Isolation of DNA for RAPD analysis from leaves of the saskatoon (Amelanchier alnifolia Nutt.) and other horticultural crops

Abstract

Three DNA extraction procedures were examined to determine which might yield DNA from saskatoon leaves suitable for randomly amplified polymorphic DNA (RAPD) analysis. The three methods examined were: (1) the miniprep procedure and (2) the modified miniprep for difficult species, both described by Dellaporta et al. (1983), and (3) a slightly modified protocol of Doyle and Doyle (1990). Only the modified method of Doyle and Doyle (1990) consistently yielded DNA suitable for polymerase chain reaction (PCR) amplification, regardless of plant growing conditions or leaf age. The recovery of high molecular weight DNA with an average yield of 53 mg g−1 fresh weight of leaf material was possible using this procedure. Reproducible RAPD markers used to distinguish among saskatoon cultivars were generated. This same procedure also yielded PCR-amplifiable DNA from various other perennial, woody species and herbaceous plant material. Key words: DNA isolation; RAPD analysis; horticultural crops.