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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S4g

DEVELOPMENT AND CHARACTERIZATION OF MICROSATELLITE LOCI IN ARTICHOKE (CYNARA CARDUNCULUS VAR. SCOLYMUS L.)

PORTIS E.*, ACQUADRO A.*, SABA E.**, LANTERI S.*

*) Di.Va.P.R.A. settore Genetica Agraria, Università di Torino.

**) Dipartimento di Scienze agronomiche e Genetica vegetale agraria, Università di Sassari.

artichoke, microsatellite, sequence database, enriched library, M-AFLP

Globe artichoke (C. cardunculus L. var. scolymus = Cynara scolymus L.) is a perennial and predominantly cross pollinated species native to the Mediterranean basin, where the cultivated cardoon C. cardunculus L. var. altilis DC and at least seven other wild Cynara taxa are present.

Italy is the richest source of primary cultivated ‘gene pool’, as harbours many distinct clonal cultivars, best adapted to local environments and tastes. Today commercial production is mainly based on perennial cultivation of vegetatively propagated clones, which are usually highly heterozygous and segregate widely when progeny tested.

Studies based on the application of molecular markers to artichoke genome are extremely limited. In previous studies we applied RAPD markers for the analysis of genetic variation and ISSR and AFLP markers for the fingerprinting of selected clones within the varietal type ‘Spinoso sardo’.

Here we report our results on the development of microsatellites, or simple sequence repeats (SSRs). Because of their high variability, SSRs have proven to be an extremely valuable tool for genome mapping as well as for population genetics studies and conservation/management of germplasm.

Three approaches have been followed: search in sequence database, enriched library and M-AFLP (Microsatellite – AFLP).

The twenty Cynara sequences that were present in the EMBL and GeneBank nucleic acid databases were analysed using the microsatellite motif identification program Sputnik. Four microsatellite motifs were found and primer designed on flanking regions.

A biotin/streptavidin capture technique for (CA)₁₀ and (CT)₁₀ motifs was used for the isolation of SSR directly from the artichoke genome. Twenty nine transformed Eco strain INVaF’ colonies gave a positive signal when hybridized with digoxigenin-labelled probes. Twelve clones were confirmed to contain SSR motifs by PIMA (PCR-based Isolation of Microsatellite Arrays) using (GT)₁₀H or (GA)₁₀H primers in combination with SP6 and T7 primers. Five microsatellite loci were detected and primer designed on flanking region.

Among 16 M-AFLP primer combinations tested (obtained by combining four 5’ anchored ISSR primers and four Eco primers with three selective nucleotides), 10 originated clear electrophoretic patterns. Twenty five bands, containing putative microsatellites, were detected, eluted, re-amplified and observed as pure fragments on agarose gel. After cloning and sequencing, they were found to include repetitive motifs and primers were designed for 15 selected loci on one flanking region.

The developed SSRs were tested for polymorphism in 15 artichoke accessions which are representative of the genetic variation within cultivated varietal types, in three accession of cultivated cardoon and one accession of wild cardoon.