THE ALISEI FAMILY OF GYPSY-LIKE RETROTRANSPOSONS IN THE NORWAY SPRUCE GENOME

¹ Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Universita' di Udine, Via delle Scienze 208, I-33100 Udine, Italy

² E.I. duPont de Nemours and Co., DuPont Agricultural Products, Biotechnology Research, Delaware Technology Park, PO Box 6104, Newark, DE 19714-6104 USA

Norway spruce (Picea abies K.) has, as other Gymnosperms, a very large genome (C=30x10⁹bp), 75 % of which is constituted by repetitive DNA. In order to characterise this repetitive fraction, we isolated more than 100 repetitive sequences, by screening small inserts genomic libraries with total genomic DNA of Norway spruce. Homology searches of sequence databases showed that a large fraction of these repetitive sequences belonged to Gypsy and Copia retrotransposable elements. We analysed in detail a Gypsy-like family that was subsequently called Alisei. First we sequenced some PCR fragments obtained with primers designed inside the pol gene, then the entire coding domain obtained by Long Range PCR using primers taken from an LTR fragment. Moreover, some genomic clones derived from the screening of a large insert genomic library were completely or partially sequenced, showing colinearity with the element obtained via Long Range PCR. We completely sequenced 24 Alisei LTRs (sequence divergence between 2.12-12.85%) obtained from genomic clones. The LTRs are 288-343bp long and show polymorphism for a tandem duplication of 44-53bp. We constructed a consensus sequence (internal domain 5206 bp) representative of the Alisei family based on all the sequences obtained. We were also able to reconstruct the putative aminoacid sequence of the Alisei pol gene, and on the basis of the homology with other Gypsy-like elements, we could identify the protease, reverse transcriptase, RNAse H and integrase domains. Dot blot analysis demonstrates that about 2.75 % of the genome is constituted of Alisei elements. A fragment of the pol gene corresponding to the integrase domain was amplified from three different genera of the Pinaceae and sequenced. This confirmed the presence of the Alisei family across the Pinaceae and produced a phylogenetic tree compatible with a vertical transmission of these elements during the Pinaceae evolution. A marker system that allows the detection of the presence or absence of the elements in multiple locations in the genome has also been developed.