An enlarged set of consensus primers for the study of organelle DNA in plants

the chloroplast genome (cpDNA) in population genetics studies of plants. Indeed, despite its low substitution rate (Wolfe et al. 1987), its typically uniparental mode of transmission, and hence clonal mode of evolution is a unique feature of cytoplasmic genomes of great interest for evolution studies. The plant mitochondrial genome (mtDNA) shares these characteristics, with however, an even slower substitution rate, but is much less studied because it varies enormously in size and gene arrangement (Palmer 1992). Consensus primers which are homologous to the most conserved coding regions of cpDNA or mtDNA but amplify the more variable noncoding regions are likely therefore to be very useful. Here, we greatly enlarge a previous list of primers published in this journal (Demesure et al. 1995). A total of seven pairs of cpDNA primers and nine pairs of mtDNA primers are described. Moreover, the degree of conservation of the whole set (16 and 12 pairs of cpDNA and mtDNA primers, respectively) is tested by amplification on five species, three angiosperms (two dicotyledons and one monocotyledon), a pine (gymnosperm) and a fern (pteridophyte), i.e. on a broader taxonomic range than in the former study. The primers are also evaluated by comparison with sequences available in the molecular databases. The primers were designed in order to be as consensual as possible, although the chloroplast genome of tobacco (i.e. a dicotyledon) was used as reference, and full homology with dicotyledon sequences was also given priority in the case of the mtDNA primers. Their design was facilitated by the availability of an increasing number of complete sequences in the molecular databases. Conserved coding sequences flanking the regions to be amplified were identified with the computer software B L A S T (Altschul et al. 1990). The cpDNA primers are all located in the relatively variable large single-copy region (Palmer 1985). For the mtDNA primers, introns of genes available in the molecular databases (e.g. subunits 1, 4, 5 and 7 of NADH dehydrogenase) were selected. Finally, the exact position and length of the primers were chosen according to their thermodynamic parameters using the Oligo Primer Analysis Software version 4.1 (Rychlik et al. 1990). The annealing temperatures were then manually optimized with a PHC3 (Techne) apparatus and then more rapidly with a Robocycler Gradient 96 (Stratagene). The list of the new pairs of primers is given in Table 1 along with their sequence (from 5′ to 3′) and annealing temperature. Because optimal PCR conditions can vary according to the thermocycler, the temperatures given here are only indicative. The degree of conservation of the whole set is described in Table 2. First, the exact position of the primers and the expected length of the fragment is provided for a reference sequence. Then, the list of species that were successfully amplified and the list of the species retrieved from databases that are likely to amplify, as judged from the orientation and homology of their sequences compared with those of the primers, are given. The 28 pairs of primers were directly tested by PCR on five species: Quercus robur L. (pedunculate oak, Fagaceae, Dicotyledons, Angiosperms), Ilex aquifolium L. (holly, Aquifoliaceae, Dicotyledons, Angiosperms), Arundinaria japonica (bamboo, Poaceae, Monocotyledons, Angiosperms), Pinus pinaster Ait. (maritime pine, Pinaceae, Gymnosperms) and Polystichum filix-mas (L.) Roth (male fern, Aspidiaceae, Pteridophytes). The cpDNA primers were also checked against the complete chloroplast sequences of two cereals, Zea mays (X86563) and Oryza sativa (X15901), of a gymnosperm, Pinus thunbergii (D17510), and of a bryophyte, Marchantia polymorpha (X04465). The mtDNA primers were checked against the only complete mtDNA sequence available to date, i.e. that of Marchantia polymorpha (M68929). This was completed by making comparisons with mtDNA genomic sequences of variable sizes, as far as these sequences encompassed the positions of both primers from a pair. Total DNA was extracted following the procedure described in Dumolin et al. (1995). The amplifications were performed as detailed in Demesure et al. (1995). The fragments were separated on 1% agarose gels and stained with ethidium bromide. PRIMER NOTE