The quest for new molecular markers continues because, despite the availability of PCR primers for numerous plastid introns or intergenic spacer regions (IGSs), many botanists are still unable to resolve relationships within their study group. Additionally, structural rearrangements limit the utility of some primer combinations. The availability of complete plant plastid genome sequences has grown substantially over the past 10 years. It is now possible to design a wide array of primers for use across most flowering plants based on their predicted utility. It is true that whole genome sequencing is more available and less expensive than ever before, but many of us will continue to rely on comparative sequencing for a limited number of genic regions. Shaw et al. (2005) reviewed many of the non-coding regions used in prior studies, and went on to develop primers for additional chloroplast regions (Shaw et al. 2007). I designed primers for dozens of additional non-coding regions in the large and small single copy regions of the chloroplast genome based on the complete chloroplast genomes of 16 flowering plants. The primers were designed for universal Angiosperm application, but may be useful for non-flowering plants as well. If you find this information useful, please cite this webpage and share with your colleagues. Click to learn more about research and graduate educational opportunities at Rancho Santa Ana Botanic Garden. Revised Methods: Primer Design–Sixteen flowering plant chloroplast genomes representing a diversity of overall gene sequence order were downloaded from GenBank. Homologous gene sequences were aligned in Se-Al version 2.0a11 (Rambaut 1996). Primers were designed based on simultaneous viewing of the Se-Al file and an Oligo 4.02 (National Biosciences, Inc., Plymouth, MN, USA) file, using a single sequence from the pool in Oligo. Primers are anchored in coding regions and were designed to have a minimum number of hair-pins and primer-primer interactions, annealing temperature between 50 and 64ºC, and a 3’ GC clamp if possible. A number of the regions coincide with regions also published by Shaw et al. (2007) and others, but the primers were designed independently. Primer Evaluation–Complete chloroplast genomes from the primer design section above were imported into Sequencher version 4.9 (Gene Codes, Ann Arbor, MI, USA). A separate Sequencher file containing the named primer sequences was imported into the existing file containing the full genome sequence, and assembly was attempted. Initial assembly parameters that differed from the default values were: * Clean Data, Maximum Loop-out=0, Minimum Match Percentage=95, Minimum Overlap=15. Subsequent rounds of assembly reverted to *Dirty Data, Minimum Match Percentage=95. This was repeated until the Minimum Match Percentage was 80. Position of a primer relative to the genomic data was noted. Number of mismatch bases was also noted. Note: mismatches in the last 5 bases on the 3’ end of the primer sequence will require modification of the primer prior to use! For a small number of taxa, multiple chloroplast genomes are available for different taxa (species, subspecies, varieties, etc.) within the same genus. Complete chloroplast genomes for those organisms were imported into Sequencher, aligned manually (using a parsimony criterion for gap alignment) and the number of inferred substitutions and insertion/deletion events (indels) was noted, along with sequence length. These data provide an estimate of the utility of the regions for inferring phylogenies across a diversity of plant lineages. Monocot genomes for Oryza (N=3) and Acorus (N=2) were compared, along with unpublished data for three species of Yucca (generously provided by Michael McKain and James Leebens-Mack (University of Georgia, Athens). Non-monocot Angiosperm taxa examined included Nicotiana (N=3), Oenothera (N=5), and Gossypium (N=3). As additional complete chloroplast genomes become available, the utility of the primers for those taxa will be evaluated. As of 12/1/2011, comparisons were made for Nelumbo (N=2), Eucalyptus (N=2), Olea (N=5), and Populus (N=2). Results of these comparisons are available on the DOCUMENTS page.
LITERATURE CITED Rambaut, A. 1996. Se-Al (v2.0a11) Sequence Alignment Editor. Available at http://evolve.zoo.ox.ac.uk/. University of Oxford, Oxford, UK. Shaw, J., E.B. Lickey, E.E. Schilling, and R.L. Small. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in Angiosperms: the tortoise and the hare III. American Journal of Botany 94: 275–28 |