Dataset Information

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

ABSTRACT: This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

SUBMITTER: Dubey A

PROVIDER: S-EPMC3111231 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

Dubey Anuja A Farmer Andrew A Schlueter Jessica J Cannon Steven B SB Abernathy Brian B Tuteja Reetu R Woodward Jimmy J Shah Trushar T Mulasmanovic Benjamin B Kudapa Himabindu H Raju Nikku L NL Gothalwal Ragini R Pande Suresh S Xiao Yongli Y Town Chris D CD Singh Nagendra K NK May Gregory D GD Jackson Scott S Varshney Rajeev K RK

DNA research : an international journal for rapid publication of reports on genes and genomes 20110512 3

This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights seve ...[more]

PMID: 21565938

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Project description:Pigeonpea is a major source of dietary protein to the vegetarian population of the Indian sub-continent. Crop improvement to mitigate biotic and abiotic stresses for realization of its potential yield and bridging yield gap is the need of the hour. Availability of limited genomic resources in the cultivated germplasm, however, is a serious bottleneck towards successful molecular breeding for the development of superior genotypes in pigeonpea. In view of this, improvement of pigeonpea can be attempted through transgenesis or by exploiting genetic resources from its wild relatives. Pigeonpea wild relatives are known to be bestowed with agronomic traits of importance; discovery and deployment of genes from them can provide a lucrative option for crop improvement. Understanding molecular signatures of wild relatives would not only provide information about the mechanism behind desired traits but also enable us to extrapolate the information to cultivated pigeonpea. The present study deals with the characterization of leaf transcriptomes of Cajanus cajan and one of its wild relatives, Cajanus platycarpus. Illumina sequencing revealed 0.11 million transcripts in both the species with an annotation of 0.09 million (82%) transcripts using BLASTX. Comparative transcriptome analyses on the whole, divulged cues about the wild relative being vigilant and agile. Gene ontology and Mapman analysis depicted higher number of transcripts in the wild relative pertaining to signaling, transcription factors and stress responsive genes. Further, networking between the differentially expressed MapMan bins demonstrated conspicuous interactions between different bins through 535 nodes (512 Genes and 23 Pathways) and 1857 edges. The authenticity of RNA-seq analysis was confirmed by qRT-PCR. The information emanating from this study can provide valuable information and resource for future translational research including genome editing to alleviate varied stresses. Further, this learning can be a platform for in-depth investigations to decipher molecular mechanisms for mitigation of various stresses in the wild relative.

Project description:BackgroundPigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping.ResultsIn this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥ 18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population.ConclusionWe developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

Project description:Genome assembly of short reads from large plant genomes remains a challenge in computational biology despite major developments in next generation sequencing. Of late several draft assemblies have been reported in sequenced plant genomes. The reported draft genome assemblies of Cajanus cajan have different levels of genome completeness, a large number of repeats, gaps, and segmental duplications. Draft assemblies with portions of genome missing are shorter than the referenced original genome. These assemblies come with low map accuracy affecting further functional annotation and the prediction of gene components as desired by crop researchers. Genome coverage, i.e., the number of sequenced raw reads mapped onto a certain location of the genome is an important quality indicator of completeness and assembly quality in draft assemblies. The present work aimed to improve the coverage in reported de novo sequenced draft genomes (GCA_000340665.1 and GCA_000230855.2) of pigeonpea, a legume widely cultivated in India. The two recently sequenced assemblies, A1 and A2 comprised 72% and 75% of the estimated coverage of the genome, respectively. We employed an assembly reconciliation approach to compare the draft assemblies and merge them, filling the gaps by employing an algorithm size sorting mate-pair library to generate a high quality and near complete assembly with enhanced contiguity. The majority of gaps present within scaffolds were filled with right-sized mate-pair reads. The improved assembly reduced the number of gaps than those reported in draft assemblies resulting in an improved genome coverage of 82.4%. Map accuracy of the improved assembly was evaluated using various quality metrics and for the presence of specific trait-related functional genes. Employed pair-end and mate-pair local libraries helped us to reduce gaps, repeats, and other sequence errors resulting in lengthier scaffolds compared to the two draft assemblies. We reported the prediction of putative host resistance genes against Fusarium wilt disease by their performance and evaluated them both in wet laboratory and field phenotypic conditions.

Dataset Information

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

Publications

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

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