Amplified Fragment Length Microsatellites (AFLM) might be used to develop microsatellite markers in organisms with limited amounts of DNA applied to Arbuscular Mycorrhizal (AM) fungi

Developing microsatellite markers for organisms with limited amounts of DNA can be difficult because sequence information is needed. To overcome this problem in the arbuscular mycorrhizal (AM) fungi Glomus etunicalum and Gigaspora gigantea, global amplification of the gerlomes of each species was performed with linker-adaptor-PCR from single spores. Amplified fragments were enriched for microsatellite motifs with 5'-biotinylated oligonucleotides and recovered by magnetic streptavidin beads. The recovered fragments were reamplified and separated on denaturing polyacrylamide gels, and 16 selected bands were excised, cloned and sequenced. Seven microsatellite motifs were detected from six clones (efficiency rate of 43.8%). Primers were designed for all putative microsatellite loci and most were successfully amplified from three single-spore preparations and from pools of five, 10 and 20 spores after global amplification. This approach, termed amplified fragment-length micosatellites (AFLM), might aid investigations of organisms that cannot or are not readily cultured in vitro and where DNA is a limiting factor for genetic studies. However, the technique also can be used to isolate microsatellite loci in any organism.