Increased Informativeness of RAPD Analysis by Detection of Microsatellite Motifs

Abstract

The recently developed random-amplified microsatellite polymorphism (RAMPO) technique detects second-level amplification products that are useful as molecular markers. In the first step of the procedure, genomic DNA is amplified with a single arbitrary or microsatellite-complementary primer. PCR products are then electrophoretically separated, photographed, blotted and hybridized to a 32P-labeled microsatellite probe. Autoradiography reveals highly reproducible, polymorphic, probedependent fingerprints, which are different from the ethidium bromide staining patterns. In this paper, we report the successful application of various mono-, tri- and tetranucleotide repeat motifs as RAMPO probes. We also compare the efficiency of arbitrary vs. microsatellite primers for the generation of RAMPO patterns. Repeated rehybridization to different probes has expanded the information contained in a single random-amplified polymorphic DNA (RAPD) gel at least fivefold. Pattern complexity varies with the length and sequence of the probe. Application of the technique to a genetic relatedness study in the genus Dioscorea (yam) yielded highly informative markers, mainly at an interspecific level.