DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species Because single-locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole-chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource-effective and does not yet offer the speed of analysis provided by single-locus barcodes to unspecialized laboratory facilities Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super-barcode We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single-locus barcodes and super-barcodes for efficient plant identification Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels

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Plant DNA barcoding: From gene to genome

Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2

Background 
DNA barcoding promises to revolutionize the way taxonomists work, facilitating species identification by using small, standardized portions of the genome as substitutes for morphology The concept has gained considerable momentum in many animal groups, but the higher plant world has been largely recalcitrant to the effort In plants, efforts are concentrated on various regions of the plastid genome, but no agreement exists as to what kinds of regions are ideal, though most researchers agree that more than one region is necessary One reason for this discrepancy is differences in the tests that are used to evaluate the performance of the proposed regions Most tests have been made in a floristic setting, where the genetic distance and therefore the level of variation of the regions between taxa is large, or in a limited set of congeneric species


Methodology and Principal Findings 
Here we present the first in-depth coverage of a large taxonomic group, all 86 known species (except two doubtful ones) of crocus Even six average-sized barcode regions do not identify all crocus species This is currently an unrealistic burden in a barcode context Whereas most proposed regions work well in a floristic context, the majority will – as is the case in crocus – undoubtedly be less efficient in a taxonomic setting However, a reasonable but less than perfect level of identification may be reached – even in a taxonomic context


Conclusions/Significance 
The time is ripe for selecting barcode regions in plants, and for prudent examination of their utility Thus, there is no reason for the plant community to hold back the barcoding effort by continued search for the Holy Grail We must acknowledge that an emerging system will be far from perfect, fraught with problems and work best in a floristic setting

How many loci does it take to DNA barcode a crocus

Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference. The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats, short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability. A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential, the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.

Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Background 
The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task.


Methodology and Principal Findings 
We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome- ITS, and three from plastid genome- trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation.


Conclusions/Significance 
We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case.

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Universal plant DNA barcode loci may not work in complex groups: a case study with Indian berberis species.

The DNA barcode potential of three regions (the nuclear ribosomal ITS and the plastid psbA-trnH and trnT-trnL intergenic spacers) was investigated for the plant genus Aspalathus L. (Fabaceac: Crotalarieae). Aspalathus is a large genus (278 species) that revealed low levels of DNA variation in phylogenetic studies. In a 51-species dataset for the psbA-trnH and ITS regions, 45%, and 16% of sequences respectively were identical to the sequence of at least one other species, with two species undiscriminated even when the two regions were combined. In contrast, trnT-trnL, discriminated between all species in this dataset. In a larger ITS and trnT-trnL dataset. including a further 82 species. 7 species in five pairwise comparisons remained Undiscriminated when the two regions were combined. Four of the five pairs of species not discriminated by sequence data were readily distinguished using a combination of qualitative and quantitative morphological data. The difficulty of barcoding in this group is increased by the presence of intraspecific variation in all three regions studied. In the case of psbA-trnH, three intraspecific samples had a sequence identical to at least one other species. Overall, psbA-trnH. currently a candidate for plant barcoding, was the least discriminatory region in our study.

Anne Horn

Papers

DNA barcoding of a large genus, Aspalathus L. (Fabaceae)