Microsatellite Libraries Enriched for Several Microsatellite Sequences in Plants

Simple sequence repeats (SSRs), or microsatellites, have been isolated and characterized from numerous animal and plant species (6,7). These sequences have been shown to be highly polymorphic between individuals within populations or closely related genotypes. This, together with the fact that they can be converted into a simple polymerase chain reaction (PCR)based assay, has led to their widespread use as molecular markers of genetic diversity (6). The use of microsatellites has been limited in plants, however, by the costs involved in isolating large numbers from the target species. Recently, a number of papers have reported the isolation of microsatellite clones by an enrichment procedure (1,2,4,5). These reports have all resulted in the isolation of a single type of microsatellite. However, as little is known about the level of polymorphism of individual microsatellite sequences, it is possible that this approach will result in markers that do not detect the level of polymorphism often required in diversity studies. Furthermore, the targeting of a single microsatellite species may produce only a small number of clones necessitating the construction of further libraries in the future. To eliminate these problems, we have significantly modified the enrichment technique (2,5) to yield clones that contain a variety of microsatellites. These clones can then be characterized by either direct sequencing or colony hybridization using specific microsatellite oligonucleotides. Interestingly, the technique also yields a high percentage of clones containing more than one type of microsatellite. Results to date suggest that the clones produced by this procedure are sufficiently polymorphic to be used in population genetics and/or breeding studies. Preparation of reagents for microsatellite enrichment. The following oligonucleotides can be used to enrich for DNA fragments containing microsatellites: [GA]15, [GT]15, [AT]15, [GC]15, [CAA]10, [CATA]10, [ATT]10, [GATA]10, [GCC]10 and [ATAG]10. Fifty nanograms of each oligonucleotide in 3× SSC (45 mM sodium citrate, pH 7.0 and 450 mM NaCl) are pooled in a total volume of 80 μL, spotted onto a 0.5-cm2 piece of Hybond N+ (Amersham, Arlington Heights, IL, USA) and air-dried for 1 h. The dry membrane is UV-treated for 30 s using a 260-nm transilluminator. Weakly bound oligonucleotides are washed off the membrane by washing twice in 10 mL of hybridization buffer (50% formamide, 3× standard saline citrate [SSC], 25 mM Na-phosphate, pH 7.0, and 0.5% sodium dodecyl sulfate [SDS]) at 45°C for 2 days. The membranes are then stored at -20°C until required. Preparation of genomic DNA. One microgram of genomic DNA is digested with 2 units of RsaI in a volume of 20 μL for 1 h at 37°C. Fifty nanograms