iPBS: a universal method for DNA fingerprinting and retrotransposon isolation

• Published in: Theoretical and applied genetics 2010-11, Vol.121 (8), p.1419-1430
• Main Authors: Kalendar, Ruslan, Antonius, Kristiina, Smýkal, Petr, Schulman, Alan H
• Format: Article
• Language: English
• Subjects: Agriculture; Animals; Base Sequence; Binding Sites; Biochemistry; Biodiversity; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Classical genetics, quantitative genetics, hybrids; Cloning; Computational Biology; Criminal investigation; DNA; DNA Fingerprinting - methods; DNA polymerases; DNA Primers - metabolism; DNA testing; Fundamental and applied biological sciences. Psychology; Genes; Genetics of eukaryotes. Biological and molecular evolution; Genic rearrangement. Recombination. Transposable element; Genome - genetics; Genomes; Genomics; Life Sciences; Malus - genetics; Methods; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Original Paper; Plant Biochemistry; Plant Breeding/Biotechnology; Plant Genetics and Genomics; Polymorphism; Polymorphism, Genetic; Pteridophyta, spermatophyta; Retroelements - genetics; Retrovirus; Terminal Repeat Sequences - genetics; Transfer RNA; Vegetals; Zea mays - genetics
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/s00122-010-1398-2

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.