Isolation of Ty1-copia putative LTR sequences and their use as a tool to analyse genetic diversity in Olea europaea

A DNA fragment putatively belonging to Ty1-copia retrotransposons was isolated from genomic DNA of Olea europaea L., by PCR, using degenerated primers deduced from sequence databases. This fragment includes portions of reverse transcriptase and RNAseH of a copia-like element and has been extended, using chromosome walking techniques, to retrotransposon ends, i.e. the long terminal repeats (LTRs). Ten putative LTR sequences were isolated, and sequence analysis showed that they belong to three different copia subgroups. Putative LTR sequences were evaluated in slot blot hybridisations as to their copy number: only three sequences (Oec04, Oec06 and Oec07), belonging to the same subgroup, were shown to be middle repetitive, with a redundancy ranging from 2.0 x 10(3) to 2.4 x 10(3) copies per genome. Southern blot experiments using Oec06 as probe evidenced hybridisation patterns with many weak bands and heavy background smear, indicating genomic interspersion of related sequences. Oec06 was also used to design a primer and to generate REtroelement Microsatellite Amplified Polymorphism (REMAP) molecular markers to distinguish different cultivars of Olea europaea. A total of 108 bands, of which 100 were polymorphic, were obtained. Genetic similarities among cultivars were calculated according to Simple Matching Similarity Index and used to build a dendrogram based on the UPGMA. A comparison between polymorphism based only on retrotransposon sequences and on inter-simple-sequence-repeats (ISSR) showed that polymorphism associated with the former was much higher than that shown with the latter. Our results demonstrate that REMAP markers can be useful in evaluating Olea europaea genetic diversity and in classifying cultivars.