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United States Department of Agriculture

Agricultural Research Service

Title: Effects of Plant Tissue and DNA Purification Method on Rapd Based Genetic Fingerprinting Analysis in (Daucus Carota L)

Authors
item Boiteux, L S - DEPT OF HORT UW MADISON
item Fonseca, M E N - DEPT OF HORT UW MADISON
item Simon, Philipp

Submitted to: Journal of American Society of Horticulture Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 1, 1998
Publication Date: N/A

Interpretive Summary: DNA is widely used to evaluate genetic variation in plants and animals. The results of experiments utilizing DNA can vary due to both the method of DNA extraction as well as the source tissue used for extraction. In this study we evaluated seven extraction methods for six tissues of carrot plants to determine which ones worked best. We found that the best method was a standard one. Several carrot tissues were equally well-suited for DNA extraction, although seed and flower tissues were difficult for obtaining repeatable and reliable results. This research has a potential positive impact for geneticists and biotechnologists studying carrot genetics.

Technical Abstract: Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting performance using sex tissues obtained from inbred carrot (Daucus carota L.) Lines. Evaluations included: 1)DNA yield, 2)DNA purity (A260nm/A280nm ratio), 3) DNA utility (cleavage with Hind lll), 4) DNA integrity (banding pattern of uncleaved DNA), and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and method examined for the total amount of DNA. A CTAB buffer method (using organic solvents) gave the best results in DNA yield, purity, and utility when compared with the other six non-organic methods. Of the tissues examined, flowers were highest in DNA yield. For most of the preparations, the DNA migrated as high-molecula weight bands in agarose gels with no traces of degradation. DNA preparations were not consistently accessible to Hind lll cleavage in all tissue/method combinations. Uncut DNA was observed chiefly in flowers and lyophilized leaves suggesting a tissue-specific adverse effect on restriction endonuclease performance. Differences in both amplicon intensity and number were observed across tissues and methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD based fingerprinting yielding consistently higher number of more intense amplicon when compared to the other tissues. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA indication that some RAPD amplicons/markers may not present satisfactory levels of reproducibility.

Last Modified: 4/23/2014
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