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De novo assembly and characterisation of the field pea transcriptome using RNA-Seq.

Sudheesh S, Sawbridge TI, Cogan NO, Kennedy P, Forster JW, Kaur S - BMC Genomics (2015)

Bottom Line: Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources.This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis.In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, Bundoora, VIC, 3083, Australia. shimna.sudheesh@ecodev.vic.gov.au.

ABSTRACT

Background: Field pea (Pisum sativum L.) is a cool-season grain legume that is cultivated world-wide for both human consumption and stock-feed purposes. Enhancement of genetic and genomic resources for field pea will permit improved understanding of the control of traits relevant to crop productivity and quality. Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources. The objective of this study was to perform transcriptome sequencing and characterisation from two genotypes of field pea that differ in terms of seed and plant morphological characteristics.

Results: Transcriptome sequencing was performed with RNA templates from multiple tissues of the field pea genotypes Kaspa and Parafield. Tissue samples were collected at various growth stages, and a total of 23 cDNA libraries were sequenced using Illumina high-throughput sequencing platforms. A total of 407 and 352 million paired-end reads from the Kaspa and Parafield transcriptomes, respectively were assembled into 129,282 and 149,272 contigs, which were filtered on the basis of known gene annotations, presence of open reading frames (ORFs), reciprocal matches and degree of coverage. Totals of 126,335 contigs from Kaspa and 145,730 from Parafield were subsequently selected as the reference set. Reciprocal sequence analysis revealed that c. 87% of contigs were expressed in both cultivars, while a small proportion were unique to each genotype. Reads from different libraries were aligned to the genotype-specific assemblies in order to identify and characterise expression of contigs on a tissue-specific basis, of which 87% were expressed in more than one tissue, while others showed distinct expression patterns in specific tissues, providing unique transcriptome signatures.

Conclusion: This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis. In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

No MeSH data available.


Related in: MedlinePlus

Computational pipeline for the functional annotation and classification of the field pea transcriptome
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Fig1: Computational pipeline for the functional annotation and classification of the field pea transcriptome

Mentions: A workflow detailing the process of annotation and classification of the field pea transcriptome is shown in Fig. 1. All assembled contigs were searched against the non-redundant (nr) protein database maintained by NCBI using BLASTX [34] under the threshold parameter of E-value < 10−10. Any contigs that showed significant matches to non-plant databases were excluded from further analysis. For further assembly annotation, the contigs were utilised for similarity searches against the NCBI nucleotide (nt) database, genomes and coding DNA sequences (CDS) of M. truncatula (medicago v3.5) [4], chickpea [35] and soybean [36] using BLASTN with a threshold E-value of < 10−10 to capture any genomic sequences that may have been missed by BLASTX analysis. In order to obtain the final transcriptome set, results from the nr database were preferentially selected followed by those from the nt and other legume databases, where necessary. Both Kaspa and Parafield contigs were queried by BLASTN analysis against the pea chloroplast genome sequence (NCBI RefSeq NC_014057.1) in order to identify chloroplast-derived sequences.Fig. 1


De novo assembly and characterisation of the field pea transcriptome using RNA-Seq.

Sudheesh S, Sawbridge TI, Cogan NO, Kennedy P, Forster JW, Kaur S - BMC Genomics (2015)

Computational pipeline for the functional annotation and classification of the field pea transcriptome
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4537571&req=5

Fig1: Computational pipeline for the functional annotation and classification of the field pea transcriptome
Mentions: A workflow detailing the process of annotation and classification of the field pea transcriptome is shown in Fig. 1. All assembled contigs were searched against the non-redundant (nr) protein database maintained by NCBI using BLASTX [34] under the threshold parameter of E-value < 10−10. Any contigs that showed significant matches to non-plant databases were excluded from further analysis. For further assembly annotation, the contigs were utilised for similarity searches against the NCBI nucleotide (nt) database, genomes and coding DNA sequences (CDS) of M. truncatula (medicago v3.5) [4], chickpea [35] and soybean [36] using BLASTN with a threshold E-value of < 10−10 to capture any genomic sequences that may have been missed by BLASTX analysis. In order to obtain the final transcriptome set, results from the nr database were preferentially selected followed by those from the nt and other legume databases, where necessary. Both Kaspa and Parafield contigs were queried by BLASTN analysis against the pea chloroplast genome sequence (NCBI RefSeq NC_014057.1) in order to identify chloroplast-derived sequences.Fig. 1

Bottom Line: Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources.This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis.In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, Bundoora, VIC, 3083, Australia. shimna.sudheesh@ecodev.vic.gov.au.

ABSTRACT

Background: Field pea (Pisum sativum L.) is a cool-season grain legume that is cultivated world-wide for both human consumption and stock-feed purposes. Enhancement of genetic and genomic resources for field pea will permit improved understanding of the control of traits relevant to crop productivity and quality. Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources. The objective of this study was to perform transcriptome sequencing and characterisation from two genotypes of field pea that differ in terms of seed and plant morphological characteristics.

Results: Transcriptome sequencing was performed with RNA templates from multiple tissues of the field pea genotypes Kaspa and Parafield. Tissue samples were collected at various growth stages, and a total of 23 cDNA libraries were sequenced using Illumina high-throughput sequencing platforms. A total of 407 and 352 million paired-end reads from the Kaspa and Parafield transcriptomes, respectively were assembled into 129,282 and 149,272 contigs, which were filtered on the basis of known gene annotations, presence of open reading frames (ORFs), reciprocal matches and degree of coverage. Totals of 126,335 contigs from Kaspa and 145,730 from Parafield were subsequently selected as the reference set. Reciprocal sequence analysis revealed that c. 87% of contigs were expressed in both cultivars, while a small proportion were unique to each genotype. Reads from different libraries were aligned to the genotype-specific assemblies in order to identify and characterise expression of contigs on a tissue-specific basis, of which 87% were expressed in more than one tissue, while others showed distinct expression patterns in specific tissues, providing unique transcriptome signatures.

Conclusion: This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis. In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

No MeSH data available.


Related in: MedlinePlus