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      Transcriptome Characterization and Sequencing-Based Identification of Salt-Responsive Genes in Millettia pinnata, a Semi-Mangrove Plant

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          Abstract

          Semi-mangroves form a group of transitional species between glycophytes and halophytes, and hold unique potential for learning molecular mechanisms underlying plant salt tolerance. Millettia pinnata is a semi-mangrove plant that can survive a wide range of saline conditions in the absence of specialized morphological and physiological traits. By employing the Illumina sequencing platform, we generated ∼192 million short reads from four cDNA libraries of M. pinnata and processed them into 108 598 unisequences with a high depth of coverage. The mean length and total length of these unisequences were 606 bp and 65.8 Mb, respectively. A total of 54 596 (50.3%) unisequences were assigned Nr annotations. Functional classification revealed the involvement of unisequences in various biological processes related to metabolism and environmental adaptation. We identified 23 815 candidate salt-responsive genes with significantly differential expression under seawater and freshwater treatments. Based on the reverse transcription–polymerase chain reaction (RT–PCR) and real-time PCR analyses, we verified the changes in expression levels for a number of candidate genes. The functional enrichment analyses for the candidate genes showed tissue-specific patterns of transcriptome remodelling upon salt stress in the roots and the leaves. The transcriptome of M. pinnata will provide valuable gene resources for future application in crop improvement. In addition, this study sets a good example for large-scale identification of salt-responsive genes in non-model organisms using the sequencing-based approach.

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          Most cited references 66

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          Mapping and quantifying mammalian transcriptomes by RNA-Seq.

          We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41-52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3' untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 x 10(5) distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.
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            Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.

            We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process. Blast2GO is freely available via Java Web Start at http://www.blast2go.de. http://www.blast2go.de -> Evaluation.
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              Genome sequencing in microfabricated high-density picolitre reactors.

              The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
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                Author and article information

                Journal
                DNA Res
                DNA Res
                dnares
                dnares
                DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
                Oxford University Press
                1340-2838
                1756-1663
                April 2012
                19 February 2012
                19 February 2012
                : 19
                : 2
                : 195-207
                Affiliations
                [1 ]College of Life Science, simpleShenzhen University , Shenzhen 518060, China
                [2 ]Institute of Genetics and Developmental Biology, simpleChinese Academy of Sciences , Beijing 100101, China
                [3 ]Biotechnology Research Institute, simpleChinese Academy of Agricultural Sciences , Beijing 100081, China
                Author notes
                [* ]To whom correspondence should be addressed. Tel. +86 10-64886859 (W.Z.); +86 10-82106143 (R.H.); +86 755-26558941 (Y.Z.). Fax. +86 755-26534274 (Y.Z.). Email: wkzhang@ 123456genetics.ac.cn (W.Z.); rfhuang@ 123456caas.net.cn (R.H.); yzzheng@ 123456szu.edu.cn (Y.Z.)
                [†]

                These authors contributed equally to this work.

                Edited by Satoshi Tabata

                dss004
                10.1093/dnares/dss004
                3325082
                22351699
                © The Author 2012. Published by Oxford University Press on behalf of Kazusa DNA Research Institute

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Genetics

                illumina sequencing, transcriptome characterization, salt-responsive genes

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