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High-throughput sequencing and de novo transcriptome assembly of Swertia japonica to identify genes involved in the biosynthesis of therapeutic metabolites

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Abstract

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Here, we report potential transcripts involved in the biosynthesis of therapeutic metabolites in Swertia japonica , the first report of transcriptome assembly, and characterization of the medicinal plant from Swertia genus.

Abstract

Swertia genus, representing over 170 plant species including herbs such as S. chirata, S. hookeri, S. longifolia, S. japonica, among others, have been used as the traditional medicine in China, India, Korea, and Japan for thousands of years. Due to the lack of genomic and transcriptomic resources, little is known about the molecular basis involved in the biosynthesis of characteristic key bioactive metabolites. Here, we performed deep-transcriptome sequencing for the aerial tissues and the roots of S. japonica, generating over 2 billion raw reads with an average length of 101 bps. Using a combined approach of three popular assemblers, de novo transcriptome assembly for S. japonica was obtained, yielding 81,729 unigenes having an average length of 884 bps and N50 value of 1452 bps, of which 46,963 unigenes were annotated based on the sequence similarity against NCBI-nr protein database. Annotation of transcriptome assembly resulted in the identification of putative genes encoding all enzymes from the key therapeutic metabolite biosynthesis pathways. Transcript abundance analysis, gene ontology enrichment analysis, and KEGG pathway enrichment analysis revealed metabolic processes being up-regulated in the aerial tissues with respect to the roots of S. japonica. We also identified 37 unigenes as potential candidates involved in the glycosylation of bioactive metabolites. Being the first report of transcriptome assembly and annotation for any of the Swertia species, this study will be a valuable resource for future investigations on the biosynthetic pathways of therapeutic metabolites and their regulations.

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Acknowledgments

This study, in part, was supported by the “Health and Labour Sciences Research Grant” on the enhancement of “Comprehensive Medicinal Plant Database” by the Grants-in-Aid for the Scientific Research of the Japan Society for the Promotion of Science (JSPS), and by the “Strategic Priority Research Promotion Program” of the Chiba University. HT was partially supported by MEXT KAKENHI (no. 221S0002). The super-computing resources were provided by the National Institute of Genetics (NIG), and Research Organization of Information and Systems, Japan. The computing resources were provided by the Medical Mycology Research Center, Chiba University, Japan. We also thank Mr. Tsutomu Hosouchi, and Ms. Sayaka Shinpo from the Kazusa DNA Research Institute for technical support in Illumina sequencing. We would also like to thank Dr. Ashfaq Mahmood, Megha from Chiba University, Dr. Gourvendu Saxena from the National University of Singapore, and two anonymous reviewers for their helpful and critical comments to improve this manuscript.

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, 260-8675, Japan

    Amit Rai, Michimi Nakamura, Kazuki Saito & Mami Yamazaki

  2. Medical Mycology Research Center, Chiba University, Chiba, Japan

    Hiroki Takahashi

  3. Department of Research and Development, Kazusa DNA Research Institute, Chiba, Japan

    Hideyuki Suzuki

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  1. Amit Rai
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  2. Michimi Nakamura
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  3. Hiroki Takahashi
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  6. Mami Yamazaki
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Correspondence to Amit Rai or Mami Yamazaki.

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Communicated by P. P. Kumar.

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Fig. S1 Summary for the InterProScan results of the S. japonica transcriptome assembly (EPS 1687 kb)

299_2016_2021_MOESM2_ESM.eps

Fig. S2 Distribution of the FPKM values of unigenes across the aerial tissues and the roots of S. japonica. The number of unigenes with the transcript expression levels in the FPKM values within different ranges for the aerial tissues and the roots of S. japonica is shown here (EPS 844 kb)

299_2016_2021_MOESM3_ESM.eps

Fig. S3 Distribution of unigenes, differentially expressed in the aerial tissues with respect to the roots in S. japonica, to the KEGG pathways database. A number of differentially expressed unigenes assigned to KEGG pathways are shown here. Red and green colour bar represents up- or down-regulated unigenes in the aerial tissues with respect to the roots of S. japonica (EPS 1352 kb)

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Fig. S4 Proposed pathways for the biosynthesis of mangiferin, and expression of unigenes assigned to the different enzyme coding genes in the S. japonica. Biosynthetic pathways for mangiferin, and candidate unigenes of S. japonica annotated as the enzyme coding genes at different biochemical reactions, and their expression levels are shown here. Red or green colour represents high or low expression levels, respectively, for a specific unigenes across the aerial tissues and the roots of S. japonica, while asterisks identify the false discovery rate, (FDR) <0.05, with the DESeq2 package. Abbreviation: DAHPS (3-deoxy-d-arabinoheptulosonate-7-phosphate synthase), DHQS (3-dehydroquinate synthase), DHQD (3-dehydroquinate dehydratase), SDH shikimate dehydrogenase), SAK (shikimate kinase), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), CS (chorismate synthase), CM (chorismate mutase), ADT (prephenate dehydratase), PAT (aspartate–prephenate aminotransferase), PHAT (phenylalanine-histidine aminotransferase), ADH (arogenate dehydrogenase), PAL (phenylalanine ammonia lyase), TAL (tyrosine ammonia lyase), C4H (trans-cinnamate 4-hydroxylase), and C3H (p-coumarate 3-hydroxylase) (EPS 2056 kb)

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Table S1 Output summary of the Trimmomatic program-based pre-processing of raw reads of the aerial tissues and the roots of S. japonica (DOCX 12 kb)

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Table S2 Blastx search summary for the unigenes of the S. japonica transcriptome assembly with sequence similarity against NCBI-non-redundant (nr) database (XLSX 5674 kb)

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Table S3 InterProScan result summary for the unigenes of the S. japonica transcriptome assembly with a hit against InterPro protein signature databases (XLSX 1255 kb)

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Table S4 Summary of S. japonica transcriptome assembly annotation with assigned gene ontology terms, EC numbers, and KEGG pathways annotation based on Blastx and InterProScan search results using Blast2GO program (XLSX 9805 kb)

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Table S5 List of KEGG pathways identified, and the number of unigenes assigned from the S. japonica transcriptome assembly (XLSX 11 kb)

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Table S6 Identified simple sequence repeats (SSRs) motifs and their frequencies in the S. japonica transcriptome assembly (XLSX 23 kb)

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Table S7 Transcript expression values (FPKM values) for all unigenes for the aerial tissues and the roots of S. japonica (XLSX 2133 kb)

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Table S8 Annotation summary and transcript expression values for the unigenes annotated as UDP-glycosyltransferase and up-regulated in the aerial tissues with respect to the roots of S. japonica (XLSX 15 kb)

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Table S9 Annotation summary and transcript expression values for the unigenes annotated as UDP-glycosyltransferase and down-regulated in the aerial tissues with respect to the roots of S. japonica (XLSX 19 kb)

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Data set S1 Nucleotide sequences for the unigenes of S. japonica, annotated as enzyme coding genes associated with the methylerythritol phosphate (MEP) biosynthesis pathways (DOCX 19 kb)

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Data set S2 Nucleotide sequences for the unigenes of S. japonica, annotated as enzyme coding genes associated with the mevalonate (MVA) biosynthesis pathways (DOCX 19 kb)

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Data set S3 Nucleotide sequences for the unigenes of S. japonica, annotated as enzyme coding genes associated with the secoiridoid biosynthesis pathways (DOCX 26 kb)

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Data set S4 Nucleotide sequences for the unigenes of S. japonica, annotated as enzyme coding genes associated with the mangiferin biosynthetic pathways (DOCX 31 kb)

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Data set S5 Nucleotide sequences for the unigenes up-regulated in the aerial tissues with respect to the roots of the S. japonica, and annotated as UDP-glycosyltransferase coding genes (DOCX 27 kb)

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Data set S6 Nucleotide sequences for the unigenes down-regulated in the aerial tissues with respect to the roots of the S. japonica, and annotated as UDP-glycosyltransferase coding genes (DOCX 38 kb)

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Rai, A., Nakamura, M., Takahashi, H. et al. High-throughput sequencing and de novo transcriptome assembly of Swertia japonica to identify genes involved in the biosynthesis of therapeutic metabolites. Plant Cell Rep 35, 2091–2111 (2016). https://doi.org/10.1007/s00299-016-2021-z

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