Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5′-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hot-spot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp. Received: 17 March 1999 / Accepted: 20 July 1999     

Genetic diversity in European Pisum germplasm collections

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.     

Analysis of PDR1 retrotransposon insertions in the pea (Pisum Sativum L.)

We research polymorphism of PDR1 insertion grades, lines, and mutants of peas by means of seven pairs of specific RBIP-primers. Seven PDR1 insertions loci are identified in the studied forms of peas. We establish the high level of polymorphism of PDR1 retrotransposon inserts with primers RBIP2, RBIP1794, and RBIP64, that has made 92, 82.98 and 78%, respectively. The genetic analysis in a population of F2 hybrids (Chil 15 × WL1238) is conducted. RBIP700 polymorphic fragment is localized on a genetic map of peas in the third group of coupling.     

A non-LTR retroelement extinction in Spermophilus tridecemlineatus

The typical mammalian genome is dominated by two types of transposable elements (TEs), the autonomous and non-autonomous non-LTR retrotransposons, i.e. LINEs and SINEs, and with few exceptions there is a sole active LINE family (L1). During an ongoing investigation of TEs in rodents we determined that overall transposon activity has been steadily declining in Spermophilus tridecemlineatus. More specifically, the typically ubiquitous L1 activity of mammals has decreased drastically within the last 26MY. Indeed, only three L1 insertions with intact ORF1 sequences were readily identifiable and no intact ORF2 sequences were identified. The last L1 and SINE insertions date to ~5.3MYA and 4MYA, respectively. Based on our inability to computationally identify recently inserted L1 elements we suggest that S. tridecemlineatus is experiencing a quiescence or extinction of non-LTR retrotransposon activity. Such a finding represents only the fourth instance of a loss of non-LTR retrotransposon activity identified in mammals and, as such, represents an important additional data point to guide our understanding of LINE dynamics in eutherians.     

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum

The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and inter-species relationships within Pisum. Received: 23 January 2000 / Accepted: 24 March 2000     

Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis

Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in Pisum sativum have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co-dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.     

Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R₁ lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000–16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula . This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula , and could be a primary choice for other plant species with large genomes.     

Transposable elements reveal the impact of introgression,rather than transposition,in Pisum diversity,evolution, and domestication

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.     

Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP)

Retrotransposons are present in high copy number in many plant genomes. They show a considerable degree of sequence heterogeneity and insertional polymorphism, both within and between species. We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for the generation of molecular markers in barley. The method produces amplified fragments containing a Bare–1-like retrotransposon long terminal repeat (LTR) sequence at one end and a flanking host restriction site at the other. The level of polymorphism is higher than that revealed by amplified fragment length polymorphism (AFLP) in barley. Segregation data for 55 fragments, which were polymorphic in a doubled haploid barley population, were analysed alongside an existing framework of some 400 other markers. The markers showed a widespread distribution over the seven linkage groups, which is consistent with the distribution of the Bare–1 class of retrotransposons in the barley genome based on in situ hybridisation data. The potential applicability of this method to the mapping of other multicopy sequences in plants is discussed.     

Genomic instability associated with human LINE-1 rétrotransposition

LINE-1 (L1) retrotransposon accounts for approximately 17 % of the human genome. Because of the great number of identical copies, L1 can be implicated in genomic rearrangements associated with events of homologous recombination between heterologous sites. Moreover, even if the vast majority of the L1 elements are inactive, some are still able to mobilize themselves by retrotransposition. Thus, L1 is regarded as an insertional mutagenic agent. Moreover, recent works have shown that active retrotransposons were able to mobilize other sequences to generate retro-pseudogenes or to amplify other repeated sequences. Finally, L1 has been associated recently with new genomic rearrangements generated upon insertions such as large genomic deletions. L1 then can be considered as a major factor that has affected and shaped the human genome through several mechanisms.     

Parasitism and the retrotransposon life cycle in plants: a hitchhiker's guide to the genome

LTR (long terminal repeat) retrotransposons are the main components of higher plant genomic DNA. They have shaped their host genomes through insertional mutagenesis and by effects on genome size, gene expression and recombination. These Class I transposable elements are closely related to retroviruses such as the HIV by their structure and presumptive life cycle. However, the retrotransposon life cycle has been closely investigated in few systems. For retroviruses and retrotransposons, individual defective copies can parasitize the activity of functional ones. However, some LTR retrotransposon groups as a whole, such as large retrotransposon derivatives and terminal repeats in miniature, are non-autonomous even though their genomic insertion patterns remain polymorphic between organismal accessions. Here, we examine what is known of the retrotransposon life cycle in plants, and in that context discuss the role of parasitism and complementation between and within retrotransposon groups.     

Analysis of genes associated with retrotransposons in the rice genome

Retrotransposons comprise a significant fraction of the rice genome. Despite their prevalence, the effects of retrotransposon insertions are not well understood, especially with regard to how they affect the expression of genes. In this study, we identified one-sixth of rice genes as being associated with retrotransposons, with insertions either in the gene itself or within its putative promoter region. Among genes with insertions in the promoter region, the likelihood of the gene being expressed was shown to be directly proportional to the distance of the retrotransposon from the translation start site. In addition, retrotransposon insertions in the transcribed region of the gene were found to be positively correlated with the presence of alternative splicing forms. Furthermore, preferential association of retrotransposon insertions with genes in several functional classes was identified. Some of the retrotransposons that are part of full-length cDNA (fl-cDNA) contribute splice sites and give rise to novel exons. Several interesting trends concerning the effects of retrotransposon insertions on gene expression were identified. Taken together, our data suggests that retrotransposon association with genes have a role in gene regulation. The data presented in this study provides a foundation for experimental studies to determine the role of retrotransposons in gene regulation.     

Osmotic shock improves <Emphasis Type="Italic">Tnt1</Emphasis> transposition frequency in <Emphasis Type="Italic">Medicago truncatula</Emphasis> cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.     

Genomewide comparative analysis of the highly abundant transposable element DINE-1 suggests a recent transpositional burst in Drosophila yakuba

DINE-1 (Drosophila interspersed element) is the most abundant repetitive sequence in the Drosophila genome derived from transposable elements. It comprises >1% of the Drosophila melanogaster genome (DMG) and is believed to be a relic from an ancient transpositional burst that occurred approximately 5-10 MYA. We performed a genomewide comparison of the abundance, sequence variation, and chromosomal distribution of DINE-1 in D. melanogaster and D. yakuba. Unlike the highly diverged copies in the DMG (pairwise distance approximately 15%), DINE-1's in the Drosophila yakuba genome (DYG) have diverged by only 3.4%. Moreover, the chromosomal distribution of DINE-1 in the two species is very different, with a significant number of euchromatic insertions found only in D. yakuba. We propose that these different patterns are caused by a second transpositional burst of DINE-1's in the D. yakuba genome approximately 1.5 MYA. On the basis of the sequence of these recently transposed copies, we conclude that DINE-1 is likely to be a family of nonautomomous DNA transposons. Analysis of the chromosomal distribution of two age groups of DINE-1's in D. yakuba indicates that (1) there is a negative correlation between recombination rates and the density of DINE-1's and (2) younger copies are more evenly distributed in the chromosome arms, while older copies are mostly located near the centromere regions. Our results fit the predictions of a selection-transposition balance model. Our data on whole-genome comparison of a highly abundant TE among Drosophila sibling species demonstrate the unexpectedly dynamic nature of TE activity in different host genomes.     

Retrotransposon characterisation and fingerprinting of apple clones by S-SAP markers

Retrotransposons have been found to comprise the most common class of transposable elements in eukaryotes and to occur in high copy number in plant genomes. Several of these elements have been sequenced and were found to display a high degree of heterogeneity and insertional polymorphism, both within and between species. The dispersion, ubiquity and prevalence of retrotransposons in plant genomes provide an excellent basis for the development of marker systems and, hence, may be good molecular candidates in distinguishing among apple clones, when they represent bud mutations of the original variety, considering that the random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) used thus far in fingerprinting analyses have failed to meet discrimination expectations. The technique called sequence-specific amplified polymorphism (S-SAP), which makes it possible to identify dominant markers for the detection of variation in the DNA flanking the retrotransposon insertion site, was used in the present study to distinguish several clones of the cultivars ‘Gala’ and ‘Braeburn’ in apple fingerprinting. Moreover, our results suggest that the bud mutations, which have generated new patented varieties of ‘Gala’ and ‘Braeburn’, appear to derive from retrotransposon insertion.