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      The Complete Chloroplast Genome Sequences of Aconitum pseudolaeve and Aconitum longecassidatum, and Development of Molecular Markers for Distinguishing Species in the Aconitum Subgenus Lycoctonum

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          Abstract

          Aconitum pseudolaeve Nakai and Aconitum longecassidatum Nakai, which belong to the Aconitum subgenus Lycoctonum, are distributed in East Asia and Korea. Aconitum species are used in herbal medicine and contain highly toxic components, including aconitine. A. pseudolaeve, an endemic species of Korea, is a commercially valuable material that has been used in the manufacture of cosmetics and perfumes. Although Aconitum species are important plant resources, they have not been extensively studied, and genomic information is limited. Within the subgenus Lycoctonum, which includes A. pseudolaeve and A. longecassidatum, a complete chloroplast (CP) genome is available for only one species, Aconitum barbatum Patrin ex Pers. Therefore, we sequenced the complete CP genomes of two Aconitum species, A. pseudolaeve and A. longecassidatum, which are 155,628 and 155,524 bp in length, respectively. Both genomes have a quadripartite structure consisting of a pair of inverted repeated regions (51,854 and 52,108 bp, respectively) separated by large single-copy (86,683 and 86,466 bp) and small single-copy (17,091 and 16,950 bp) regions similar to those in other Aconitum CP genomes. Both CP genomes consist of 112 unique genes, 78 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes. We identified 268 and 277 simple sequence repeats (SSRs) in A. pseudolaeve and A. longecassidatum, respectively. We also identified potential 36 species-specific SSRs, 53 indels, and 62 single-nucleotide polymorphisms (SNPs) between the two CP genomes. Furthermore, a comparison of the three Aconitum CP genomes from the subgenus Lycoctonum revealed highly divergent regions, including trnK- trnQ, ycf1- ndhF, and ycf4-cemA. Based on this finding, we developed indel markers using indel sequences in trnK- trnQ and ycf1- ndhF. A. pseudolaeve, A. longecassidatum, and A. barbatum could be clearly distinguished using the novel indel markers AcoTT ( Aconitum trnK- trnQ) and AcoYN ( Aconitum ycf1- ndhF) . These two new complete CP genomes provide useful genomic information for species identification and evolutionary studies of the Aconitum subgenus Lycoctonum.

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          Using MUMmer to identify similar regions in large sequence sets.

          The MUMmer sequence alignment package is a suite of computer programs designed to detect regions of homology in long biological sequences. Version 2.1 makes several improvements to the package, including: increased speed and reduced memory requirements; the ability to handle both protein and DNA sequences; the ability to handle multiple sequence fragments; and new algorithms for clustering together basic matches. The system is particularly efficient at comparing highly similar sequences, such as alternative versions of fragment assemblies or closely related strains of the same bacterium.
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            Chloroplast microsatellites: new tools for studies in plant ecology and evolution.

            The nonrecombinant, uniparentally inherited nature of organelle genomes makes them useful tools for evolutionary studies. However, in plants, detecting useful polymorphism at the population level is often difficult because of the low level of substitutions in the chloroplast genome, and because of the slow substitution rates and intramolecular recombination of mtDNA. Chloroplast microsatellites represent potentially useful markers to circumvent this problem and, to date, studies have demonstrated high levels of intraspecific variability. Here, we discuss the use of these markers in ecological and evolutionary studies of plants, as well as highlighting some of the potential problems associated with such use.
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              The Complete Chloroplast Genome Sequence of the Medicinal Plant Salvia miltiorrhiza

              Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.
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                Author and article information

                Journal
                Molecules
                Molecules
                molecules
                Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry
                MDPI
                1420-3049
                21 November 2017
                November 2017
                : 22
                : 11
                : 2012
                Affiliations
                K-herb Research Center, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea; pik6885@ 123456kiom.re.kr (I.P.); sgyang81@ 123456kiom.re.kr (S.Y.); serparas@ 123456kiom.re.kr (G.C.); ukgene@ 123456kiom.re.kr (W.J.K.)
                Author notes
                [* ]Correspondence: bcmoon@ 123456kiom.re.kr ; Tel.: +82-42-868-9530
                Author information
                https://orcid.org/0000-0002-5374-6274
                Article
                molecules-22-02012
                10.3390/molecules22112012
                6150344
                29160852
                3821b48f-82fa-4be1-9b50-25a267e005fb
                © 2017 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 October 2017
                : 19 November 2017
                Categories
                Article

                lycoctonum,plastid,ranunculaceae,medicinal plant,species identification

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