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      De novo assembly and characterization of the root transcriptome of Aegilops variabilis during an interaction with the cereal cyst nematode

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          Abstract

          Background

          Aegilops variabilis No.1 is highly resistant to cereal cyst nematode (CCN). However, a lack of genomic information has restricted studies on CCN resistance genes in Ae. variabilis and has limited genetic applications in wheat breeding.

          Results

          Using RNA-Seq technology, we generated a root transcriptome at a sequencing depth of 4.69 gigabases of Ae. variabilis No. 1 from a pooled RNA sample. The sample contained equal amounts of RNA extracted from CCN-infected and untreated control plants at three time-points. Using the Trinity method, nearly 52,081,238 high-quality trimmed reads were assembled into a non-redundant set of 118,064 unigenes with an average length of 500 bp and an N50 of 599 bp. The total assembly was 59.09 Mb of unique transcriptome sequences with average read-depth coverage of 33.25×. In BLAST searches of our database against public databases, 66.46% (78,467) of the unigenes were annotated with gene descriptions, conserved protein domains, or gene ontology terms. Functional categorization further revealed 7,408 individual unigenes and three pathways related to plant stress resistance.

          Conclusions

          We conducted high-resolution transcriptome profiling related to root development and the response to CCN infection in Ae. variabilis No.1. This research facilitates further studies on gene discovery and on the molecular mechanisms related to CCN resistance.

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          Most cited references31

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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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            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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              Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx

              Background New methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora. Results More than 600,000 reads produced in a single 454 sequencing run were assembled into ~40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified ~11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed ~8,500 pairs of orthologs and ~100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing. Conclusion The methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.
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                Author and article information

                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2012
                11 April 2012
                : 13
                : 133
                Affiliations
                [1 ]Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China
                [2 ]Graduate University of the Chinese Academy of Sciences, Beijing, China
                Article
                1471-2164-13-133
                10.1186/1471-2164-13-133
                3439707
                22494814
                b1321f77-6c9b-4708-84ef-2bb7ecbb1c3c
                Copyright ©2012 Xu 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
                : 15 August 2011
                : 11 April 2012
                Categories
                Research Article

                Genetics
                Genetics

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