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      RNA-Seq Analysis and De Novo Transcriptome Assembly of Jerusalem Artichoke ( Helianthus tuberosus Linne)

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          Abstract

          Jerusalem artichoke ( Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Genic microsatellite markers in plants: features and applications.

            Expressed sequence tag (EST) projects have generated a vast amount of publicly available sequence data from plant species; these data can be mined for simple sequence repeats (SSRs). These SSRs are useful as molecular markers because their development is inexpensive, they represent transcribed genes and a putative function can often be deduced by a homology search. Because they are derived from transcripts, they are useful for assaying the functional diversity in natural populations or germplasm collections. These markers are valuable because of their higher level of transferability to related species, and they can often be used as anchor markers for comparative mapping and evolutionary studies. They have been developed and mapped in several crop species and could prove useful for marker-assisted selection, especially when the markers reside in the genes responsible for a phenotypic trait. Applications and potential uses of EST-SSRs in plant genetics and breeding are discussed.
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              Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

              Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.
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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
                2014
                6 November 2014
                : 9
                : 11
                : e111982
                Affiliations
                [1 ]Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
                [2 ]Animal Material Engineering, Gyeongnam National University of Science and Technology, Jinju, Korea
                The Chinese University of Hong Kong, Hong Kong
                Author notes

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

                Conceived and designed the experiments: HSC J-HJ WYJ SSL. Performed the experiments: WYJ SSL HSK SRM JSM SYK J-HJ. Analyzed the data: HSC WYJ SSL CWK. Contributed reagents/materials/analysis tools: WYJ SSL. Contributed to the writing of the manuscript: HSC J-HJ WYJ.

                Article
                PONE-D-14-20749
                10.1371/journal.pone.0111982
                4222968
                25375764
                0676d061-fc29-4cc1-ba51-f81240eed3b9
                Copyright @ 2014

                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.

                History
                : 9 May 2014
                : 9 October 2014
                Page count
                Pages: 15
                Funding
                This work was supported by KRIBB Research Initiative Program, The Cabbage Genomics assisted breeding supporting center (CGsC) research programs funded by the Ministry for Food, Agriculture, Forestry and Fisheries of the Korean Government, The Next Generation of Bio Green 21 Project, The National Center for GM Crops (PJ009043) from RDA to HSC, and Bio-industry Technology Development Program (No.310006-5), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea to J-HJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biotechnology
                Plant Biotechnology
                Plant Genomics
                Plant Science
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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