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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

ISOLATION OF POLYMORPHIC MICROSATELLITES IN P. PRATENSIS L. BY USING THE NEW MICROSATELLITE-AFLP (M-AFLP)PROCEDURE

ALBERTINI E.*, BERTOLI F., MARCONI G., FALCINELLI M.

Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia

* emidalb@tiscalinet.it; web.tiscalinet.it\emidioalbertini

SSR, AFLP, Poa pratensis, M-AFLP

Microsatellites, or simple sequence repeats (SSRs) are valuable markers for plant and animal breeding due to their high polymorphism information content compared to AFLP markers and other forms of single nucleotide polymorphisms (SNPS). The development of locus-specific SSR markers requires the isolation and characterization of individual loci, a process involving the construction and screening of classical or enriched DNA libraries with microsatellite-specific probes, followed by DNA sequencing of positive clones. The recovery rate of useful SSRs is generally low due to non-specific amplification and monomorphic loci. These procedures are time consuming and very expensive and limit the routine application of SSRs in the genetic study of non-commercial species and for identifying markers located in chromosomal region of interest. Several techniques that generate multi-locus SSR fingerprints are able to utilize SSRs as molecular markers without the expense of single locus isolation. Most of them are unfortunately dominant. Here a microsatellite-AFLP (M-AFLP) as a method for simultaneous co-amplification of microsatellite and AFLP markers is presented. M-AFLP is based on the use of an AFLP primer (Eco-RI+3 primer) in combination with a primer consisting of a microsatellite repeat sequence anchored at the 5’ end by a random sequence (RAMP primer). Amplification with these primers, using AFLP pre-amplification mixtures as starting material, under AFLP selective amplification conditions, results in microsatellite-enriched fingerprints. We report the isolation of 50 microsatellites obtained by using this approach in Poa pratensis L.. The isolated M-AFLP have been cloned, sequenced and converted into conventional SSR assays based on flanking PCR primers. Microsatellite-AFLP provides a valuable technique for rapid development of informative SSRs randomly across the genome and for their routine detection even in complex species such as P. pratensis.