Abstract
BackgroundAt present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species.Methodology/Principal FindingsWe scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnSUGA-trnGUCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnSUGA-trnGUCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species.Significance/ConclusionsChloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.
Highlights
At present, techniques for studying the molecular phylogeny of plants rely heavily on chloroplast genome sequence data
Techniques for studying the molecular phylogeny of plants rely heavily on chloroplast genome sequence data. This is because the chloroplast genome has a simple and stable genetic structure, it is haploid, there are no recombination, it is generally uniparentally transmitted, and universal primers can be used to amplify target sequences
Another important reason is the ease of Polymerase chain reactions (PCR) amplification and sequencing of chloroplast genes, despite some intrinsic problems similar to those encountered when using animal mitochondrial DNA [1]
Summary
Techniques for studying the molecular phylogeny of plants rely heavily on chloroplast genome sequence data This is because the chloroplast genome has a simple and stable genetic structure, it is haploid, there are no (or very rare) recombination, it is generally uniparentally transmitted, and universal primers can be used to amplify target sequences. Many fragments of coding regions, introns, and intergenic spacers, including atpB, atpB-rbcL, matK, ndhF, rbcL, rpl, rps4-trnS, rps, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels [2,3,4,5,6,7] These regions often lack variations in closely related species, especially those that have diverged recently in evolution. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species
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