EVALUATION OF RAPD ANALYSIS FOR CULTIVAR IDENTIFICATION OF PERSIMMONS

The effectiveness of RAPD analysis for cultivar identification of pers immons (Diospyros kaki) was evaluated by using 10 base primers (Operon Technologies, CA). A protocol of reproducible RAPD analysis for persi mmon was established by determining suitable buffer and optimum concen trations of DNA polymerase, MgCl2, primer, and dNTP. The 12.5 mu l rea ction mixture, found to be reliable for RAPD for persimmon, contains 5 0 mM Tris-HCl (pH 8.9), 20 mM NaCl, 1.0% Triton-X 100, 0.1% gelatin as a buffer solution (Levi ct al., 1993), 0.06 units(.)mu l(-1) AmpliTaq DNA polymerase, 2.0 mM MgCl2, 0.28 mu M primer, and 0.1 mM each of dN TP per reaction. Among these elements, the kinds of buffer and concent rations of ampliTaq DNA polymerase and MgCl2 were important for reprod ucibility of RAPD. Amplification was performed by 45 cycles of 1 min a t 94 degrees C, 1 min at 45 degrees C, 2 min at 72 degrees C in the Pe rkin Elmer Cetus DNA Thermal Cycler. 5 ng(.)mu l(-1) DNA were used as a template. By this reproducible protocol, we selected 2 primers (OPA- 06 and OPA-08) among 20 primers (OPA-01 to OPA-20) as effective ones f or cultivar identification of persimmons. The fifteen cultivars tested were completely distinguishable from each other by RAPDs, using OPA-0 6 or OPA-08. Furthermore, two bud mutants of 'Hiratanenashi', i.e. 'To newase' and 'Sugitawase', showed different DNA patterns with a few add itional minor bands by RAPD, using OPA-06 primer. It may be possible t o identify persimmon sports by this method. In addition, polymorphisms among 11 Diospyros species were observed by RAPD, using OPA-10 primer . The same method revealed little polymorphisms among intraspecific le vels of D. kaki or D. lotus. The potential of RAPD analysis for persim mon identification is discussed.