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Establishment and analysis of a reference transcriptome for Spodoptera frugiperda.

Legeai F, Gimenez S, Duvic B, Escoubas JM, Gosselin Grenet AS, Blanc F, Cousserans F, Séninet I, Bretaudeau A, Mutuel D, Girard PA, Monsempes C, Magdelenat G, Hilliou F, Feyereisen R, Ogliastro M, Volkoff AN, Jacquin-Joly E, d'Alençon E, Nègre N, Fournier P - BMC Genomics (2014)

Bottom Line: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome.While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts.Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1333, DGIMI, Montpellier, France. nicolas.negre@univ-montp2.fr.

ABSTRACT

Background: Spodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides.

Results: In this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family--ribosomal proteins--as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied.

Conclusion: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

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Related in: MedlinePlus

Content of the reference transcriptome. A. Barplot representing the percentages of multiple hit reads, unmapped reads and uniquely mapped reads, as provided by Bowtie, when aligning an RNAseq library against either the Sf_454_clusters assembly or the Sf_TR2012b assembly. The percentages obtained are the average of four independent experiments. B. Pie chart representing the number and percentage of contigs from Sf_TR2012b grouped by their best blastx hit against nr. Number of contigs and percentage of the total are represented. C. Synthetic table representing the number of genes found per family in the Sf_TR2012b assembly. The number of full and partial transcripts for Hox-domain proteins is irrelevant because the only conserved part is the homeodomain itself.
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Fig1: Content of the reference transcriptome. A. Barplot representing the percentages of multiple hit reads, unmapped reads and uniquely mapped reads, as provided by Bowtie, when aligning an RNAseq library against either the Sf_454_clusters assembly or the Sf_TR2012b assembly. The percentages obtained are the average of four independent experiments. B. Pie chart representing the number and percentage of contigs from Sf_TR2012b grouped by their best blastx hit against nr. Number of contigs and percentage of the total are represented. C. Synthetic table representing the number of genes found per family in the Sf_TR2012b assembly. The number of full and partial transcripts for Hox-domain proteins is irrelevant because the only conserved part is the homeodomain itself.

Mentions: First, we wanted to evaluate whether the last step of assembly, combining the short reads with the long reads sequences, was improving the quality of our transcriptome. We evaluated this improvement by aligning independent Illumina libraries on the different reference transcriptome assemblies using the Bowtie software [7]. We used 4 .fastq files containing sequences for whole larvae RNA extracted in experimental conditions, which were unrelated to our transcriptome project (N. Volkoff, personal communication), to align the reads against Sf_TR2012b and Sf_GATC_clusters. For all 4 samples, the average of unmapped reads is around 20% when aligned on both assemblies (Sf_GATC_Clusters and Sf_TR2012b) (Figure 1A). However we greatly improved the percentage of uniquely mapped reads from 22% to 45% while decreasing the number of multiple reads from 58% to 32% (Figure 1A). This indicates that our second step of assembly was particularly effective at eliminating most of the redundancy that one could expect from an organism that is far from being isogenic.Figure 1


Establishment and analysis of a reference transcriptome for Spodoptera frugiperda.

Legeai F, Gimenez S, Duvic B, Escoubas JM, Gosselin Grenet AS, Blanc F, Cousserans F, Séninet I, Bretaudeau A, Mutuel D, Girard PA, Monsempes C, Magdelenat G, Hilliou F, Feyereisen R, Ogliastro M, Volkoff AN, Jacquin-Joly E, d'Alençon E, Nègre N, Fournier P - BMC Genomics (2014)

Content of the reference transcriptome. A. Barplot representing the percentages of multiple hit reads, unmapped reads and uniquely mapped reads, as provided by Bowtie, when aligning an RNAseq library against either the Sf_454_clusters assembly or the Sf_TR2012b assembly. The percentages obtained are the average of four independent experiments. B. Pie chart representing the number and percentage of contigs from Sf_TR2012b grouped by their best blastx hit against nr. Number of contigs and percentage of the total are represented. C. Synthetic table representing the number of genes found per family in the Sf_TR2012b assembly. The number of full and partial transcripts for Hox-domain proteins is irrelevant because the only conserved part is the homeodomain itself.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150953&req=5

Fig1: Content of the reference transcriptome. A. Barplot representing the percentages of multiple hit reads, unmapped reads and uniquely mapped reads, as provided by Bowtie, when aligning an RNAseq library against either the Sf_454_clusters assembly or the Sf_TR2012b assembly. The percentages obtained are the average of four independent experiments. B. Pie chart representing the number and percentage of contigs from Sf_TR2012b grouped by their best blastx hit against nr. Number of contigs and percentage of the total are represented. C. Synthetic table representing the number of genes found per family in the Sf_TR2012b assembly. The number of full and partial transcripts for Hox-domain proteins is irrelevant because the only conserved part is the homeodomain itself.
Mentions: First, we wanted to evaluate whether the last step of assembly, combining the short reads with the long reads sequences, was improving the quality of our transcriptome. We evaluated this improvement by aligning independent Illumina libraries on the different reference transcriptome assemblies using the Bowtie software [7]. We used 4 .fastq files containing sequences for whole larvae RNA extracted in experimental conditions, which were unrelated to our transcriptome project (N. Volkoff, personal communication), to align the reads against Sf_TR2012b and Sf_GATC_clusters. For all 4 samples, the average of unmapped reads is around 20% when aligned on both assemblies (Sf_GATC_Clusters and Sf_TR2012b) (Figure 1A). However we greatly improved the percentage of uniquely mapped reads from 22% to 45% while decreasing the number of multiple reads from 58% to 32% (Figure 1A). This indicates that our second step of assembly was particularly effective at eliminating most of the redundancy that one could expect from an organism that is far from being isogenic.Figure 1

Bottom Line: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome.While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts.Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1333, DGIMI, Montpellier, France. nicolas.negre@univ-montp2.fr.

ABSTRACT

Background: Spodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides.

Results: In this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family--ribosomal proteins--as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied.

Conclusion: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

Show MeSH
Related in: MedlinePlus