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Published June 1994 | metadata_only
Journal Article

Characterization of Random Amplified Polymorphic DNA (RAPD) Products from Xanthomonas campestris and Some Comments on the Use of RAPD Products in Phylogenetic Analysis


As part of our research to determine phylogenetic relationships of organisms within the phytobacterial species Xanthomonas campestris, we have examined the use of the random amplified polymorphic DNA (RAPD) technique. The objective of this aspect of our research was to determine if a valid cladistic character analysis could be carried out by direct comparison of RAPD products separated on ethidium bromide-stained agarose gels. RAPD products were amplified from 47 Xanthomonas campestris DNA templates using a single oligonucleotide primer. These RAPD products were compared and variation was characterized by Southern analysis of both RAPD products and genomic DNA of the 47 bacterial strains using two cloned RAPD products as probes. Analysis of the data set revealed that the RAPD products were not necessarily homologous or independent, crucial prerequisites for characters to be analyzed in a cladistic phylogenetic analysis. It has been commonly assumed that RAPD variation occurs due to insertion/deletion events or alterations in the primer binding site. Within our data set, we demonstrate absence phenotypes arising from the apparent absence of corresponding loci and also due to the preferred synthesis of alternative RAPD products from unrelated loci. These different types of variation are a reflection of different types of genotypic variation, and direct examination of RAPD products did not allow us to distinguish by which mechanism a particular absence phenotype arose. Although this may not be important for phenetic analyses, for analyses of homologous characters using a cladistic approach it is critical. We also detected unrelated, co-migrating RAPD products and multiple related RAPD products within reaction mixtures. These could both contribute to errors in estimates of similarity, important in any phylogenetic analysis. All of these characteristics of RAPD products should be taken into consideration when RAPD products are used for phylogenetic comparisons.

Additional Information

© 1994 by Academic Press, Inc. Received May 12, 1993; revised January 14, 1994. Available online 26 April 2002. We thank Dr. John Hartung, USDA, Beltsville, MD, for providing several purified X. campestris DNA samples; Dr. Alan Poplawsky University of Idaho, for providing the xanthomonadin clone; and the Mycogen Corp. for generously supplying many of the X campestris strains in this study. We also thank Darlene Salman for expert technical assistance; Dr. Jennifer Weller, Dr. Scott Williams, Dr. David Hillis, and Dr. Ray Seidler for critically reviewing various versions of the manuscript; and John Jenkins for useful discussions on the theory and practice of phylogenetic systematics. J.S.C. was supported in part by a gift from the Mycogen Corp. Work supported by NSF Grants BIR 9120006 and DIR 8809640.

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August 22, 2023
August 22, 2023