Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 2.17

ANALYSIS OF GYPSY- AND COPIA-LIKE RETROTRANSPOSONS IN THE OLIVE GENOME AND THEIR USE FOR STUDYING GENETIC DIVERSITY

T. GIORDANI, P. MAESTRINI, A. CAVALLINI, L. NATALI

Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Università di Pisa, Pisa

lnatali@agr.unipi.it

genome evolution, Olea europaea, olive, retrotransposons

Retrotransposons, or their remnants, usually represent a major fraction of interspersed repetitive DNA in eukaryotes, especially in plant species. It is generally accepted that retrotransposons have played an important role in plant genome evolution. Their amplification and dispersion contributed to genome plasticity perhaps allowing plants the adaptive responses to environmental stresses.

Analysis of the genomic repetitive component, especially retrotransposable elements, can be useful for studying important features of olive culture, such as the determination of phylogenetic relationships in the genus Olea and its close genera and the accurate identification of olive (Olea europaea L.) cultivars.

Since retrotransposons (particularly retrotransposons with long terminal repeats, LTR) are usually interspersed in different genomic regions, they may be used to generate molecular markers based on the amplification of retrotransposon flanking sequences, using as primers oligonucleotides designed on LTRs in conjunction with primers containing 3’-anchored microsatellites, i.e. short simple sequences (dinucleotides). The use of retrotransposon sequences to generate molecular markers depends on the availability of conserved LTR regions. In species in which complete retroelements are not available, it is necessary to extend retrotransposon fragments towards 5’- or 3’-ends to isolate putative LTRs. Then, since retroelement sequences are subject to high mutation rates during evolution, the conservation and the redundancy of putative LTR within the genome is to be tested before applying molecular marker techniques.

We have isolated DNA fragments belonging to Ty3-gypsy and Ty1-copia retrotransposons by PCR, using degenerated primers deduced from sequence databases. These fragments include portions of retrotranscriptase and RNAse H of a copia-like element, and portions of retrotranscriptase, RNAse H and integrase of a gypsy-like element. Such fragments have been extended using chromosome walking techniques, to retrotransposon 3’-extremities, i.e. the long terminal repeats (LTRs). Copia- and gypsy-like putative LTR sequences were quantitatively analysed in slot blot hybridisations to evaluate copy number and resulted medium-highly repeated. Southern blot experiments evidenced different hybridisation patterns, i.e., with typical smears and/or heavy hybridisation bands, indicating redundancy of the sequence and its genomic interspersion or with a few light hybridisation bands, indicating the degeneration of the retroelement. Using this approach and sequence comparison, retroelement subfamilies were characterised.

The LTR sequences belonging to different subfamilies can be used to design primers and generate molecular markers. Preliminary experiments showed polymorphism among different cultivars of Olea europaea.