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      De Novo Assembly of Chickpea Transcriptome Using Short Reads for Gene Discovery and Marker Identification

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          Abstract

          Chickpea ranks third among the food legume crops production in the world. However, the genomic resources available for chickpea are still very limited. In the present study, the transcriptome of chickpea was sequenced with short reads on Illumina Genome Analyzer platform. We have assessed the effect of sequence quality, various assembly parameters and assembly programs on the final assembly output. We assembled ∼107million high-quality trimmed reads using Velvet followed by Oases with optimal parameters into a non-redundant set of 53 409 transcripts (≥100 bp), representing about 28 Mb of unique transcriptome sequence. The average length of transcripts was 523 bp and N50 length of 900 bp with coverage of 25.7 rpkm (reads per kilobase per million). At the protein level, a total of 45 636 (85.5%) chickpea transcripts showed significant similarity with unigenes/predicted proteins from other legumes or sequenced plant genomes. Functional categorization revealed the conservation of genes involved in various biological processes in chickpea. In addition, we identified simple sequence repeat motifs in transcripts. The chickpea transcripts set generated here provides a resource for gene discovery and development of functional molecular markers. In addition, the strategy for de novo assembly of transcriptome data presented here will be helpful in other similar transcriptome studies.

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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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            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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              Genome Structure of the Legume, Lotus japonicus

              The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.
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                Author and article information

                Journal
                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
                February 2011
                7 January 2011
                7 January 2011
                : 18
                : 1
                : 53-63
                Affiliations
                simpleNational Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg , New Delhi 110 067, India
                Author notes
                [* ]To whom correspondence should be addressed. Tel. +91-11-26735182; Fax. +91-11-26741658. E-mail: mjain@ 123456nipgr.res.in

                Edited by Kazuo Shinozaki

                Article
                dsq028
                10.1093/dnares/dsq028
                3041503
                21217129
                0b3f3736-8f9b-44bb-b9ca-6d54e96b884a
                © The Author 2011. 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/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 September 2010
                : 2 November 2010
                Categories
                Full Papers

                Genetics
                de novo assembly,transcriptome,short read,next generation sequencing,chickpea
                Genetics
                de novo assembly, transcriptome, short read, next generation sequencing, chickpea

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