Identification of conserved regions in the plastid genome: implications for DNA barcoding and biological function

Abstract

All oligonucleotides of the sugarcane chloroplast genome that are conserved in one or more of 36 other completed plastid genomes have been identified by computer-assisted sequence comparison. These regions are of interest because they (i) are indicative of strong selection pressures to maintain specific nucleotide sequences that may yield insights into plastid biology and (ii) can be used as priming sites for amplifying intervening sequences that represent potential DNA barcodes for species identification. The majority of conserved sites are located in the inverted repeat (IR) region, but several sites in the single copy region (predominantly in tRNA and psa/psb genes) are conserved among chloroplasts of all higher plants examined here. Of particular interest are protein coding regions that have been conserved at the nucleotide level, as these may be involved in transcript regulation. This analysis also provides the basis for rational design of a DNA barcode for plastids, and several potential barcode regions have been identified. In particular, two oligonucleotides of length 33 and 25, and separated by approximately 362 nucleotides, are found in all cyanobacteria, red, brown and green algae, as well as diatoms, euglenids, apicomplexans and land plants that have been examined to date. Their widespread occurrence makes the intervening sequence a universal marker for all photosynthetic lineages. Analysis of 160 GenBank accessions illustrates that this region discriminates many algae at the species level, but lacks sufficient variation among the more recently diverged land plants to serve as a single DNA barcode for this taxon. However, this marker should be particularly useful for the DNA barcoding of algal lineages and lichens, as well as for environmental sampling. More rapidly evolving regions of the plastid genome also identified here serve as a starting point to design and test barcodes for more narrowly defined lineages, including the more recently diverged angiosperms.