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Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus.

Obermeier C, Hosseini B, Friedt W, Snowdon R - BMC Genomics (2009)

Bottom Line: The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant.Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Justus Liebig University Giessen, Department of Plant Breeding, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. christian.obermeier@agrar.uni-giessen.de

ABSTRACT

Background: Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.

Results: Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development.

Conclusion: This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.

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Statistically enriched Gene Ontology terms level 2 at p ≤ 0.05 in B. napus seeds for LongSAGE tag-matched A. thaliana gene loci at 23 DAP, compared with level 2 GO terms for all other A. thaliana gene loci. The plot was produced using the online tool WEGO (Ye et al., 2006).
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Figure 1: Statistically enriched Gene Ontology terms level 2 at p ≤ 0.05 in B. napus seeds for LongSAGE tag-matched A. thaliana gene loci at 23 DAP, compared with level 2 GO terms for all other A. thaliana gene loci. The plot was produced using the online tool WEGO (Ye et al., 2006).

Mentions: A total of 7,208 of the 8,255 matched genes were expressed at 23 DAP, while 4,875 genes were expressed at 35 DAP (Table 2). 3,828 of 8,255 genes (46.4%) were expressed at both time-points. Figure 1 gives a comparison of GO categories that are statistically over-represented for the genes expressed at 23 DAP compared to all other A. thaliana genes. From the 490 genes matched by statistically significant differentially expressed tags between 23 and 35 DAP, 194 were up-regulated at 23 DAP and 296 were up-regulated at 35 DAP. Statistically enriched GO terms found for the differentially expressed genes either up-regulated at 23 or up-regulated at 35 DAP were calculated using GOEAST [20] and details of the results are provided in additional file 2: Enriched_GO_terms_490_differentially_expressed_genes.xls. Within the category 'Biological Process', the GO terms 'developmental process', 'localization' and 'metabolic process' are enriched at both time-points. Generally, GO terms that are statistically enriched at the highly general information level 2 are similar for the two time-points. Comparison of statistically enriched GO terms for 490 differentially expressed genes at 23 and 35 DAP reveals their involvement in seed maturation, regulation of meristem organization and photomorphogenesis, seed coat development, water and fluid transport, cell-cell signalling, cell wall modification and glycerolipid, neutral lipid, acylglycerol, triglyceride, and carbohydrate metabolic processes at 23 DAP, and in protein processing, protein targeting, photosynthesis, fatty acid biosynthesis, lipid localization, storage and lipid metabolic processes at 35 DAP.


Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus.

Obermeier C, Hosseini B, Friedt W, Snowdon R - BMC Genomics (2009)

Statistically enriched Gene Ontology terms level 2 at p ≤ 0.05 in B. napus seeds for LongSAGE tag-matched A. thaliana gene loci at 23 DAP, compared with level 2 GO terms for all other A. thaliana gene loci. The plot was produced using the online tool WEGO (Ye et al., 2006).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719671&req=5

Figure 1: Statistically enriched Gene Ontology terms level 2 at p ≤ 0.05 in B. napus seeds for LongSAGE tag-matched A. thaliana gene loci at 23 DAP, compared with level 2 GO terms for all other A. thaliana gene loci. The plot was produced using the online tool WEGO (Ye et al., 2006).
Mentions: A total of 7,208 of the 8,255 matched genes were expressed at 23 DAP, while 4,875 genes were expressed at 35 DAP (Table 2). 3,828 of 8,255 genes (46.4%) were expressed at both time-points. Figure 1 gives a comparison of GO categories that are statistically over-represented for the genes expressed at 23 DAP compared to all other A. thaliana genes. From the 490 genes matched by statistically significant differentially expressed tags between 23 and 35 DAP, 194 were up-regulated at 23 DAP and 296 were up-regulated at 35 DAP. Statistically enriched GO terms found for the differentially expressed genes either up-regulated at 23 or up-regulated at 35 DAP were calculated using GOEAST [20] and details of the results are provided in additional file 2: Enriched_GO_terms_490_differentially_expressed_genes.xls. Within the category 'Biological Process', the GO terms 'developmental process', 'localization' and 'metabolic process' are enriched at both time-points. Generally, GO terms that are statistically enriched at the highly general information level 2 are similar for the two time-points. Comparison of statistically enriched GO terms for 490 differentially expressed genes at 23 and 35 DAP reveals their involvement in seed maturation, regulation of meristem organization and photomorphogenesis, seed coat development, water and fluid transport, cell-cell signalling, cell wall modification and glycerolipid, neutral lipid, acylglycerol, triglyceride, and carbohydrate metabolic processes at 23 DAP, and in protein processing, protein targeting, photosynthesis, fatty acid biosynthesis, lipid localization, storage and lipid metabolic processes at 35 DAP.

Bottom Line: The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant.Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Justus Liebig University Giessen, Department of Plant Breeding, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. christian.obermeier@agrar.uni-giessen.de

ABSTRACT

Background: Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.

Results: Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development.

Conclusion: This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.

Show MeSH
Related in: MedlinePlus