DNA amplification fingerprinting of centipedegrass

The DNA amplification fingerprinting (DAF) method was used to characterize five cultivars of centipedegrass. The technology was optimized for centipedegrass through experiments testing the parameters for each reagent. The optimal reaction mixture was found to be: 200 μM each dNTP, 1x buffer stock, 3 μM primer (octomer), 0.05-0.15 ng/μl volume template, 1.5 mM MgCl₂, and 0.3 units/μl reaction enzyme (AmpliTaq Stoffel fragment polymerase).

After optimization, five centipedegrass cultivars (Tennessee Hardy', Tennessee Tuff, 'Oklawn', 'Centennial' and Tifton common') were separated using DAF with primers 8.6d (GTAACGCC) and 8.6i (GTTACGCC). Studies of endonuclease digestion prior to amplification found the following endonucease-primer combinations were successful in separating centipedegrass cultivars: MspI-8.6i, HinfI, MspI-8.6h and HinfI, AluI, RsaI-8.6h.

Amplification patterns were scored and a phylogenetic analysis was conducted to explore relationships of the five cultivars. The analysis found that the cultivar Tennessee Hardy' is significantly different from the other four cultivars, which have several potential relationships.

A technique to isolate products from silver-stained polyacrylamide gels was developed and 5 products were isolated. Restriction fragment length polymorphism (RFLP) analysis was done on the centipedegrass cultivars using two of these isolation products as probes without further purification by subcloning. One was subcloned and the results compared. The results were similar enough to indicate that further purification by subcloning was not necessary in order to make probes by the method used.