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Transcriptome Analysis of the Emerald Ash Borer (EAB), Agrilus planipennis: De Novo Assembly, Functional Annotation and Comparative Analysis.

Duan J, Ladd T, Doucet D, Cusson M, vanFrankenhuyzen K, Mittapalli O, Krell PJ, Quan G - PLoS ONE (2015)

Bottom Line: The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific.This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date.Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

View Article: PubMed Central - PubMed

Affiliation: Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, Sault Ste. Marie, Ontario, Canada; Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.

ABSTRACT

Background: The Emerald ash borer (EAB), Agrilus planipennis, is an invasive phloem-feeding insect pest of ash trees. Since its initial discovery near the Detroit, US- Windsor, Canada area in 2002, the spread of EAB has had strong negative economic, social and environmental impacts in both countries. Several transcriptomes from specific tissues including midgut, fat body and antenna have recently been generated. However, the relatively low sequence depth, gene coverage and completeness limited the usefulness of these EAB databases.

Methodology and principal findings: High-throughput deep RNA-Sequencing (RNA-Seq) was used to obtain 473.9 million pairs of 100 bp length paired-end reads from various life stages and tissues. These reads were assembled into 88,907 contigs using the Trinity strategy and integrated into 38,160 unigenes after redundant sequences were removed. We annotated 11,229 unigenes by searching against the public nr, Swiss-Prot and COG. The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific. Differential gene expression revealed that 290 unigenes are expressed during larval molting and 3,911 unigenes during metamorphosis from larvae to pupae, respectively (FDR< 0.01 and log2 FC>2). In addition, 1,167 differentially expressed unigenes were identified from larval and adult midguts, 435 unigenes were up-regulated in larval midgut and 732 unigenes were up-regulated in adult midgut. Most of the genes involved in RNA interference (RNAi) pathways were identified, which implies the existence of a system RNAi in EAB.

Conclusions and significance: This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date. Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

No MeSH data available.


Related in: MedlinePlus

Correlation between the gene expression ratios obtained from RNA-Seq data and qRT-PCR.(A) Expression ratios (Log2FC) obtained by RNA-seq and qRT-PCR. The expression ratio change was calculated by the 2-ΔΔCT method. TEF-1α was used as a reference gene to normalize the qRT-PCR data. Error bars represent the standard error of the mean (n = 4). Investigated genes were listed in S5 Table, including 1, EABT36748; 2, EABT26334; 3, EABT1664; 4, EABT37717; 5, EABT755; 6, EABT22472; 7, EABT11324; 8, EABT14053; 9, EABT30570; 10,EABT27511; 11, EABT36884; 12, EABT35689; 13, EABT19583; 14, EABT23189; 15, EABT23473; 16, EABT4817; 17, EABT14338; 18, EABT16135; 19, EABT21639; 20, EABT4315; 21, EABT37729; 22, EABT33854; 23, EABT7214 and 24, EABT36743. (B) Lineage analysis between RNA-Seq and qRT-PCR. The Log2FC obtained by qRT-PCR (x-axis) are plotted against the Log2FC obtained by RNA-Seq (y-axis).
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pone.0134824.g007: Correlation between the gene expression ratios obtained from RNA-Seq data and qRT-PCR.(A) Expression ratios (Log2FC) obtained by RNA-seq and qRT-PCR. The expression ratio change was calculated by the 2-ΔΔCT method. TEF-1α was used as a reference gene to normalize the qRT-PCR data. Error bars represent the standard error of the mean (n = 4). Investigated genes were listed in S5 Table, including 1, EABT36748; 2, EABT26334; 3, EABT1664; 4, EABT37717; 5, EABT755; 6, EABT22472; 7, EABT11324; 8, EABT14053; 9, EABT30570; 10,EABT27511; 11, EABT36884; 12, EABT35689; 13, EABT19583; 14, EABT23189; 15, EABT23473; 16, EABT4817; 17, EABT14338; 18, EABT16135; 19, EABT21639; 20, EABT4315; 21, EABT37729; 22, EABT33854; 23, EABT7214 and 24, EABT36743. (B) Lineage analysis between RNA-Seq and qRT-PCR. The Log2FC obtained by qRT-PCR (x-axis) are plotted against the Log2FC obtained by RNA-Seq (y-axis).

Mentions: In order to validate the RNA-seq expression analysis, we selected 24 differentially-expressed unigenes and verified their expression between different developmental stages and tissues by qRT-PCR. As shown in Fig 7A and S5 Table, fold-changes obtained by qRT-PCR were compared with those from the RNA-Seq expression analysis. A high correlation coefficient of R = 0.96 was observed between qRT-PCR and RNA-Seq expression data. Linear regression analysis of the correlation (Fig 7B) shows an R2 (goodness of fit) value of 0.93, with a corresponding slope of 1.03, suggesting a strong positive correlation between qRT-PCR and RNA-Seq data. These results confirm that fold-changes by qRT-PCR were consistent with the fold changes obtained by RNA-Seq.


Transcriptome Analysis of the Emerald Ash Borer (EAB), Agrilus planipennis: De Novo Assembly, Functional Annotation and Comparative Analysis.

Duan J, Ladd T, Doucet D, Cusson M, vanFrankenhuyzen K, Mittapalli O, Krell PJ, Quan G - PLoS ONE (2015)

Correlation between the gene expression ratios obtained from RNA-Seq data and qRT-PCR.(A) Expression ratios (Log2FC) obtained by RNA-seq and qRT-PCR. The expression ratio change was calculated by the 2-ΔΔCT method. TEF-1α was used as a reference gene to normalize the qRT-PCR data. Error bars represent the standard error of the mean (n = 4). Investigated genes were listed in S5 Table, including 1, EABT36748; 2, EABT26334; 3, EABT1664; 4, EABT37717; 5, EABT755; 6, EABT22472; 7, EABT11324; 8, EABT14053; 9, EABT30570; 10,EABT27511; 11, EABT36884; 12, EABT35689; 13, EABT19583; 14, EABT23189; 15, EABT23473; 16, EABT4817; 17, EABT14338; 18, EABT16135; 19, EABT21639; 20, EABT4315; 21, EABT37729; 22, EABT33854; 23, EABT7214 and 24, EABT36743. (B) Lineage analysis between RNA-Seq and qRT-PCR. The Log2FC obtained by qRT-PCR (x-axis) are plotted against the Log2FC obtained by RNA-Seq (y-axis).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526369&req=5

pone.0134824.g007: Correlation between the gene expression ratios obtained from RNA-Seq data and qRT-PCR.(A) Expression ratios (Log2FC) obtained by RNA-seq and qRT-PCR. The expression ratio change was calculated by the 2-ΔΔCT method. TEF-1α was used as a reference gene to normalize the qRT-PCR data. Error bars represent the standard error of the mean (n = 4). Investigated genes were listed in S5 Table, including 1, EABT36748; 2, EABT26334; 3, EABT1664; 4, EABT37717; 5, EABT755; 6, EABT22472; 7, EABT11324; 8, EABT14053; 9, EABT30570; 10,EABT27511; 11, EABT36884; 12, EABT35689; 13, EABT19583; 14, EABT23189; 15, EABT23473; 16, EABT4817; 17, EABT14338; 18, EABT16135; 19, EABT21639; 20, EABT4315; 21, EABT37729; 22, EABT33854; 23, EABT7214 and 24, EABT36743. (B) Lineage analysis between RNA-Seq and qRT-PCR. The Log2FC obtained by qRT-PCR (x-axis) are plotted against the Log2FC obtained by RNA-Seq (y-axis).
Mentions: In order to validate the RNA-seq expression analysis, we selected 24 differentially-expressed unigenes and verified their expression between different developmental stages and tissues by qRT-PCR. As shown in Fig 7A and S5 Table, fold-changes obtained by qRT-PCR were compared with those from the RNA-Seq expression analysis. A high correlation coefficient of R = 0.96 was observed between qRT-PCR and RNA-Seq expression data. Linear regression analysis of the correlation (Fig 7B) shows an R2 (goodness of fit) value of 0.93, with a corresponding slope of 1.03, suggesting a strong positive correlation between qRT-PCR and RNA-Seq data. These results confirm that fold-changes by qRT-PCR were consistent with the fold changes obtained by RNA-Seq.

Bottom Line: The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific.This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date.Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

View Article: PubMed Central - PubMed

Affiliation: Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, Sault Ste. Marie, Ontario, Canada; Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.

ABSTRACT

Background: The Emerald ash borer (EAB), Agrilus planipennis, is an invasive phloem-feeding insect pest of ash trees. Since its initial discovery near the Detroit, US- Windsor, Canada area in 2002, the spread of EAB has had strong negative economic, social and environmental impacts in both countries. Several transcriptomes from specific tissues including midgut, fat body and antenna have recently been generated. However, the relatively low sequence depth, gene coverage and completeness limited the usefulness of these EAB databases.

Methodology and principal findings: High-throughput deep RNA-Sequencing (RNA-Seq) was used to obtain 473.9 million pairs of 100 bp length paired-end reads from various life stages and tissues. These reads were assembled into 88,907 contigs using the Trinity strategy and integrated into 38,160 unigenes after redundant sequences were removed. We annotated 11,229 unigenes by searching against the public nr, Swiss-Prot and COG. The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific. Differential gene expression revealed that 290 unigenes are expressed during larval molting and 3,911 unigenes during metamorphosis from larvae to pupae, respectively (FDR< 0.01 and log2 FC>2). In addition, 1,167 differentially expressed unigenes were identified from larval and adult midguts, 435 unigenes were up-regulated in larval midgut and 732 unigenes were up-regulated in adult midgut. Most of the genes involved in RNA interference (RNAi) pathways were identified, which implies the existence of a system RNAi in EAB.

Conclusions and significance: This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date. Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

No MeSH data available.


Related in: MedlinePlus