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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

Details of the selection process for field pea contigs. K - Kaspa transcriptome and P - Parafield transcriptome
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Fig5: Details of the selection process for field pea contigs. K - Kaspa transcriptome and P - Parafield transcriptome

Mentions: In summary, a total of 80,592 contigs from Kaspa and 88,487 from Parafield were annotated and characterised using the similarity searches as described. However, a large proportion of contigs from both cultivars (47,058 from Kaspa and 60,280 from Parafield) still remained uncharacterised. These sub-sets were further evaluated and searched for the presence of ORFs. This process identified an additional 23,800 contigs from Kaspa (Additional file 8A) and 28,047 from Parafield (Additional file 8B) which contained a START and STOP codon with minimum sequence length of 100 bp. An additional 16,178 contigs from Kaspa and 20,602 from Parafield were identified in the reciprocal searches (Fig. 5). A final set of contigs (126,335 contigs in Kaspa and 145,730 contigs in Parafield) were compiled after further selection of contigs from the remaining sub-set based on level of coverage (≥10×), although this threshold requirement prevented discovery of lowly expressed novel contigs in field pea. All filtered contigs from both Kaspa and Parafield assemblies were deposited in DDBJ/EMBL/GenBank (accession numbers, Kaspa - GCMF00000000, GCMG00000000, GCMH00000000, GCMI00000000, GCMJ00000000, GCMK00000000 and GCML00000000 and Parafield - GCKA00000000, GCMM00000000, GCMN00000000, GCMO00000000, GCMP00000000 and GCMQ00000000).Fig. 5


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)

Details of the selection process for field pea contigs. K - Kaspa transcriptome and P - Parafield transcriptome
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Details of the selection process for field pea contigs. K - Kaspa transcriptome and P - Parafield transcriptome
Mentions: In summary, a total of 80,592 contigs from Kaspa and 88,487 from Parafield were annotated and characterised using the similarity searches as described. However, a large proportion of contigs from both cultivars (47,058 from Kaspa and 60,280 from Parafield) still remained uncharacterised. These sub-sets were further evaluated and searched for the presence of ORFs. This process identified an additional 23,800 contigs from Kaspa (Additional file 8A) and 28,047 from Parafield (Additional file 8B) which contained a START and STOP codon with minimum sequence length of 100 bp. An additional 16,178 contigs from Kaspa and 20,602 from Parafield were identified in the reciprocal searches (Fig. 5). A final set of contigs (126,335 contigs in Kaspa and 145,730 contigs in Parafield) were compiled after further selection of contigs from the remaining sub-set based on level of coverage (≥10×), although this threshold requirement prevented discovery of lowly expressed novel contigs in field pea. All filtered contigs from both Kaspa and Parafield assemblies were deposited in DDBJ/EMBL/GenBank (accession numbers, Kaspa - GCMF00000000, GCMG00000000, GCMH00000000, GCMI00000000, GCMJ00000000, GCMK00000000 and GCML00000000 and Parafield - GCKA00000000, GCMM00000000, GCMN00000000, GCMO00000000, GCMP00000000 and GCMQ00000000).Fig. 5

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