A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms

Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)(n) and (T)(n) repeats (n greater than or equal to 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.