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      De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

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          Abstract

          Background

          De novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species ( E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs.

          Results

          We present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions.

          Conclusions

          De novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants.

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          Most cited references27

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          The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

          The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.
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            The transcriptional landscape of the yeast genome defined by RNA sequencing.

            The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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              Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing.

              We present a de novo assembly of a eukaryote transcriptome using 454 pyrosequencing data. The Glanville fritillary butterfly (Melitaea cinxia; Lepidoptera: Nymphalidae) is a prominent species in population biology but had no previous genomic data. Sequencing runs using two normalized complementary DNA collections from a genetically diverse pool of larvae, pupae, and adults yielded 608,053 expressed sequence tags (mean length = 110 nucleotides), which assembled into 48,354 contigs (sets of overlapping DNA segments) and 59,943 singletons. BLAST comparisons confirmed the accuracy of the sequencing and assembly, and indicated the presence of c. 9000 unique genes, along with > 6000 additional microarray-confirmed unannotated contigs. Average depth of coverage was 6.5-fold for the longest 4800 contigs (348-2849 bp in length), sufficient for detecting large numbers of single nucleotide polymorphisms. Oligonucleotide microarray probes designed from the assembled sequences showed highly repeatable hybridization intensity and revealed biological differences among individuals. We conclude that 454 sequencing, when performed to provide sufficient coverage depth, allows de novo transcriptome assembly and a fast, cost-effective, and reliable method for development of functional genomic tools for nonmodel species. This development narrows the gap between approaches based on model organisms with rich genetic resources vs. species that are most tractable for ecological and evolutionary studies.
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                Author and article information

                Journal
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2010
                1 December 2010
                : 11
                : 681
                Affiliations
                [1 ]Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa
                [2 ]Bioinformatics and Computational Biology Unit, Department of Biochemistry, University of Pretoria, Pretoria, 0002, South Africa
                Article
                1471-2164-11-681
                10.1186/1471-2164-11-681
                3053591
                21122097
                d701ed0f-6a66-4660-b7ff-65f40bd5a6bb
                Copyright ©2010 Mizrachi et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 30 May 2010
                : 1 December 2010
                Categories
                Research Article

                Genetics
                Genetics

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