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Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery.

Kaur S, Cogan NO, Pembleton LW, Shinozuka M, Savin KW, Materne M, Forster JW - BMC Genomics (2011)

Bottom Line: When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space.A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, La Trobe University Research and Development Park, Bundoora, Australia.

ABSTRACT

Background: Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality.

Results: Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.

Conclusions: A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

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Pie-chart representation of GO annotation results from lentil consensus sequences for Molecular Function, with a total number of gene counts of 119,316. Details are as for Figure 4.
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Figure 5: Pie-chart representation of GO annotation results from lentil consensus sequences for Molecular Function, with a total number of gene counts of 119,316. Details are as for Figure 4.

Mentions: The consensus sequences were also compared against Arabidopsis thaliana database and 7,476 unique matches were identified, including 3,941 contigs and 3,535 singletons (Additional file 5). All unique matches were annotated and gene ontology (GO) terms were further assigned corresponding to a total of 34,034 gene counts and 44,734 annotation counts (Figures 4-6). The intracellular component category of the cellular component classification class contributed the largest proportion of all annotations (17%), followed by the cytoplasmic component (13%), chloroplast component (11%), membrane component (10%), other cellular component (10%), nuclear component (8%) and plasma membrane component (8%) categories. Other components such as plastid, cytosol, mitochondria, ER, golgi apparatus, cell wall, ribosome and extracellular components were represented at proportions less than 5% of total (Figure 4). Among the molecular function classification class, the enzyme activity, transferase activity, binding activity, hydrolase activity, molecular function, nucleotide binding and protein binding categories included the majority of detected matches (Figure 5). In the biological processes classification class, cellular (25%) and metabolic processes (22%) constituted the major categories, followed by protein metabolism (9%), unknown biological processes (9%), developmental processes (5%), stress response (5%), transport (5%) and cell organization and biogenesis (4%) (Figure 6).


Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery.

Kaur S, Cogan NO, Pembleton LW, Shinozuka M, Savin KW, Materne M, Forster JW - BMC Genomics (2011)

Pie-chart representation of GO annotation results from lentil consensus sequences for Molecular Function, with a total number of gene counts of 119,316. Details are as for Figure 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113791&req=5

Figure 5: Pie-chart representation of GO annotation results from lentil consensus sequences for Molecular Function, with a total number of gene counts of 119,316. Details are as for Figure 4.
Mentions: The consensus sequences were also compared against Arabidopsis thaliana database and 7,476 unique matches were identified, including 3,941 contigs and 3,535 singletons (Additional file 5). All unique matches were annotated and gene ontology (GO) terms were further assigned corresponding to a total of 34,034 gene counts and 44,734 annotation counts (Figures 4-6). The intracellular component category of the cellular component classification class contributed the largest proportion of all annotations (17%), followed by the cytoplasmic component (13%), chloroplast component (11%), membrane component (10%), other cellular component (10%), nuclear component (8%) and plasma membrane component (8%) categories. Other components such as plastid, cytosol, mitochondria, ER, golgi apparatus, cell wall, ribosome and extracellular components were represented at proportions less than 5% of total (Figure 4). Among the molecular function classification class, the enzyme activity, transferase activity, binding activity, hydrolase activity, molecular function, nucleotide binding and protein binding categories included the majority of detected matches (Figure 5). In the biological processes classification class, cellular (25%) and metabolic processes (22%) constituted the major categories, followed by protein metabolism (9%), unknown biological processes (9%), developmental processes (5%), stress response (5%), transport (5%) and cell organization and biogenesis (4%) (Figure 6).

Bottom Line: When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space.A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, La Trobe University Research and Development Park, Bundoora, Australia.

ABSTRACT

Background: Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality.

Results: Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.

Conclusions: A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

Show MeSH
Related in: MedlinePlus