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      How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

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          Abstract

          Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL (UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200–300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.

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          Most cited references 13

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          A DNA barcode for land plants.

            (2009)
          DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.
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            A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region

            Background A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Methodology/Principal Findings Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. Conclusions/Significance A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.
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              DNA extraction from Pleistocene bones by a silica-based purification method.

               S Pääbo,  M Höss (1993)
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                28 August 2012
                : 7
                : 8
                Affiliations
                [1 ]Royal Botanic Garden Edinburgh, Inverleith Row, Edinburgh, United Kingdom
                [2 ]Natural History Museum, Cromwell Road, London, United Kingdom
                [3 ]Biosystematics Group, Wageningen University, Wageningen, The Netherlands
                [4 ]Universidad de Los Andes, Apartado Aéreo, Bogotá, Colombia
                [5 ]Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom
                University of Florence, Italy
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FB, JR, MS, RC TS. Performed the experiments: TS. Analyzed the data: TS MS. Wrote the paper: TS, FB, JR, MS RC.

                Article
                PONE-D-12-16930
                10.1371/journal.pone.0043808
                3429509
                22952770

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 9
                Funding
                The work was funded by the European Community Research Infrastructures program under FP7 call ‘Synthesis of Systematic Resources’, grant number 226506-CP-CSA-Infra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Ecology
                Biodiversity
                Evolutionary Biology
                Evolutionary Systematics
                Molecular Systematics
                Phylogenetics
                Organismal Evolution
                Plant Evolution
                Molecular Cell Biology
                Nucleic Acids
                DNA
                DNA amplification
                Plant Science
                Plant Taxonomy

                Uncategorized

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