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RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne).

Jung WY, Lee SS, Kim CW, Kim HS, Min SR, Moon JS, Kwon SY, Jeon JH, Cho HS - PLoS ONE (2014)

Bottom Line: In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR.Exsiting genetic and genomic data for H. tuberosus are scarce.The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

View Article: PubMed Central - PubMed

Affiliation: Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea; Animal Material Engineering, Gyeongnam National University of Science and Technology, Jinju, Korea.

ABSTRACT
Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

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Gene Ontology (GO) classification of the assembled loci.The results of BLASTX searches against the Phytozome database were used for GO term mapping and annotation. The number and ratio of sequences assigned to level 2 GO terms from GO subcategories including biological process, molecular process, molecular function, and cellular component are shown (BP: biological process, CC: Cellular Component, MF: Molecular Function).
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pone-0111982-g002: Gene Ontology (GO) classification of the assembled loci.The results of BLASTX searches against the Phytozome database were used for GO term mapping and annotation. The number and ratio of sequences assigned to level 2 GO terms from GO subcategories including biological process, molecular process, molecular function, and cellular component are shown (BP: biological process, CC: Cellular Component, MF: Molecular Function).

Mentions: We used GO term enrichment analysis to classify the functions of the assembled H. tuberosus loci [44]. The BLASTX similarity search results for the 66,322 H. tuberosus loci were imported into the Phytozome database for GO mapping and annotation with TAIR information. Sequence annotations associated with 19,848 loci (29.93%) were categorized into the three main GO ontologies: biological process (BP), cellular component (CC), and molecular function (MF) (Figure 2). In total, 7,589, 8,685 and 8,510 loci were assigned GO terms from the BP, CC, and MF categories, respectively. The GO terms were summarized into 49 subcategories with GO classifications at level 2. In the BP category, the dominant subcategories assigned to H. tuberosus loci were as follows: ‘Primary metabolic process’ (15.19%), ‘Cellular metabolic process’ (14.75%), ‘Response to stress’ (6.76%), ‘Nitrogen compound metabolic process’ (5.33%) and ‘multicellular organismal development’ (4.08%). In the CC category, ‘Cell part’ (21.61%), ‘Intracellular’ (13.81%), ‘Intracellular part’ (13.49%), ‘Intracellular organelle’ (12.33%), and ‘Membrane-bounded organelle’ (11.89%) were the dominant subcategories. Finally, ‘Nucleotide binding’ (22.12%), ‘Protein binding’ (20.45%), ‘Nucleoside binding’ (18.94%), ‘Transferase activity’ (15.80%), and ‘Hydrolase activity’ (12.17%) were dominant in the MF category. These annotations indicated that extensive membrane metabolic activity occurred in H. tuberous in the sampled tissues. The loci were analyzed further for GO-category enrichment relative to Plant GO slim categories using AgriGO [43]. The H. tuberosus loci contained 71 significantly enriched (FDR ≤ 0.01) functional GO terms in the BP category, including top five terms (“cellular process”, GO:0009987; “cellular metabolic process”, GO:0044237; “metabolic process”, GO:0008152, “primary metabolic process”, GO:0044238, and “response to stimulus”, GO:0050896, respectively). The GO term “cellular, macromolecule, nitrogen compound and primary metabolic process” was highly enriched (FDR≤1.0E-40), and enriched daughter terms included “nucleobase, nucleoside, nucleotide and nucleic acid metabolic process” (GO:0006139), “cellular macromolecule metabolic process” (GO:0044260), “macromolecule modification” (GO:0043412), “carbohydrate metabolic process” (GO:0005975; including several loci with fructan 1,2-beta-fructan 1-fructosyltransferase, invertase, hexokinase, sucrose synthase, sucrose phosphate synthase, starch synthase, starch branching enzyme, and beta glucosidase sequences), and “cellular biosynthetic process” (GO:00044249; sucrose 1F-beta-D-fructosyltransferase). These results suggest that gene expression in H. tuberosus is geared towards carbohydrate metabolism, cellular biosynthetic processes, and macromolecule modification functions. This expression enrichment concurs with biosynthetic analysis results indicating that inulin accumulation occurs at the time of tuber initiation [4], [19]. An additional enriched GO term was “protein modification process” (GO:0006464). This included loci with cyclophilin, FKBP-type peptidyl-prolyl cis-trans isomerase, CONSTANS-like 4, heat shock protein 7, chaperones protein chaperone, and transferase sequences. As in the MF category, loci were associated with 16 significantly enriched GO terms. These included the level two terms “catalytic activity” (GO:0003824), “binding” (GO:0005488), “transporter activity” (GO:0005215), and “receptor activity” (GO:0004872), the level three terms “protein binding” (GO:0005515), “transferase activity” (GO:0016740), and “hydrolase activity” (GO:0016787), and the level four terms “transferase activity, transferring phosphorus-containing groups” (GO:0016772) and “hydrolase activity, acting on acid anhydrides” (GO:0016817, including several fructosyltransferase loci). The most significantly enriched of these was the level two term “catalytic activity”. In the CC category, the GO terms “cytoplasmic part” (GO:0044444), “interacellular membrane-bounded organelle” (GO:0043231), “interacellular organelle part” (GO:0044446) and their daughter terms (“plastid”, “Golgi apparatus”, “cytosol” and “vacuole”) were highly enriched (FDR≤1.0E-60). These enrichments correspond with the involvement of storage organelles in tuber inulin accumulation. The “vacuole” term was also found to be significantly enriched in tuber samples. The H. tuberosus annotation results were similar to those from the potato and sweet potato transcriptomes [57]–[60]. The majority of the sequenced H. tuberosus loci were associated with fundamental regulatory and metabolic processes in the membrane.


RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne).

Jung WY, Lee SS, Kim CW, Kim HS, Min SR, Moon JS, Kwon SY, Jeon JH, Cho HS - PLoS ONE (2014)

Gene Ontology (GO) classification of the assembled loci.The results of BLASTX searches against the Phytozome database were used for GO term mapping and annotation. The number and ratio of sequences assigned to level 2 GO terms from GO subcategories including biological process, molecular process, molecular function, and cellular component are shown (BP: biological process, CC: Cellular Component, MF: Molecular Function).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4222968&req=5

pone-0111982-g002: Gene Ontology (GO) classification of the assembled loci.The results of BLASTX searches against the Phytozome database were used for GO term mapping and annotation. The number and ratio of sequences assigned to level 2 GO terms from GO subcategories including biological process, molecular process, molecular function, and cellular component are shown (BP: biological process, CC: Cellular Component, MF: Molecular Function).
Mentions: We used GO term enrichment analysis to classify the functions of the assembled H. tuberosus loci [44]. The BLASTX similarity search results for the 66,322 H. tuberosus loci were imported into the Phytozome database for GO mapping and annotation with TAIR information. Sequence annotations associated with 19,848 loci (29.93%) were categorized into the three main GO ontologies: biological process (BP), cellular component (CC), and molecular function (MF) (Figure 2). In total, 7,589, 8,685 and 8,510 loci were assigned GO terms from the BP, CC, and MF categories, respectively. The GO terms were summarized into 49 subcategories with GO classifications at level 2. In the BP category, the dominant subcategories assigned to H. tuberosus loci were as follows: ‘Primary metabolic process’ (15.19%), ‘Cellular metabolic process’ (14.75%), ‘Response to stress’ (6.76%), ‘Nitrogen compound metabolic process’ (5.33%) and ‘multicellular organismal development’ (4.08%). In the CC category, ‘Cell part’ (21.61%), ‘Intracellular’ (13.81%), ‘Intracellular part’ (13.49%), ‘Intracellular organelle’ (12.33%), and ‘Membrane-bounded organelle’ (11.89%) were the dominant subcategories. Finally, ‘Nucleotide binding’ (22.12%), ‘Protein binding’ (20.45%), ‘Nucleoside binding’ (18.94%), ‘Transferase activity’ (15.80%), and ‘Hydrolase activity’ (12.17%) were dominant in the MF category. These annotations indicated that extensive membrane metabolic activity occurred in H. tuberous in the sampled tissues. The loci were analyzed further for GO-category enrichment relative to Plant GO slim categories using AgriGO [43]. The H. tuberosus loci contained 71 significantly enriched (FDR ≤ 0.01) functional GO terms in the BP category, including top five terms (“cellular process”, GO:0009987; “cellular metabolic process”, GO:0044237; “metabolic process”, GO:0008152, “primary metabolic process”, GO:0044238, and “response to stimulus”, GO:0050896, respectively). The GO term “cellular, macromolecule, nitrogen compound and primary metabolic process” was highly enriched (FDR≤1.0E-40), and enriched daughter terms included “nucleobase, nucleoside, nucleotide and nucleic acid metabolic process” (GO:0006139), “cellular macromolecule metabolic process” (GO:0044260), “macromolecule modification” (GO:0043412), “carbohydrate metabolic process” (GO:0005975; including several loci with fructan 1,2-beta-fructan 1-fructosyltransferase, invertase, hexokinase, sucrose synthase, sucrose phosphate synthase, starch synthase, starch branching enzyme, and beta glucosidase sequences), and “cellular biosynthetic process” (GO:00044249; sucrose 1F-beta-D-fructosyltransferase). These results suggest that gene expression in H. tuberosus is geared towards carbohydrate metabolism, cellular biosynthetic processes, and macromolecule modification functions. This expression enrichment concurs with biosynthetic analysis results indicating that inulin accumulation occurs at the time of tuber initiation [4], [19]. An additional enriched GO term was “protein modification process” (GO:0006464). This included loci with cyclophilin, FKBP-type peptidyl-prolyl cis-trans isomerase, CONSTANS-like 4, heat shock protein 7, chaperones protein chaperone, and transferase sequences. As in the MF category, loci were associated with 16 significantly enriched GO terms. These included the level two terms “catalytic activity” (GO:0003824), “binding” (GO:0005488), “transporter activity” (GO:0005215), and “receptor activity” (GO:0004872), the level three terms “protein binding” (GO:0005515), “transferase activity” (GO:0016740), and “hydrolase activity” (GO:0016787), and the level four terms “transferase activity, transferring phosphorus-containing groups” (GO:0016772) and “hydrolase activity, acting on acid anhydrides” (GO:0016817, including several fructosyltransferase loci). The most significantly enriched of these was the level two term “catalytic activity”. In the CC category, the GO terms “cytoplasmic part” (GO:0044444), “interacellular membrane-bounded organelle” (GO:0043231), “interacellular organelle part” (GO:0044446) and their daughter terms (“plastid”, “Golgi apparatus”, “cytosol” and “vacuole”) were highly enriched (FDR≤1.0E-60). These enrichments correspond with the involvement of storage organelles in tuber inulin accumulation. The “vacuole” term was also found to be significantly enriched in tuber samples. The H. tuberosus annotation results were similar to those from the potato and sweet potato transcriptomes [57]–[60]. The majority of the sequenced H. tuberosus loci were associated with fundamental regulatory and metabolic processes in the membrane.

Bottom Line: In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR.Exsiting genetic and genomic data for H. tuberosus are scarce.The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

View Article: PubMed Central - PubMed

Affiliation: Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea; Animal Material Engineering, Gyeongnam National University of Science and Technology, Jinju, Korea.

ABSTRACT
Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

Show MeSH
Related in: MedlinePlus