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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

Length distribution of contigs from the (a) Kaspa-specific assembly, and (b) Parafield-specific assembly
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Fig2: Length distribution of contigs from the (a) Kaspa-specific assembly, and (b) Parafield-specific assembly

Mentions: To generate a comprehensive transcriptome dataset for field pea, a total of 23 cDNA libraries were generated from the various target tissues of the two cultivars, and were sequenced using both the HiSeq 2000 and MiSeq platforms. For cv. Kaspa, a total of 432 million paired-end reads with an average read length of 100 bp were obtained from the HiSeq 2000, as compared to 4 million paired-end reads with average read length of 250 bp from the MiSeq. The comparable figures from cv. Parafield were 372 million paired end reads with an average read length of 100 bp from the HiSeq 2000, and 3.7 million paired end reads with an average read length of 250 bp from the MiSeq. Details of the sequencing outcomes for each tissue-specific library of both varieties are provided in Additional file 1. An average of 35.2 million reads were generated per tissue type. After strict quality filtering, 408 million and 352 million reads (Table 1) from Kaspa and Parafield, respectively, were used for de novo assembly. Trinity assemblies were selected for further analysis, which produced 201,317 transcripts with N50 of 781 bp (Kaspa) and 226,701 transcripts with N50 of 772 bp (Parafield) (Table 2). Further CAP3 assembly in the former resulted in 129,282 contigs, while the latter constituted 149,272 contigs (Table 2). The contig length distribution from both assemblies is shown in Fig. 2.Table 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)

Length distribution of contigs from the (a) Kaspa-specific assembly, and (b) Parafield-specific assembly
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Length distribution of contigs from the (a) Kaspa-specific assembly, and (b) Parafield-specific assembly
Mentions: To generate a comprehensive transcriptome dataset for field pea, a total of 23 cDNA libraries were generated from the various target tissues of the two cultivars, and were sequenced using both the HiSeq 2000 and MiSeq platforms. For cv. Kaspa, a total of 432 million paired-end reads with an average read length of 100 bp were obtained from the HiSeq 2000, as compared to 4 million paired-end reads with average read length of 250 bp from the MiSeq. The comparable figures from cv. Parafield were 372 million paired end reads with an average read length of 100 bp from the HiSeq 2000, and 3.7 million paired end reads with an average read length of 250 bp from the MiSeq. Details of the sequencing outcomes for each tissue-specific library of both varieties are provided in Additional file 1. An average of 35.2 million reads were generated per tissue type. After strict quality filtering, 408 million and 352 million reads (Table 1) from Kaspa and Parafield, respectively, were used for de novo assembly. Trinity assemblies were selected for further analysis, which produced 201,317 transcripts with N50 of 781 bp (Kaspa) and 226,701 transcripts with N50 of 772 bp (Parafield) (Table 2). Further CAP3 assembly in the former resulted in 129,282 contigs, while the latter constituted 149,272 contigs (Table 2). The contig length distribution from both assemblies is shown in Fig. 2.Table 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