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      Telling plant species apart with DNA: from barcodes to genomes

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          Abstract

          Land plants underpin a multitude of ecosystem functions, support human livelihoods and represent a critically important component of terrestrial biodiversity—yet many tens of thousands of species await discovery, and plant identification remains a substantial challenge, especially where material is juvenile, fragmented or processed. In this opinion article, we tackle two main topics. Firstly, we provide a short summary of the strengths and limitations of plant DNA barcoding for addressing these issues. Secondly, we discuss options for enhancing current plant barcodes, focusing on increasing discriminatory power via either gene capture of nuclear markers or genome skimming. The former has the advantage of establishing a defined set of target loci maximizing efficiency of sequencing effort, data storage and analysis. The challenge is developing a probe set for large numbers of nuclear markers that works over sufficient phylogenetic breadth. Genome skimming has the advantage of using existing protocols and being backward compatible with existing barcodes; and the depth of sequence coverage can be increased as sequencing costs fall. Its non-targeted nature does, however, present a major informatics challenge for upscaling to large sample sets.

          This article is part of the themed issue ‘From DNA barcodes to biomes’.

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          Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach

          We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data—mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.
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            Use of DNA barcodes to identify flowering plants.

            Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.
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              Applications of next generation sequencing in molecular ecology of non-model organisms.

              As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.
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                Author and article information

                Journal
                Philos Trans R Soc Lond B Biol Sci
                Philos. Trans. R. Soc. Lond., B, Biol. Sci
                RSTB
                royptb
                Philosophical Transactions of the Royal Society B: Biological Sciences
                The Royal Society
                0962-8436
                1471-2970
                5 September 2016
                5 September 2016
                : 371
                : 1702 , Theme issue ‘From DNA barcodes to biomes’ compiled and edited by Paul D. N. Hebert, Mehrdad Hajibabaei and Peter M. Hollingsworth
                : 20150338
                Affiliations
                [1 ]Royal Botanic Garden Edinburgh , 20A Inverleith Row, Edinburgh EH3 5LR, UK
                [2 ]Germplasm Bank of Wild Species, Kunming Institute of Botany , Chinese Academy of Sciences, 132 Lanhei Road, Heilongtan, Kunming, Yunnan 650201, People's Republic of China
                [3 ]Department of Botany and Plant Biotechnology, University of Johannesburg , Auckland park, Johannesburg PO Box 524, South Africa
                [4 ]Ashworth Laboratories, Institute of Evolutionary Biology, University of Edinburgh , Edinburgh EH9 3FL, UK
                Author notes

                One contribution of 16 to a theme issue ‘ From DNA barcodes to biomes’.

                Author information
                http://orcid.org/0000-0003-0602-0654
                http://orcid.org/0000-0002-8746-6617
                Article
                rstb20150338
                10.1098/rstb.2015.0338
                4971190
                27481790
                26ff2253-08b1-49e3-b381-d15dea139bde
                © 2016 The Authors.

                Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

                History
                : 1 June 2016
                Funding
                Funded by: European Union;
                Award ID: SYNTHESYS 3
                Funded by: Natural Environment Research Council, http://dx.doi.org/10.13039/501100000270;
                Award ID: NE/L011336/1
                Funded by: Basic Research Programme of China;
                Award ID: 2014CB954100
                Funded by: Scottish Government's Rural and Environment Science and Analytical Services Division;
                Funded by: Chinese Academy of Sciences, http://dx.doi.org/10.13039/501100002367;
                Award ID: 2009-LSFGBOWS-01
                Categories
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                Custom metadata
                September 5, 2016

                Philosophy of science
                plant dna barcoding,next-generation sequencing,genome skimming,species discrimination,hybrid baits

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