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      Genomic sequencing and microsatellite marker development for Boswellia papyrifera, an economically important but threatened tree native to dry tropical forests

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          Abstract

          The world famous aromatic resin Frankincense is tapped from natural populations of Boswellia trees. Most of these populations have been shrinking rapidly over recent decades. To help guide conservation efforts for imperilled species of this genus, we developed 46 genetic markers for Boswellia papyrifera. Several of these were cross-transferable to other Boswellia species that occur in Ethiopia and Yemen. We also identified genes involved in the biosynthesis of the terpenes and terpenoids that are major constituents of frankincense.

          Abstract

          Microsatellite (or simple sequence repeat, SSR) markers are highly informative DNA markers often used in conservation genetic research. Next-generation sequencing enables efficient development of large numbers of SSR markers at lower costs. Boswellia papyrifera is an economically important tree species used for frankincense production, an aromatic resinous gum exudate from bark. It grows in dry tropical forests in Africa and is threatened by a lack of rejuvenation. To help guide conservation efforts for this endangered species, we conducted an analysis of its genomic DNA sequences using Illumina paired-end sequencing. The genome size was estimated at 705 Mb per haploid genome. The reads contained one microsatellite repeat per 5.7 kb. Based on a subset of these repeats, we developed 46 polymorphic SSR markers that amplified 2–12 alleles in 10 genotypes. This set included 30 trinucleotide repeat markers, four tetranucleotide repeat markers, six pentanucleotide markers and six hexanucleotide repeat markers. Several markers were cross-transferable to Boswellia pirrotae and B. popoviana. In addition, retrotransposons were identified, the reads were assembled and several contigs were identified with similarity to genes of the terpene and terpenoid backbone synthesis pathways, which form the major constituents of the bark resin.

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          Most cited references50

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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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            Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.

            Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.
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              Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences.

              The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.
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                Author and article information

                Journal
                AoB Plants
                AoB Plants
                aobpla
                aobpla
                AoB Plants
                Oxford University Press
                2041-2851
                2015
                7 January 2015
                : 7
                : plu086
                Affiliations
                [1 ]Wageningen UR Plant Breeding, Wageningen University and Research Center , PO Box 386, NL-6700 AJ Wageningen, The Netherlands
                [2 ]Center for Ecosystem Studies, Forest Ecology and Forest Management Group, Wageningen University and Research Center , PO Box 47, NL-6700 AA Wageningen, The Netherlands
                [3 ]Wondo Genet College of Forestry and Natural Resources , PO Box 128, Shashemene, Ethiopia
                Author notes
                [* ]Corresponding author's e-mail address: rene.smulders@ 123456wur.nl

                Associate Editor: Kermit Ritland

                Article
                plu086
                10.1093/aobpla/plu086
                4433549
                25573702
                f7ec866a-220c-4a2e-8f29-87a12a62753b
                Published by Oxford University Press on behalf of the Annals of Botany Company.

                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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 July 2014
                : 8 December 2014
                Page count
                Pages: 11
                Categories
                1006
                1009
                1013
                Research Articles

                Plant science & Botany
                conservation genetics,resin,ssr,terpene biosynthesis,terpenoid,tropical dry forest.

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