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      A chloroplast genomic strategy for designing taxon specific DNA mini-barcodes: a case study on ginsengs

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          Abstract

          Background

          Universal conventional DNA barcodes will become more and more popular in biological material identifications. However, in many cases such as processed medicines or canned food, the universal conventional barcodes are unnecessary and/or inapplicable due to DNA degradation. DNA mini-barcode is a solution for such specific purposes. Here we exemplify how to develop the best mini-barcodes for specific taxa using the ginseng genus ( Panax) as an example.

          Results

          The chloroplast genome of P. notoginseng was sequenced. The genome was compared with that of P. ginseng. Regions of the highest variability were sought out. The shortest lengths which had the same discrimination powers of conventional lengths were considered the best mini-barcodes. The results showed that the chloroplast genome of P. notoginseng is 156,387 bp. There are only 464 (0.30%) substitutions between the two genomes. The intron of rps16 and two regions of the coding gene ycf1, ycf1a and ycf1b, evolved the quickest and served as candidate regions. The mini-barcodes of Panax turned out to be 60 bp for ycf1a at a discrimination power of 91.67%, 100 bp for ycf1b at 100%, and 280 bp for rps16 at 83.33%.

          Conclusions

          The strategy by searching the whole chloroplast genomes, identifying the most variable regions, shortening the focal regions for mini-barcodes are believed to be efficient in developing taxon-specific DNA mini-barcodes. The best DNA mini-barcodes are guaranteed to be found following this strategy.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12863-014-0138-z) contains supplementary material, which is available to authorized users.

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          Highly Variable Chloroplast Markers for Evaluating Plant Phylogeny at Low Taxonomic Levels and for DNA Barcoding

          Wenpan Dong, Jing Liu, Jing-Jing Yu … (2012)
          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 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/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.
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            Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

            Ki-Joong Kim, Hae-Lim Lee (2004)
            The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.
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              A universal DNA mini-barcode for biodiversity analysis

              Isabelle Meusnier, Gregory Singer, Jean-François Landry … (2008)
              Background The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed). Results We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens. Conclusion In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.
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                Author and article information

                Contributors
                Wenpan Dong: wpdong@ibcas.ac.cn
                Han Liu: loolarloo@gmail.com
                Chao Xu: xuchao@ibcas.ac.cn
                Yunjuan Zuo: zuoyunjuan@tom.com
                Zhongjian Chen: 08765173@163.com
                Shiliang Zhou: slzhou@ibcas.ac.cn
                Journal
                Journal ID (nlm-ta): BMC Genet
                Journal ID (iso-abbrev): BMC Genet
                Title: BMC Genetics
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1471-2156
                Publication date (Electronic): 20 December 2014
                Publication date PMC-release: 20 December 2014
                Publication date Collection: 2014
                Volume: 15
                Issue: 1
                Electronic Location Identifier: 138
                Affiliations
                [ ]State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093 China
                [ ]Institute of Sanqi Research, Wenshan College, Wenshan, 663000 Yunnan China
                [ ]Shanghai Chenshan Plant Science Research Center, the Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, Shanghai, 201602 China
                Article
                Publisher ID: 138
                DOI: 10.1186/s12863-014-0138-z
                PMC ID: 4293818
                PubMed ID: 25526752
                SO-VID: 5d31aa48-5970-46a4-8e99-2bd627558e59
                Copyright © © Dong et al.; licensee BioMed Central. 2014
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 11 October 2014
                Date accepted : 26 November 2014
                Categories
                Subject: Research Article
                Custom metadata
                issue-copyright-statement © The Author(s) 2014

                ScienceOpen disciplines: Genetics
                Keywords: panax,chloroplast genome,dna mini-barcode,ycf1
                Data availability:
                ScienceOpen disciplines: Genetics
                Keywords: panax, chloroplast genome, dna mini-barcode, ycf1

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