Smith J J, Scott-Craig J S, Leadbetter J R, Bush G L, Roberts D L, Fulbright D W
NSF/Center for Microbial Ecology, Michigan State University, East Lansing 48824.
Mol Phylogenet Evol. 1994 Jun;3(2):135-45. doi: 10.1006/mpev.1994.1016.
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.
作为我们确定植物细菌野油菜黄单胞菌属内生物体系统发育关系研究的一部分，我们研究了随机扩增多态性DNA（RAPD）技术的应用。我们这部分研究的目的是确定通过直接比较在溴化乙锭染色的琼脂糖凝胶上分离的RAPD产物，是否能够进行有效的分支特征分析。使用单个寡核苷酸引物从47个野油菜黄单胞菌DNA模板中扩增RAPD产物。对这些RAPD产物进行比较，并使用两个克隆的RAPD产物作为探针，通过对47个细菌菌株的RAPD产物和基因组DNA进行Southern分析来表征变异。数据集分析表明，RAPD产物不一定是同源的或独立的，而这是在分支系统发育分析中进行特征分析的关键前提条件。通常认为RAPD变异是由于插入/缺失事件或引物结合位点的改变而发生的。在我们的数据集中，我们证明了缺失表型的出现是由于相应位点明显缺失，以及由于从无关位点优先合成替代RAPD产物。这些不同类型的变异反映了不同类型的基因型变异，直接检查RAPD产物无法让我们区分特定缺失表型是由哪种机制产生的。虽然这对表征分析可能不重要，但对于使用分支方法分析同源特征来说至关重要。我们还在反应混合物中检测到不相关的、共迁移的RAPD产物以及多个相关的RAPD产物。这些都可能导致相似性估计的误差，而相似性估计在任何系统发育分析中都很重要。当使用RAPD产物进行系统发育比较时，应考虑RAPD产物的所有这些特征。
