Mapping of sequence-tagged sites in rice by single strand conformation polymorphism

Abstract

The conditions for efficient single-strand conformation polymorphism (SSCP) detection were examined for its application to mapping of DNA regions in the rice genome. Temperature for electrophoresis and glycerol concentrations in gel affected SSCP patterns significantly. The optimal detection conditions for SSCP also depends on the nucleotide sequences of fragments analyzed. Fragments over 300 bp show complicated patterns depending on their nucleotide sequences and were not suitable for SSCP analysis. Seventy primer pairs were designed from the sequence data available to amplify DNA regions as sequence tagged sites (STSs), and 39 of these STSs were found to generate SSCP between japonica rice (Nipponbare) and indica rice (Kasalath) in at least one of the experimental conditions. The maps of DNA fragments amplified from 186 F2-plant DNAs with 17 primer pairs were successfully determined. This direct mapping method of the amplified DNA fragments with PCR is simple and quite sensitive, and can be used to set markers in the gap regions of a genetic linkage map.