Molecular markers are essential in plant and animal breeding and biodiversity applications, in human forensics, and for map-based cloning of genes. The long terminal repeat (LTR) retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their "copy and paste" life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Nevertheless, up to a year has been required to develop a retrotransposon marker system in a new species, involving cloning and sequencing steps as well as the development of custom primers. Here, we describe a novel PCR-based method useful both as a marker system in its own right and for the rapid isolation of retrotransposon termini and full-length elements, making it ideal for "orphan crops" and other species with underdeveloped marker systems. The method, iPBS amplification, is based on the virtually universal presence of a tRNA complement as a reverse transcriptase primer binding site (PBS) in LTR retrotransposons. The method differs from earlier retrotransposon isolation methods because it is applicable not only to endogenous retroviruses and retroviruses, but also to both Gypsy and Copia LTR retrotransposons, as well as to non-autonomous LARD and TRIM elements, throughout the plant kingdom and to animals. Furthermore, the inter-PBS amplification technique as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence knowledge.
We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle.pol III ͉ genome evolution ͉ transcription ͉ transposable element R etrotransposons, excepting SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements), resemble retroviruses in their structure and intracellular life cycle. They are ubiquitous in the genomes of plants, animals, and fungi and account for Ͼ50% of large plant genomes (1, 2). Their life cycle comprises transcription of genomic copies, translation of their encoded proteins, packaging of the transcripts into virus-like particles, reverse transcription, and targeting of the cDNA copy to the nucleus for integration into the genome (3, 4). The transcriptional signals for RNA polymerase II (pol II) are found in the long terminal repeats (LTRs) at either end of the element, flanking the priming sites for reverse transcription and the coding domain specifying the proteins needed for replication and integration [supporting information (SI) Fig. S1].In addition to the classical retrotransposons, several well conserved nonautonomous groups have been discovered that lack all or part of their coding capacity (5). The BARE2 elements cannot express the capsid protein GAG (6), and Morgane lacks most of its coding capacity (7). The TRIM (terminal repeat retrotransposon in miniature) and LARD (large retrotransposon derivative) elements (Fig. S1) entirely lack reading frames for retrotransposon proteins (8-12). The TRIM elements are composed of 100-to 250-bp LTRs, priming sites for reverse transcriptase, and a small intervening segment. Evidence for past mobility suggests that they are activated by transcomplementation (10). These have been found in at least 13 species from four plant families (9, 10).Here, we describe a group of TRIM elements, which w...
Establishing the genotypic distribution in natural plant populations is a n important part of ecological studies concerning plant growth, reproduction and turn-over. Restriction enzyme-digested DNA samples, isolated from 24 plants of a natural Rubus idaeus population, were analysed with DNA fingerprinting using the M13 repeat sequence as well as a synthetic (AC)/(TG) polydinucleotide as hybridization probes. All the examined samples exhibit unique DNA fingerprint patterns, suggesting that vegetative reproduction may be considerably more restricted in wild R. idaeus populations than previously assumed. By comparison, all samples of the apomictic blackberry species Rubus nessensis, collected on the same location, were completely identical.
The purpose of the study was to support the selection process of the most valuable currant and gooseberry accessions cultivated in Northern Europe, in order to establish a decentralized core collection and, following the selection, to ensure sufficient genetic diversity in the selected collection. Molecular analyses of the material from nine project partners were run at seven different laboratories. The results were first analysed for each partner separately, and then combined to ensure sufficient genetic diversity in the core collection.
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