LTR retrotransposon, RTPOSON, might have an impact on genome evolution, genic innovation, and genetic variation.

LTR-retrotransposon activation, IRAP marker development and its potential in genetic diversity assessment of masson pine (Pinus massoniana)

Activation of retrotransposon is the pivotal factor for the genesis of genetic polymorphism. Retrotransposon-based molecular markers are excellent tools for detecting genetic diversity and genomic changes associated with retrotransposon activity. The scarcity of retrotransposon long terminal repeat (LTR) sequence limits the application of retrotransposon-based molecular marker systems. In this study, retrotransposon 3′-LTR segments were firstly isolated and characterized from the masson pine genome using a genome walking strategy, subsequently inter-retrotransposon amplified polymorphism (IRAP) markers were explored for genetic diversity assessment. These were shown to have clearly distinguishable amplification bands and high levels of polymorphism for masson pine. Dendrogram of our IRAP data highlighted that IRAP markers for individual elements are distinguishable and will shed light on the usage in genetic diversity studies of the masson pine. Transcription activation of Ty1-copia and Ty3-gypsy group retrotransposons in masson pine was investigated with exposure to various abiotic stresses. The insertional polymorphism and the transposition activation were detected by IRAP. Results revealed that none of the analyzed materials in the course of 2-month trials displayed fingerprint changes.

Isolation, identification, and characterization of genomic LTR retrotransposon sequences from masson pine (Pinus massoniana)

Long terminal repeat (LTR) retrotransposons are ubiquitous in the plant kingdom and play an important role in plant genome evolution. Masson pine (Pinus massoniana) is one of the most economically important forest trees. However, the masson pine genome is poorly understood. Conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy LTR retrotransposons were amplified from masson pine using degenerate primers. Sequence analysis showed less heterogeneity among Ty1-copia group retrotransposons than among Ty3-gypsy group retrotransposons. Phylogenetic analysis demonstrated that Ty1-copia and Ty3-gypsy RT sequences had considerable homology with other species, indicating that both vertical transmission and horizontal transmission may probably be the source of LTR retrotransposons in masson pine. Southern dot blot hybridization results suggested that both types of LTR retrotransposons are present in the genome of masson pine with high copy number. These results contribute to further understanding about the organization and evolution of the masson pine genome and provide fundamental genetic information for the utilization of retrotransposons in masson pine.

LTR-retrotransposon diversity and transcriptional activation under phytoplasma stress in Ziziphus jujuba

Degenerate primers were used to amplify the conserved domains of the Ty1-copia and Ty3-gypsy reverse transcriptase (RT) genes of the long terminal repeat retrotransposons in the jujube (Ziziphus jujuba Mill.) genome. Sequence analysis showed that 18 of the 50 Ty1-copia unique sequences and 28 of the 38 Ty3-gypsy unique sequences possessed stop codons, frameshifts, or both. The overall sequence divergence in jujube was higher in the Ty3-gypsy than the Ty1-copia group, in contrast to the results from other plants. Our results showed that sequence heterogeneity during vertical transmission could be a major influence on the evolution of copia- and gypsy-type retroelements in the jujube genome. In order to elucidate the transcriptional activity under phytoplasma stress of these two classes of RTs, RT-PCR primers were designed to amplify products corresponding to either the Ty3-gypsy or Ty1-copia RT domains from total RNA extracted from leaves or flowers of plants infected by phytoplasma. Amplification products were present for Ty3-gypsy RT in both tissues during infection; however, no RT-PCR products were amplified using the Ty1-copia RT domain primers in phytoplasma-infected leaves or flowers. RT-PCR amplification from total RNA extracted from leaves or flowers of healthy plants did not yield either of the RT fragments. The Ty3-gypsy RT-PCR products were confirmed to originate from the Z. jujuba genome. Our results indicated that the gypsy group of retrotransposons was autonomous retroelements. This is the first report characterizing the Ty1-copia and Ty3-gypsy groups of retrotransposons in the Z. jujuba genome.

Ty1-copia group retrotransposons and the evolution of retroelements in several angiosperm plants: evidence of horizontal transmission.

The phylogenetic relationships among thirty-seven new Ty1-copia group retrotransposons in seven angiosperm plants were examined by reverse transcriptase and ribonuclease H sequence analysis. Distribution pattern of the retrotransposons of closely related plant species generally reflects a close phylogenetic relationship. In contrast, we found that several retrotransposon sequences from the same genome exhibited a high degree of divergence and had a relatively high degree of identity versus retrotransposon sequences from widely divergent species, including an ancestral phytopathogen fungus. This finding supports the hypothesis that the horizontal transmission from phytopatogen organism to the host flowering plants could have played a role in the evolutionary dynamics of Ty1-copia group retrotransposons.

Isolation and characterization of Ty1-copia group of LTRs in genome of three species of Datura: D. innoxia, D. stramonium and D. metel.

Retrotransposons (RT) constitute a major fraction of plant genome. They are implicated in evolution and sequence organization. These elements have been proposed to have major role in evolution and variation in genome size. The sequence information of these RT regions in terms of divergence and conservation could be utilized for determining the interrelationship among various copia retrotransposons within the genome. In order to assess the diversity of Ty1-copia group of retroelements, reverse transcriptase (RT) sequence was amplified from genomes of three medicinally important Datura species: D. innoxia, D. stramonium and D. metel using the primers derived from two conserved domains of RT region. A total of twenty one independent amplicons from RT regions were cloned, sequenced and compared. The intra-family divergence at amino acid level ranged from 4 to 52%. Though intra-family RT sequences are conserved, no two sequences are identical. Southern blot hybridization suggested that Ty1-copia-like retrotransposons are dispersed throughout the Datura genome. The results indicate a high degree of heterogeneity among the Ty1-copia group of retroelements in Datura species.

Genome-wide detection of Ty1-copia and Ty3-gypsy group retrotransposons in Japanese apricot ( Prunus mume Sieb. et Zucc.)

The conserved domains of reverse transcriptase (RT) genes of approximately 260 bp for Ty1-copia and 430 bp for Ty3-gypsy groups of long terminal repeat (LTR) retrotransposons were amplified from Japanese apricot ( Prunus mume Sieb. et Zucc.) using degenerate oligonucleotide primers. Sequence analysis showed that 32.3% of Ty1-copia and 27.5% of Ty3-gypsy RT sequences possessed stop codons and/or frameshifts, and all sequences were AT-rich. Ty1-copia retrotransposon has higher heterogeneity than Ty3-gypsy retrotransposon, but the latter has a higher copy number revealed by southern dot blot hybridization. Phylogenetic analysis illustrated that some of the clones were more closely related to the representative elements present in other plant species than to other clones of Japanese apricot. Transcription was not detected by reverse transcription-polymerase chain reaction amplification for either Ty1-copia or Ty3-gypsy retrotransposons in young leaves of plants treated with UV light and 2,4-dichlorophenoxyacetic acid either individually or in both combinations, even though the ratios of dN/dS of the open reading frames among members of each subgroup of both group retrotransposons were less than 1. This is the first report on the presence of RT sequences of Ty1-copia and Ty3-gypsy group retrotransposons in Japanese apricot genome. Keywords: Prunus mume , retrotransposon, heterogeneity, copy number, transcriptional activity

Characterization of heterogeneity in Ty1-copia group retrotransposons in chickpea (Cicer arietinum L.)

Retrotransposons represent a major fraction of the plant genome and play a significant role in the molecular evolution through sequence re-organization. In order to access the diversity in Ty1-copia group of retrotransposons in chickpea, reverse transcriptase (RT) conserved domain specific primers were selected to amplify RT conserved sequences. Thirty-six amplified fragments were cloned and characterized. On the basis of deduced amino acid homology among them, these sequences were grouped into five families. These sequence families showed from 34 to 81% inter-family homology at the amino acid level. Although these sequences belong to a highly conserved region no two sequences were identical. The results show that there is a high degree of heterogeneity among the Ty1-copia group of retroelements in chickpea. The genomic Southern hybridization with one of the reverse transcriptase sequences as a probe shows the presence of a large population of the Ty1-copia group of retrotransposons in chickpea.

ISOLATION AND CHARACTERIZATION OF LTR RETROTRANSPOSONS FROM STRAWBERRY GENOME

Strawberry (Fragaria spp.) is a kind of herbaceous perennial plant that propagates vegetatively. The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of LTR retrotransposons were amplified from the cultivated and wild strawberry genomes. In the cultivated strawberry, sequence analysis of clones demonstrated that 5 of 19 Ty1-copia group unique sequences and 2 of 10 Ty3-gypsy unique sequences possessed either stop codon or frameshift. Ty1-copia group sequences are highly heterogeneous (divergence ranged from 1 to 69.8%), but the Ty3-gypsy group sequences are less variable (divergence ranged from 1 to 10%). In wild strawberry, sequence analysis of clones demonstrated that 11 of 34 Ty1-copia group unique sequences and 6 of 14 Ty3-gypsy unique sequences possessed either stop codon or frameshift. Southern dot blot hybridization result suggested that both of the LTR retrotransposons are present in the genome of cultivated strawberry (Fragaria × ananassa) with high copy number (Ty1-copia group 2,875; Ty3-gypsy group 348). Both types of LTR retrotransposons contributed to 15.8% of the cultivated strawberry genome of which the contribution of Ty1-copia retrotransposons being higher (13.5%) than that of Ty3-gypsy (2.3%). RT-PCR amplification from total RNA, which was extracted from callus induced from strawberry leaves cultured on MS medium supplemented with 2,4-D, yield the RT fragments of Ty1-copia retrotransposons.

Genome‐wide Characterization of Long Terminal Repeat ‐retrotransposons in Apple Reveals the Differences in Heterogeneity and Copy Number between Ty1‐copiaand Ty3‐gypsyRetrotransposons

The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of long terminal repeat (LTR) retrotransposons were isolated from the Malus domestica genome using degenerate oligonucleotide primers. Sequence analysis showed that 45% of Ty1-copia and 63% of Ty3-gypsy RT sequences contained premature stop codons and/or indels disrupting the reading frame. High heterogeneity among RT sequences of both Ty1-copia and Ty3-gypsy group retrotransposons was observed, but Ty3-gypsy group retrotransposons in the apple genome are less heterogeneous than Ty1-copia elements. Retrotransposon copy number was estimated by dot blot hybridizations for Ty1-copia (approximately 5,000) and Ty3-gypsy (approximately 26,000). All elements of the two types of LTR retrotransposons comprise approximately 38% of the M. domestica genome, with the Ty3-gypsy group contribution being higher (33.5%) than the Ty1-copia one (4.6%). Transcription was not detected by reverse transcription-polymerase chain reaction for either Ty1-copia or Ty3-gypsy retrotransposons in the leaves of plants in vitro or in leaf explants cultured on medium supplemented with high concentration benzylaminopurine. This research reveals the differences in heterogeneity and copy number between Ty1-copia and Ty3-gypsy retrotransposons in the apple genome. Ty1-copia retrotransposon has higher heterogeneity than Ty3-gypsy retrotransposon, but the latter has a higher copy number, which implies that Ty3-gypsy retrotransposons may play a more important role in the apple genome evolution.

Isolation and characterization of genomic retrotransposon sequences from octoploid strawberry (Fragaria × ananassa Duch.)

Strawberry (Fragaria spp.) is a kind of herbaceous perennial plant that propagates vegetatively. The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of LTR retrotransposons were amplified from the cultivated strawberry (Fragaria x ananassa Duch.). Sequence analysis of clones demonstrated that 5 of 19 Ty1-copia group unique sequences and 2 of 10 Ty3-gypsy unique sequences in F. x ananassa genome possessed either stop codon or frameshift. Ty1-copia group sequences are highly heterogeneous (divergence ranged from 1 to 69.8%), but the Ty3-gypsy group sequences are less (divergence ranged from 1 to 10%). Southern dot blot hybridization result suggested that both of the LTR retrotransposons are present in the genome of cultivated strawberry with high copy number (Ty1-copia group 2,875 Ty3-gypsy group 348). RT-PCR amplification from total RNA, which was extracted from leaves of micropropagated strawberry plants, did not yield either of the RT fragments. This is the first report on the presence of RT sequences of Ty1-copia and Ty3-gypsy group retrotransposons in F. x ananassa genome.

Heterogeneity in Ty1-copia group of retroelements in chickpea (Cicer arietinum) genome

AbstractRetrotransposons constitute a major fraction of plant genomes and these elements may have played a significant role in evolution and sequence organization of genomes. In order to access the diversity of Ty1-copia group of retroelements, reverse transcriptase (RT) sequences were amplified from chickpea genome, using the primers derived from two conserved domains of RT region. Thirty-six RT regions from independent amplicons were cloned and sequenced. On the basis of homology of deduced amino acids, the RT sequences could be grouped into three major families. The intra-family divergence at amino acid level ranges from 2 to 19%. Though intra-family RT sequences were conserved but no two sequences were identical. The results indicate a high degree of heterogeneity among the Ty1-copia group of retroelements from chickpea. It was possible to isolate RT specific sequences from RNA isolated from stressed seedlings, indicating that some of the retroelements may be functional under certain stress conditions.

Genetic diversity analysis in Vicia species using retrotransposon-based SSAP markers

Twelve different Ty1-copia and Ty3-gypsy group LTR retrotransposons were compared for their usefulness in SSAP marker development in two agriculturally important Vicia species. Three of the retrotransposons, PDR1, Tps19 and Tvf4, yielded useful SSAP marker systems in V. faba, and V. narbonensis. Another, Tvf1 was a good source of SSAP markers in V. narbonensis alone. The optimized SSAP marker systems were applied to the analysis of two diverse Vicia germplasm sets. Two hundred and two polymorphic Tvf1 SSAP markers were scored in 56 V. narbonensis samples and 196 polymorphic markers derived from the other three most useful retrotransposons were scored in a collection of 20 V. faba samples. The marker data were then used to construct phylogenetic trees. The trees for both species tend to show long-branch lengths, with rather little fine structure. Some V. narbonensis accessions cluster by geographical origin but many do not and a given geographical region is often represented by multiple diverse groups in the tree, suggesting a deep and ancient structure for the diversity of V. narbonensis that spans its current geographic range. The tree for the V. faba accessions also shows very limited clustering with geographical origin and no obvious correlation between diversity and morphology-based taxonomic groupings for the species.

SIRE-1, a putative plant retrovirus is closely related to a legume TY1-copia retrotransposon family.

SIRE-1 is a potential soybean retrovirus which has a gene order similar to Ty1-copia retrotransposons but also contains an envelope-like open reading frame (ORF), which is characteristic of retroviruses. PCR and Southern analysis reveals that SIRE-1 is closely related to a legume-wide family of envelope-lacking Ty1-copia group retrotransposons which suggests that SIRE-1 was formed by the recent acquisition of an envelope gene by a Ty1-copia retrotransposon.

Phylogenetic and molecular evolutionary analyses of Ty1-copia group retrotransposons in cultivated Egyptian cotton, Gossypium barbadense L.

The Ty1-copia group retrotransposons have been characterized in cultivated Egyptian cotton, G.barbadense L., using degenerate PCR primers for their reverse transcriptase (RT) domains. Comparative nucleotide and amino acid sequences analyses showed that G. barbadense Ty1-copia RT sequences are heterogeneous and this heterogeneity is resolved into 11 distinct families. The high ratio of synonymous to nonsynonymous changes indicates that there is a strong selection for the RT domain of these families. Phylogenetic analysis revealed that two cultivated G. barbadense RT families are closely related to Ty1-copia group retrotransposons present in other plant species. In other words, these families mirror their own phylogenies rather than that of their host cultivars. On the other hand, the remaining G. barbadense RT families are closely related to their respective Gossypium species. These data show that Ty1-copia group retrotransposons tend to span species boundaries, suggesting that they existed early in plant evolution, and were diverged into heterogeneous sequences prior to modern plant species divergence.

Retrotransposon populations of Vicia 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.

Ty1-copiagroup retrotransposon families in cultivated cottonsG. barbadenseL. identified by reverse transcriptase domain analysis

The objective of the current study was to analyze Ty1-copia group retrotransposons in cultivated G. barbadense L. cottons. DNA sequence analysis of 27 partial reverse transcriptase sequences revealed that these elements are heterogeneous and this heterogeneity is resolved into 11 distinct families. Phylogenetic analyses provided strong bootstrap support for a monophyletic origin of plant Ty1-copia group retrotransposons, yet showed high diversity within and between Gossypium species. Furthermore, G. Barbadense element topologies are incongruent with Gossypium phylogeny. The high ratio of synonymous to nonsynonymous changes indicates that the reverse transcriptase domain of these families is evolving under purifying selection. The antiquity and wide distribution of Ty1-copia group retrotransposons illustrate their active role in shaping and evolution of the Gossypium genome.

Ty1-copia group retrotransposons in persimmon (Diospyros kaki Thunb.).

We cloned and characterized Ty1-copia group retrotransposons in persimmon (Diospyros kaki Thunb.). Genomic DNA or methyl jasmonate (MJA)-treated cDNA were used as templates to amplify the reverse transcriptase region of Ty1-copia group retrotransposons. About 280 bp fragments were amplified and cloned, and 97 clones were sequenced. Forty-nine clones included frameshift or the stop codon, or both. Among 48 clones containing complete reading frames, 42 clones had unique nucleotide sequences. Alignment and phylogenetic analysis of putative amino acid sequences in the 42 clones indicated that these clones (named Tdk; retroTransposon in Diospyros kaki) fell into seven subgroups and six ungrouped sequences, indicating high sequence heterogeneity in Tdk clones. Phylogenetic analysis comparing unrelated plant species shows that some Tdk clones are more closely related to Ty1-copia group retrotransposons in the orders Solanales and Sapindales rather than to other Tdk clones. Southern blot analysis using Tdk2B, Tdk4c, Tdk6Ac, Tdk12K and Tdk13G clones as probes showed that persimmon and its related species, D. lotus, D. lotus var. glabba, D. oleifera, D. rhombifolia and D. virginiana, contained multiple Tdk-like sequences, indicating that homologous elements exist in other Diospyros species.

Isolation and characterization of two novel retrotransposons of the Ty1-copia group in oat genomes.

Two repetitive sequences, As32 and As22, of 826 and 742 bp, respectively, were isolated from Avena strigosa (As genome). Databank searches revealed their high homology to different segments of the family of Ty1-copia retrotransposons. Southern hybridization showed them to be present in diploid and polyploid oat species. Polymerase chain reaction with primers designed to amplify the segment between them showed that As32 and As22 sequences are composed of two different Ty1-copia retrotransposons. The segment amplified from the pAs32 insert was 2,264 bp long and contained the entire GAG and AP domains, and more than half of the IN domain. This new element has been designated TAS-1 (transposon, A. strigosa, 1) and appears to contain a long open reading frame that encodes a polypeptide of 625 amino acids. Slot-blot and fluorescence in situ hybridization analyses revealed it to be a component of both A- and D-genome chromosomes. Further, the chromosomes involved in one C-A intergenomic translocation in A. murphyi (AC genomes), one C-D intergenomic translocation in A. byzantina cv. Kanota (ACD genomes), and two C-D intergenomic translocations in A. sativa cv. Extra Klock, were identified. Based on its physical distribution and Southern hybridization pattern, a parental retro-transposon represented by TAS-1 appears to have been active at least twice during the evolution of the genomes in species of Avena.

Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements

To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarinia, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B. mori and B. mandarina, which are part of two novel Pao-like retrotransposons, Kamikaze and Yamato, respectively. Complete characterization of these and other elements of this group reported here shows that Pao-like elements have common features that distinguish them from the other groups of LTR-retrotransposons. While the elements of the Ty1-copia group encode only one cysteine and histidine (Cys) motif in their gag-like region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the integrase domain of the retrotransposon polyprotein seems to be conserved in Pao-like elements, but the number of amino acid residues between D and E varies and is greater than 35. A comparison of the deduced amino acid sequences of the reverse transcriptase domain revealed the Pao-like elements are members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, we confirmed that the long-terminal-repeat (LTR) retrotransposons should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like groups.