Skip to main content

RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis

Plant Molecular Biology volume 77, Article number: 299 (2011) Cite this article

Abstract

Hevea brasiliensis, being the only source of commercial natural rubber, is an extremely economically important crop. In an effort to facilitate biological, biochemical and molecular research in rubber biosynthesis, here we report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain a comprehensive overview of the H. brasiliensis transcriptome. The sequencing output generated more than 12 million reads with an average length of 90 nt. In total 48,768 unigenes (mean size = 436 bp, median size = 328 bp) were assembled through de novo transcriptome assembly. Out of 13,807 H. brasiliensis cDNA sequences deposited in Genbank of the National Center for Biotechnology Information (NCBI) (as of Feb 2011), 11,746 sequences (84.5%) could be matched with the assembled unigenes through nucleotide BLAST. The assembled sequences were annotated with gene descriptions, Gene Ontology (GO) and Clusters of Orthologous Group (COG) terms. In all, 37,432 unigenes were successfully annotated, of which 24,545 (65.5%) aligned to Ricinus communis proteins. Furthermore, the annotated uingenes were functionally classified according to the GO, COG and Kyoto Encyclopedia of Genes and Genomes databases. Our data provides the most comprehensive sequence resource available for the study of rubber trees as well as demonstrates effective use of Illumina sequencing and de novo transcriptome assembly in a species lacking genomic information.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF et al (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252(5013):1651–1656

    PubMed  Article  CAS  Google Scholar 

  • Altermann E, Klaenhammer TR (2005) PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. BMC Genomics 6(1):60

    PubMed  Article  Google Scholar 

  • Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J, Corcoran K (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 18(6):630–634

    PubMed  Article  CAS  Google Scholar 

  • Chow KS, Wan KL, Isa MN, Bahari A, Tan SH, Harikrishna K, Yeang HY (2007) Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex. J Exp Bot 58(10):2429–2440

    PubMed  Article  CAS  Google Scholar 

  • Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, Brown MK, Taylor DF, Steptoe AL, Wani S, Bethel G, Robertson AJ, Perkins AC, Bruce SJ, Lee CC, Ranade SS, Peckham HE, Manning JM, McKernan KJ, Grimmond SM (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5(7):613–619

    PubMed  Article  CAS  Google Scholar 

  • Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18):3674–3676

    PubMed  Article  CAS  Google Scholar 

  • Gibbons JG, Janson EM, Hittinger CT, Johnston M, Abbot P, Rokas A (2009) Benchmarking next-generation transcriptome sequencing for functional and evolutionary genomics. Mol Biol Evol 26(12):2731–2744

    PubMed  Article  CAS  Google Scholar 

  • Haas BJ, Volfovsky N, Town CD, Troukhan M, Alexandrov N, Feldmann KA, Flavell RB, White O, Salzberg SL (2002) Full-length messenger RNA sequences greatly improve genome annotation. Genome Biol 3(6):RESEARCH0029

    Google Scholar 

  • Haas BJ, Zody MC (2010) Advancing RNA-Seq analysis. Nat Biotechnol 28(5):421–423

    PubMed  Article  CAS  Google Scholar 

  • Iseli C, Jongeneel CV, Bucher P (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. In: Proceedings/international conference on intelligent systems for molecular biology, ISMB, pp 138–148

  • Jayashree R, Rekha K, Venkatachalam P, Uratsu SL, Dandekar AM, Kumari Jayasree P, Kala RG, Priya P, Sushma Kumari S, Sobha S, Ashokan MP, Sethuraj MR, Thulaseedharan A (2003) Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene. Plant Cell Rep 22(3):201–209

    PubMed  Article  CAS  Google Scholar 

  • Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28(1):27–30

    PubMed  Article  CAS  Google Scholar 

  • Ko JH, Chow KS, Han KH (2003) Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree). Plant Mol Biol 53(4):479–492

    PubMed  Article  CAS  Google Scholar 

  • Kush A, Goyvaerts E, Chye ML, Chua NH (1990) Laticifer-specific gene expression in Hevea brasiliensis (rubber tree). Proc Natl Acad Sci USA 87(5):1787–1790

    PubMed  Article  CAS  Google Scholar 

  • Leclercq J, Lardet L, Martin F, Chapuset T, Oliver G, Montoro P (2010) The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Mull. Arg). Plant Cell Rep 29(5):513–522

    PubMed  Article  CAS  Google Scholar 

  • Li B, Ruotti V, Stewart RM, Thomson JA, Dewey CN (2010a) RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics 26(4):493–500

    PubMed  Article  Google Scholar 

  • Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, Li S, Yang H, Wang J, Wang J (2010b) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 20(2):265–272

    PubMed  Article  CAS  Google Scholar 

  • Montoro P, Rattana W, Pujade-Renaud V, Michaux-Ferriere N, Monkolsook Y, Kanthapura R, Adunsadthapong S (2003) Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium. Plant Cell Rep 21(11):1095–1102

    PubMed  Article  CAS  Google Scholar 

  • Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7):621–628

    PubMed  Article  CAS  Google Scholar 

  • Sando T, Hayashi T, Takeda T, Akiyama Y, Nakazawa Y, Fukusaki E, Kobayashi A (2009) Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy. Planta 230(1):215–225

    PubMed  Article  CAS  Google Scholar 

  • Sando T, Takaoka C, Mukai Y, Yamashita A, Hattori M, Ogasawara N, Fukusaki E, Kobayashi A (2008a) Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis. Biosci Biotechnol Biochem 72(8):2049–2060

    PubMed  Article  CAS  Google Scholar 

  • Sando T, Takeno S, Watanabe N, Okumoto H, Kuzuyama T, Yamashita A, Hattori M, Ogasawara N, Fukusaki E, Kobayashi A (2008b) Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis. Biosci Biotechnol Biochem 72(11):2903–2917

    PubMed  Article  CAS  Google Scholar 

  • Schuster SC (2008) Next-generation sequencing transforms today’s biology. Nat Methods 5(1):16–18

    PubMed  Article  CAS  Google Scholar 

  • Surget-Groba Y, Montoya-Burgos J (2010) Optimization of de novo transcriptome assembly from next-generation sequencing data. Genome Res 20(10):1432–1440

    PubMed  Article  CAS  Google Scholar 

  • Tang C, Huang D, Yang J, Liu S, Sakr S, Li H, Zhou Y, Qin Y (2010) The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree). Plant Cell Env 33(10):1708–1720

    Article  CAS  Google Scholar 

  • Tang C, Qi J, Li H, Zhang C, Wang Y (2007) A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree). J Biochem Biophys Methods 70(5):749–754

    PubMed  Article  CAS  Google Scholar 

  • Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6(5):377–382

    PubMed  Article  CAS  Google Scholar 

  • Tian WM, Shi MJ, Yu FY, Wu JL, Hao BZ, Cui KM (2003) Localized effects of mechanical wounding and exogenous jasmonic acid on the induction of secondary laticifer differentiation in relation to the distribution of jasmonic acid in Hevea brasiliensis. Acta Botanica Sinica 45(11):1366–1372

    CAS  Google Scholar 

  • Wang XW, Luan JB, Li JM, Bao YY, Zhang CX, Liu SS (2010) De novo characterization of a whitefly transcriptome and analysis of its gene expression during development. BMC Genomics 11:400

    PubMed  Article  Google Scholar 

  • Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev 10(1):57–63

    Article  CAS  Google Scholar 

  • Wilhelm BT, Marguerat S, Goodhead I, Bahler J (2010) Defining transcribed regions using RNA-seq. Nat Protoc 5(2):255–266

    PubMed  Article  CAS  Google Scholar 

  • Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34(Web Server issue):W293–W297

    Google Scholar 

Download references

Acknowledgments

We thank Beijing Genomics Institute at Shenzhen for technical assistance with data analysis. This work was supported by grants from the Project for High-Level Returned Scholar of Ministry of Personnel of P.R. China, the National Natural Science Foundation of China (No. 31060107), and the Project of Doctoral Degree Program of Minister of Education (No. 20104601110003).

Author information

Authors and Affiliations

  1. Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources/Institute of BioScience and Technology, College of Agriculture, Hainan University, Haikou, 570228, People’s Republic of China

    Zhihui Xia, Huimin Xu, Jinling Zhai, Hongli Luo, Chaozu He & Xi Huang

  2. Key Laboratory of Rubber Biology, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, 571737, Hainan, People’s Republic of China

    Dejun Li

Authors
  1. Zhihui Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huimin Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinling Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dejun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongli Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chaozu He
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xi Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Chaozu He or Xi Huang.

Additional information

Zhihui Xia, Huimin Xu and Jinling Zhai contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1. BLAST hits from NCBI Nr databases (XLS 9116 kb)

Table S2. Statistical analysis of GO classifications of H. brasiliensis unigenes. (XLS 81 kb)

Table S3. Statistical analysis of COG classifications of H. brasiliensis unigenes. (XLS 310 kb)

Table S4. Summary of annotated unigenes to the reference canonical pathways in the KEGG database. (XLS 30 kb)

Table S5. Statistical analysis of COG classifications of Hevea nucleotide sequences deposited in NCBI. (XLS 221 kb)

Supplementary material 6 (DOC 51 kb)

Table S7. BLASTX results of Hevea nucleotide sequences of NCBI against Nr databases. (XLS 3962 kb)

11103_2011_9811_MOESM8_ESM.xls

Table S8. BLASTX results of unigenes with a priority of matching with Nr proteins with known functions. Screening was performed for annotations containing the terms “hypothetical”, “predicted”, “putative” or “unknown”, which were shown only when a unigene could not be annotated to a known function. (XLS 7123 kb)

Supplementary material 9 (DOC 63 kb)

Supplementary material 10 (DOC 27 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xia, Z., Xu, H., Zhai, J. et al. RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis . Plant Mol Biol 77, 299 (2011). https://doi.org/10.1007/s11103-011-9811-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11103-011-9811-z

Keywords

  • De novo assembly
  • Hevea brasiliensis
  • RNA-Seq
  • Transcriptome