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Random amplified hybridization microsatellites (RAHM): isolation of a new class of microsatellite-containing DNA clones.

Microsatellite repeats are widely present in eukaryotic genomes (1,2) and are interspersed throughout the genomic DNA. Microsatellite markers have been developed for a variety of organisms (3,4), including plants (5-7). The regions flanking microsatellite sequences can be amplified in a PCR reaction using microsatellite primers, thus providing codominant sequence tagged sites (STS) markers. However, efforts to develop such molecular markers are quite laborious, requiring library construction, screening, and sequencing of clones. Several groups have developed alternative methods for detection (8) and isolation of flanking sequences (9,10) using PCR. Random amplified polymorphic DNA (RAPD) markers are easy to develop and less expensive to assay, but generally are genetically dominant and have a low degree of polymorphism. We have developed a new system for microsatellite DNA detection and isolation which combines RAPD amplification and oligonucleotide screening. The method is based on a prior RAPD amplification of plant genomic DNA following standard procedures (11). After scoring the RAPD polymorphisms on an agarose gel, the amplifiedDNA is transferred onto Hybond-N+ filters (Amersham Inc.) using Southern blot procedures (12). The filters are then hybridized with oligonucleotide probes carrying simple sequence repeats (SSR) labeled with Digoxigenin-ddUTP (DIG Oligonucleotide 3'-End Labeling Kit, Boehringer Mannheim), and luminescent signals detected by autoradiography, following the manufacturer's instructions. Using random decamer primers on sugar beet (Beta vulgaris L.), olive (Olea europaea L.) and sunflower (Helianthus annuus L.) DNA (Fig. lA-), we detected several bands containing microsatellites with high efficiency and accu-