Effects of plant tissue and DNA purification method on randomly amplified polymorphic DNA-based genetic fingerprinting analysis in carrot

Seven plant genomic DNA purification protocols were evaluated for genetic f ingerprinting analysis using six tissues obtained from inbred carrot (Daucu s carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DN A cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for ampli fication in a random amplified polymorphic DNA (RAPD) system. Significant d ifferences were observed among tissues and purification methods for the tot al amount of DNA, An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when comp ared with the other six nonorganic extraction methods. Of the tissues exami ned, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fres h tissue); followed by seeds (54 ng . mg(-1)), fresh leaves (48 ng . mg(-1) ), lyophilized leaves (40 ng . mg(-1)), calli (22 ng . mg(-1)), and tap roo ts (4 ng . mg(-1)). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations, Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesti ng a tissue-specific adverse effect on restriction endonuclease activity. D ifferences in RAPD band (amplicon) intensity and number were observed acros s tissues and DNA extraction methods using identical PCR conditions for RAP D, Callus was the best type of tissue for RAPD-based fingerprinting yieldin g a consistently higher number of more intense amplicons when compared to t he other tissues. In flowers and seeds, only DNA obtained with the CTAB ext raction method could be amplified, Polymorphisms deviating from genetic exp ectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. J udicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for rel iable RAPD-based DNA fingerprinting analysis in carrot. In addition, our st udies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, constru ction of genomic libraries, and gene cloning.