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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 4.48

ISOLATION OF SIMPLE SEQUENCE REPEATS FROM ARTICHOKE

Sonnante G.*, De Paolis A.**

*) CNR - Istituto del Germoplasma, Bari

**) CNR - Istituto di Scienze delle Produzioni Alimentari (ISPA), Sez. di Lecce

artichoke, SSR, microsatellites, isolation, flanking regions

Microsatellites consist of tandem repeats of mono-, di-, tri- or tetra-nucleotide patterns, also called simple sequence repeats (SSRs). These markers are frequent and usually well distributed in plant genomes. The may be used to develop locus-specific markers, thanks to the fact that the flanking regions of the microsatellite motif are usually well conserved within a species or among closely related species. Therefore, these markers find several applications in genetic mapping, genome analysis and variety identification.

A preliminary analysis of microsatellite oligonucleotides hybridization on artichoke DNA digested with a number of restriction enzymes had evidenced that some repetitive sequences produce useful polymorphisms in artichoke accessions. In order to identify and isolate microsatellite sequences in artichoke, a genomic library was constructed in lambda vector and analysed. Briefly, genomic DNA was extracted from leaves, partially digested with the restriction enzyme Sau3A1 and centrifuged on a continuous (10-40%) sucrose gradient. The fractions containing DNA fragments in the range of 4-6 Kb were collected and pooled. These fragments were used for ligation in Lambda Zap-expression vector and packed. About 500 pfu of the obtained library were screened for repetitive sequences using some oligonucleotides containing the microsatellites terminally marked with digoxigenin. A preliminary screening of the library showed some positive hybridization signals. Two positive plaques with (CAT)₈ probe were isolated and used for in vivo exicion in order to obtain the insert in the plasmid vector. The recombinant plasmid extract from this clone will be purified and finally sequenced to confirm its repetitive structure. Primers specific to microsatellite flanking regions will be synthesised and used to amplify microsatellite sequences and look for polymorphisms in wild and cultivated artichoke accessions.