Effects of Plant Tissue and DNA Purification Method on Randomly Amplified Polymorphic DNA-based Genetic Fingerprinting Analysis in Carrot

Abstract

Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh 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 roots (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 suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies 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, construction of genomic libraries, and gene cloning.