Plant & Animal Genome VI Conference

P58

A SET OF UNIVERSAL PCR PRIMERS FOR THE ANALYSIS OF SIMPLE SEQUENCE REPEAT POLYMORPHISMS IN ANGIOSPERM CHLOROPLAST GENOMES

1. Plant Molecular Biology, Biozentrum, Johann Wolfgang Goethe University, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany
2. Centre for Gene Technology, School of Biological Sciences, University of, Auckland, Private Bag 92019, Auckland, New Zealand

Short (<20 bp) runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice and pine, PCR 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 universal primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperm species where database information is limited. A total of 39 (A)_n and (T)_n repeats (n < 9) were identified in the complete 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 ten primer pairs generated PCR products from members of the Solanaceae, and eight of the ten 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. In two of three regions sequenced, the repeats responsible for the polymorphism were not those originally selected for the alignment. Our results demonstrate that universal primers targeted to chloroplast mononucleotide repeats can serve as general tools to study chloroplast variation in angiosperms.