CONSENSUS PCR PRIMERS FOR THE ANALYSIS OF MONONUCLEOTIDE REPEAT POLYMORPHISMS IN CHLOROPLAST GENOMES

We are interested in applying chloroplast markers for inheritance studies and cultivar identification in plant species, where database information is limited. To approach this problem, we constructed consensus primer pairs designed to universally amplify chloroplast microsatellites in dicotyledonous angiosperms. Three criteria were considered important for the selection and design of primer pairs, i.e. (1) sufficient conservation of primer target sequences, (2) polymorphic behaviour of amplification products, (3) a short size of amplification products to allow single-base resolution on sequencing gels.

As a first step, a total of 39 (A)_n and (T)_n repeats (n 10) were identified in the completely sequenced tobacco chloroplast genome (Shinozaki et al., 1986). For ten of these regions, consensus primer pairs flanking the repeats could be deduced from multiple alignment of orthologous DNA sequences found in the databases. Primer pairs were then used to amplify total genomic DNA from a hierarchical set of angiosperm species. PCR was performed in the presence of radio- or fluorescence-labelled dNTPs, and amplification products were separated on denaturing sequencing gels. Our results can be summarised as follows:

1. All ten consensus chloroplast microsatellite primer pairs (ccmpl to ccmp10) generated PCR products from all members of the Solanaceae, and eight of the ten (ccmp1-7 and ccmp10) were also functional in most other species. These pairs are likely to function across angiosperms in general, demonstrating that the design of universally applicable consensus primers from mononucleotide repeat flanking regions is feasible.
2. PCR products showed considerable size variation among the investigated species, genera and families. The largest size spectrum was observed with ccmp10, ranging from 91 bp for Actinidia up to 230 bp for pea and > 300 bp for the cabbage tree, Cordyline australis. The least variation was observed with primer pair ccmp6, which in fact flanks the longest mononucleotide repeat in the tobacco cpDNA. Within genera, alleles of different species were often separated by steps of 1 bp, which is consistent with a variable number of A or T residues in a mononucleotide repeat.
3. The extent of intraspecific variation was analysed in detail for three species, Nicotiana tabacum, Lycopersicon esculentum and Actinidia chinensis. PCR products from different primer/template combinations were pooled and analysed by automated

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