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Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding.

ABSTRACT: BACKGROUND: At 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 FINDINGS: We 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 trnS(UGA)-trnG(UCC), petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS(UGA)-trnG(UCC), trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (? 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/CONCLUSIONS: Chloroplast 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.

SUBMITTER: Dong W

PROVIDER: S-EPMC3325284 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding.

Dong Wenpan W Liu Jing J Yu Jing J Wang Ling L Zhou Shiliang S

PloS one 20120412 4

<h4>Background</h4>At 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.<h4>Methodology/principal findings</h4>We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 ...[more]

PMID: 22511980

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Project description:Nucleotide substitution models used in molecular phylogenetics do not account for nucleotide sequences evolving under selection, yet selection is rarely tested for. If non-neutral markers violate these models (i.e. non-independence of sites), it is expected that their reconstructed topologies be incongruent with those inferred from neutral ones and conclusions made from those phylogenies should be reexamined. Using rhodopsin as a phylogenetic marker has recently been called into question for exactly this reason. Rhodopsin is assumed to have evolved under strong positive selection for organisms that inhabit similar aquatic environments, making it unsuitable for the phylogenetics of aquatic organisms, but it is unclear what the effects of non-neutrality on phylogeny estimation are. To evaluate potential incongruence of neutral versus non-neutral markers, and the notion that rhodopsin should not be used in the molecular phylogenetics of fishes, a molecular dataset of 78 acanthomorph taxa and sequences from four nuclear, protein coding loci (including rhodopsin), were examined. Only one marker was found to be neutral while the remaining tests, for all other loci, rejected the null hypothesis of neutrality. To evaluate the possible effect(s) of positively versus negatively selected sites, the three non-neutral markers were analyzed to determine the presence of positively and negatively selected codons. To determine congruence in topology among ML trees inferred by individual neutral and non-neutral markers, as well as the combined (concatenated) dataset, tree, comparisons of distances among trees and hypothesis (topology) testing were carried out. Results of the tree distance metrics and topology testing support the notion that neutrality alone does not determine congruence in topology, and those data that are inferred to have evolved under selection should not necessarily be excluded. In addition, the number of sites inferred to have evolved under positive selection does not predict congruence with other markers or the topology inferred with the concatenated dataset.

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Dataset Information

Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding.

Publications

Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding.

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