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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

The distribution of field pea contigs against genes encoding enzymes involved in nitrogen metabolism pathways. This is a global nitrogen metabolism pathway map in which a red colour indicates genes identified in data from the present study, all of the known nitrogen metabolism genes in legumes having been identified
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Fig4: The distribution of field pea contigs against genes encoding enzymes involved in nitrogen metabolism pathways. This is a global nitrogen metabolism pathway map in which a red colour indicates genes identified in data from the present study, all of the known nitrogen metabolism genes in legumes having been identified

Mentions: In order to characterise the assembled contigs and identify active biological processes, annotated sequences were mapped to the reference biochemical pathways in the KEGG database using eudicot species such as Arabidopsis thaliana (L.) Heynh., cocao (Theobroma cacao L.), soybean, alpine strawberry (Fragaria vesca L.), grapevine (Vitis vinifera L.), potato (Solanum lycopersicum L.) and rice (Oryza sativa sp. japonica) as references. In total, 22,056 (37.3 %) contigs from Kaspa and 23,692 (37.1 %) contigs from Parafield were mapped to 157 KEGG pathways corresponding to five modules; metabolism, cellular processes, genetic information processing, environmental information processing and organismal systems (Additional file 6). Metabolic pathways were well represented, most of which were associated with biosynthesis of secondary metabolites, carbohydrate metabolism and amino acid metabolism. Furthermore, mapping of contigs against the glycolysis/gluconeogenesis pathway revealed that all of the genes involved in this pathway were present in the dataset. Another important pathway (nitrogen metabolism), which is crucial to legume species, was also analysed and revealed the presence of all known genes (Fig. 4). In addition, genes for all key enzymes required for the legume-specific isoflavonoid biosynthesis pathway were identified, using M. truncatula and chickpea as references.Fig. 4


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)

The distribution of field pea contigs against genes encoding enzymes involved in nitrogen metabolism pathways. This is a global nitrogen metabolism pathway map in which a red colour indicates genes identified in data from the present study, all of the known nitrogen metabolism genes in legumes having been identified
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: The distribution of field pea contigs against genes encoding enzymes involved in nitrogen metabolism pathways. This is a global nitrogen metabolism pathway map in which a red colour indicates genes identified in data from the present study, all of the known nitrogen metabolism genes in legumes having been identified
Mentions: In order to characterise the assembled contigs and identify active biological processes, annotated sequences were mapped to the reference biochemical pathways in the KEGG database using eudicot species such as Arabidopsis thaliana (L.) Heynh., cocao (Theobroma cacao L.), soybean, alpine strawberry (Fragaria vesca L.), grapevine (Vitis vinifera L.), potato (Solanum lycopersicum L.) and rice (Oryza sativa sp. japonica) as references. In total, 22,056 (37.3 %) contigs from Kaspa and 23,692 (37.1 %) contigs from Parafield were mapped to 157 KEGG pathways corresponding to five modules; metabolism, cellular processes, genetic information processing, environmental information processing and organismal systems (Additional file 6). Metabolic pathways were well represented, most of which were associated with biosynthesis of secondary metabolites, carbohydrate metabolism and amino acid metabolism. Furthermore, mapping of contigs against the glycolysis/gluconeogenesis pathway revealed that all of the genes involved in this pathway were present in the dataset. Another important pathway (nitrogen metabolism), which is crucial to legume species, was also analysed and revealed the presence of all known genes (Fig. 4). In addition, genes for all key enzymes required for the legume-specific isoflavonoid biosynthesis pathway were identified, using M. truncatula and chickpea as references.Fig. 4

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