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

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.

Figure 1. Comparison of assembled H. tuberosus loci with database sequences.

Species, E-value, and similarity distributions of the assembled loci against database sequences are shown. (A) Species distribution of the top BLAST hits for the assembled loci (Cut-off, E-value = 0). (B) E-value distribution of BLAST hits for the assembled loci (E-value ≤1.0e-05). (C) Similarity distribution of BLAST hits for the assembled loci.

Figure 2. 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).

Figure 3. Eukaryotic Orthologous Groups (KOG) classification of the assembled loci.

Of 66,322 loci with Nr, Phytozome and UniProt hits, 15,434 sequences with significant homologies in the KOG database (E-value ≤1.0E-5) were classified into 25 categories.

Figure 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of the assembled loci.

Locus sequences were compared using BLASTX with an E-value cut-off ≤1.0E-05 against the KEGG biological pathways database. The loci were mapped to 237 KEGG pathways. M; Metabolism, GIP; Genetic Information Processing, EIP; Environmental Information Processing, CP; Cellular Processes, OS; Organismal Systems.

Figure 5. Loci differentially expressed between tissues in H. tuberosus.

Loci were quantified and up- and down-regulated loci are shown as black and grey bars, respectively. Pairwise comparisons between tissues are shown.

Figure 6. qRT-PCR validation of loci expressed specially in five H. tuberosus tissues.

The qRT-PCR results of root-specific (A), stem-specific (B), leaf-specific (C), and tuber-specific (D) candidate loci are shown.

Figure 7. Schematic representation of the inulin biosynthesis pathway in the vacuole.

Inulin biosynthesis enzymes present in the vacuole are marked in red. Green indicates enzymes related to inulin degradation. Blue indicates enzymes related to sucrose biosynthesis. Read counts of unigenes representing enzymes were subjected to expression analysis and the results are shown as red bars (log₂). 1-SST: 1-sucrose: sucrose fructosyltransferase, 6-SFT: sucrose:sucrose fructosyltransferase, 1-FFT: 1,2-β–fructan 1F-fructosyltransferase, 6G-FFT: Fructan:fructan 6G-fructosyltransferase, FEH: fructan exohydrolase, HK: Hexokinase, SS: Sucrose synthase, SPS: Sucrose-phosphate-synthase, SPP: Sucrose-phosphate-phosphohydrolase, Suc: Sucrose, Fru: Fructose, Glu: Glucose, Inv: Invertase.