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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Polymorphisms in Fungi Using the Aflp Technique and Agarose Gels

item Vandemark, George

Submitted to: Focus
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 3, 1998
Publication Date: March 1, 1999

Interpretive Summary: The ability to clearly distinguish between individuals of a heterogeneous population has many important applications. These applications include plant and animal breeding, providing proof in the case of proprietary claims, forensic applications such as determining the identity of human, animal or plant remains and identifying the sources of biological tissues such as blood and skin. Individuals have often been distinguished based o morphological differences of traits such as in size or shape. However, these types of characteristics can bee influenced by environmental factors, the most easily conceptualized examples of this being body weight and skin color. Differences at the level of DNA sequences are better traits for distinguishing individuals because they are not influenced by environmental factors, as an individual's DNA profile is conserved over all the cells of the organism for its entire lifetime. Unfortunately, examining differences sin DNA sequences between individuals typically imvolves the use of both very expensive equipment and extremely toxic reagents. This equipment includes apparatus for determining the sequence of DNA fragments and containment facilities for the use of toxic reagents such as radioactive compounds and other carcinogens. I have developed a new protocol for distinguishing between individuals at the level of differences in DNA sequences. This protocol reduces the need for expensive equipment and does not require the use of toxic compounds such as radioactive reagents or the carcinogenic compound acrylamide. The use of this protocol will reduce both expenses and exposure to toxic compounds.

Technical Abstract: Amplified Restriction Fragment length Polymorphism (AFLP) has been widely used for the analysis of genetic diversity and recently has been effective for developing linkage maps in fungi. AFLP results have typically been visualized using radiolabeled for fluorescent-labeled primers. Both of these methods of image analysis require capital investment for either radiation containment facilities or apparatusfor automated DNA sequencing. Recently agarose gels stained with ethidium bromide were used to visualize AFLP results for different plant genomes. Here is reported a simple and rapid AFLP protocol for the analysis of fungal genomes that allows for the visualization of polymorphisms using agarose gels. Fifteen different AFLP primer pairs were used for the identification of poymorphisms between 24 is olates of the soilborne fungus Macrophomina phaseolina. All 15 primer pairs successfully amplified DNA from all 24 isolates of the fungus. The number of amplification products ranged from 14 to 29. Although fewer bands per primer pairwere resolved using this method as compared to resolution using acrylamide gels, the results were observed immediately after electrophoresis rather than having to wait 12 to 48 h for exposure of an autoradiograph. Similarly, there was no need to blot and hybridize as used for the chemiluminescent detection of AFLPs. This protocol requires less capital investment than previously reported protocols for AFLP analysis and reduces exposure to toxic reagents such as acrylamide or radioactive nucleotides.

Last Modified: 4/22/2015
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