DNA fingerprinting based on microsatellite-anchored fragment length polymorphisms, and isolation of sequence-specific PCR markers in lupin (Lupinus angustifolius L.)

We report a method of microsatellite-anchored fragment length polymorphisms for DNA fingerprinting. The method combines the concept of AFLP and the mi crosatellite-anchor primer technique. Genomic DNA was digested by one restr iction enzyme MseI. One AFLP adaptor (MseI adaptor) was ligated onto the re striction fragments. DNA fingerprints were produced by PCR using one micros atellite-anchor primer in combination with one MseI-primer. The method allo ws co-amplification of over 100 DNA fragments containing microsatellite mot ifs per PCR. Polymorphisms detected from lupin by this method included thos e arising from variation in the number of microsatellite repeat units targe ted by the microsatellite-anchor primers, from variation on the annealing s ites for the SSR-anchor primers, from insertions/deletions outside the SSR region, and from variation in restriction sites. The first three types of p olymorphisms were readily converted into sequence-specific PCR markers suit able for marker-assisted breeding.