3 Biotech

, 9:388 | Cite as

Single-nucleotide polymorphism markers within MVA and MEP pathways among Hevea brasiliensis clones through transcriptomic analysis

  • Siti Nurfazilah Abdul Rahman
  • Mohd Fahmi Abu Bakar
  • G. Veera Singham
  • Ahmad Sofiman OthmanEmail author
Short Reports


In this study, RNA sequencing of several Hevea brasiliensis clones grown in Malaysia with different annual rubber production yields and disease resistance was performed on the Illumina platform. A total of 29,862,548 reads were generated, resulting in 101,269 assembled transcripts that were used as the reference transcripts. A similarity search against the non-redundant (nr) protein databases presented 83,771 (83%) positive BLASTx hits. The transcriptome was annotated using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Pfam database. A search for putative molecular markers was performed to identify single-nucleotide polymorphisms (SNPs). Overall, 3,210,629 SNPs were detected and a total of 1314 SNPs associated with the genes involved in MVA and MEP pathways were identified. A total of 176 SNP primer pairs were designed from sequences that were related to the MVA and MEP pathways. The transcriptome of RRIM 3001 and RRIM 712 were subjected to pairwise comparison and the results revealed that there were 1262 significantly differentially expressed genes unique to RRIM 3001, 1499 significantly differentially expressed genes unique to RRIM 712 and several genes related to the MVA and MEP pathways such as AACT, HMGS, PMK, MVD, DXS and HDS were included. The results will facilitate the characterization of H. brasiliensis transcriptomes and the development of a new set of molecular markers in the form of SNPs from transcriptome assembly for the genotype identification of various rubber varieties with superior traits in Malaysia.


Hevea brasiliensis Transcriptome Single-nucleotide polymorphism MVA and MEP pathways 



This project is funded by RISDA (304/PBIOLOGI/650728/P137) awarded to Ahmad Sofiman Othman of Universiti Sains Malaysia. We thank Yue Keong Choon (Universiti Sains Malaysia) for collecting the samples used in this work and Mohd Khairul Luqman Mohd Sakaf and Khairul Nasirudin Abu Mangsor (Universiti Sains Malaysia) for assisting in analysis. We also thank MyPhD15 for providing a scholarship to the first author.

Author contributions

SNAR and MFAB carried out the field and laboratory work including data analysis. GVS and SNAR prepared the manuscript. ASO initiated the research and designed the experiment.


Rubber Industry Smallholders Development Authority (Risda) (304/PBIOLOGI/650728/P137).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

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  1. Allegre M, Argout X, Boccara M, Fouet O, Roguet Y, Bérard A, Thévenin JM, Chauveau A, Rivallan R, Clement D, Courtois B, Gramacho K, Boland-Augé A, Tahi M, Umaharan P, Brunel D, Lanaud C (2012) Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L. DNA Res 19:23–35. CrossRefPubMedGoogle Scholar
  2. Berthelot K, Lecomte S, Estevez Y, Benedicte CS, Bentaleb A, Cullin C, Deffieux A, Peruch F (2012) Rubber elongation factor (REF), a major allergen component in Hevea brasiliensis latex has amyloid properties. PLoS One 7(10):e48065. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chandra SS, Liljas A (2000) The end of the beginning: structural studies of ribosomal proteins. Curr Opin Struct Biol 10(6):633–636. CrossRefGoogle Scholar
  4. Chow KS, Mat Isa MN, Bahari A, Ghazali AK, Alias H, Zainorlina MZ, Hoh CC, Wan KL (2012) Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex. J Exp Bot 63:1863–1871. CrossRefPubMedGoogle Scholar
  5. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. CrossRefPubMedGoogle Scholar
  6. Cornish K (2001) Similarities and differences in rubber biochemistry among plant species. Phytochemistry 57(7):1123–1134. CrossRefPubMedGoogle Scholar
  7. Duan CF, Rio M, Leclercq J, Bonnot F, Oliver G, Montoro P (2010) Gene expression pattern in response to wounding, methyl jasmonate and ethylene in the bark of Hevea brasiliensis. Tree Physiol 30(10):1349–1359. CrossRefPubMedGoogle Scholar
  8. Duan C, Argout X, Gebelin V, Summo M, Dufayard JF, Leclerq J et al (2013) Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing. BMC Genomics 14:30. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gao JS, Meng Y, Sasaki N, Kanegae H, Hayashi N, Nyunoya H (2010) Characterization and cloning of TMV resistance gene N homologues from Nicotiana tabacum. Afr J Biotech 9(47):7998–8006. CrossRefGoogle Scholar
  10. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I et al (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol 29(7):644–652. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gronover CS, Wahler D, Prufer D (2011) Natural rubber biosynthesis and physic-chemical studies on plant derived latex. In: Elnashar M (ed) Biotechnology of biopolymers, InTech, Croatia, pp 75–88.
  12. Hayashi Y (2009) Production of natural rubber from Para rubber tree. Plant Biotechnology 26:67–70. CrossRefGoogle Scholar
  13. Hirakawa H, Shirasawa K, Ohyama A, Fukuoka H, Aoki K, Rothan C, Sato S, Isobe S, Tabata S (2013) Genome-wide SNP genotyping to infer the effects on gene functions in tomato. DNA Res 20(3):221–233. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Horemans N, Foyer CH, Potters G, Asard H (2000) Ascorbate function and associated transport systems in plants. Plant Physiol Biochem 38(7–8):531–540. CrossRefGoogle Scholar
  15. Hu J, Baker A, Bartel B, Linka N, Mullen RT, Reumann S, Zolman BK (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24:2279–2303. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kharel Y, Koyama T (2003) Molecular analysis of cis-prenyl chain elongating enzymes. Natural Prod Rep 20:111–118. CrossRefGoogle Scholar
  17. Kim JY, Kim WY, Kwak KJ, Oh SH, Han YS et al (2010) Glycine-rich RNA-binding proteins are functionally conserved in Arabidopsis thaliana and Oryza sativa during cold adaptation process. J Exp Bot 61(9):2317–2325. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, Miller CA, Mardis ER, Ding L, Wilson RK (2012) Varscan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22(3):568–576. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie2. Nat Methods 9:357–359PubMedPubMedCentralGoogle Scholar
  20. Lau NS, Makita Y, Kawashima M, Taylor TD, Kondo S, Othman AS, Alexander CSC, Matsui M (2016) The rubber tree genome shows expansion of gene family associated with rubber biosynthesis. Sci Rep. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659. CrossRefGoogle Scholar
  22. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25(16):2078–2079. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Li R, Wang W, Wang W, Li F, Wang Q, Xu Y, Wang S (2015) Overexpression of a cysteine proteinase inhibitor gene from Jatropha curcas confers enhanced tolerance to salinity stress. Electron J Biotechnol 18(5):368–375. CrossRefGoogle Scholar
  24. Lichtenthaler HK, Schwender J, Disch A, Rohmer M (1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Lett 400(3):271–274. CrossRefPubMedGoogle Scholar
  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Loh SC, Thottathil GP, Othman AS (2016) Identification of differentially expressed genes and signalling pathways in bark of Hevea brasiliensis seedlings associated with secondary laticifer differentiation using gene expression microarray. Plant Physiol Biochem 107:45–55. CrossRefPubMedGoogle Scholar
  27. Mantello CC, Cardoso-Silva CB, da Silva CC, de Souza LM, Junior EJS, Gonçalves PDS, Vicentini R, de Souza AP (2014) De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways. PLoS One 9(7):e102665. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mokryakova MV, Pogorelko GV, Bruskin SA, Piruzian ES, Abdeeva IA (2014) The role of peptidyl-prolyl cis/trans isomerase genes of Arabidopsis thaliana in plant defense during the course of Xanthomonas campestris infection. Russ J Genetics 50(2):140–148CrossRefGoogle Scholar
  29. Mooibroek H, Cornish K (2000) Alternatives sources of natural rubber. Appl Microbiol Biotechnol 53(4):355–365. CrossRefPubMedGoogle Scholar
  30. Putranto RA, Duan C, Kuswanhadi TC, Rio M, Piyatrakul P et al (2015) Ethylene response factors are controlled by multiple harvesting stresses in Hevea brasiliensis. PLoS One 10(4):e0123618. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Rahman AYA, Usharraj AO, Misra BB, Thottathil GP, Jayasekaran K, Feng Y et al (2013) Draft genome sequence of the rubber tree Hevea brasiliensis. BMC Genomics 14:75. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Rohmer M, Seemann M, Horbach S, Bringer-Meyer S, Sahms H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118(11):2564–2566. CrossRefGoogle Scholar
  34. Saha T, Priyadarshan PM (2012) Genomics of Hevea rubber. In: Schnell RJ, Priyadarshan PM (eds) Genomics of tree crops. Springer, New York, pp 261–298. CrossRefGoogle Scholar
  35. Sakdapipanich JT (2007) Structural characterization of natural rubber based on recent evidence from selective enzymatic treatments. J Biosci Bioeng 103(4):287–292. CrossRefPubMedGoogle Scholar
  36. Sando T, Takeno S, Watanabe N, Okumoto H, Kuzuyama T, Yamashita A, Hattori M, Ogasawara N, Fukusaki E, Kobayashi A (2008) 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. CrossRefPubMedGoogle Scholar
  37. Spurgeon SL, Porter JW (1981) Biosynthesis of isoprenoid compounds. 1:1–46Google Scholar
  38. Trapnell C et al (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562PubMedPubMedCentralGoogle Scholar
  39. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC et al (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40:e115. CrossRefPubMedPubMedCentralGoogle Scholar
  40. van Beilen JB, Poirier Y (2007) Establishment of new crops for the production of natural rubber. Trends Biotechnol 25(11):522–529. CrossRefPubMedGoogle Scholar
  41. Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17(9):847–848. CrossRefPubMedGoogle Scholar
  42. Zhu W, Wang L, Dong Z, Chen X, Song F, Liu N, Yang H, Fu J (2016) Comparative transcriptome analysis identifies candidate genes related to skin color differentiation in red tilapia. Sci Rep 6:31347. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Siti Nurfazilah Abdul Rahman
    • 1
  • Mohd Fahmi Abu Bakar
    • 2
  • G. Veera Singham
    • 3
  • Ahmad Sofiman Othman
    • 1
    Email author
  1. 1.School of Biological SciencesUniversiti Sains MalaysiaGelugorMalaysia
  2. 2.Faculty of Bioresources and Food IndustryUniversiti Sultan Zainal AbidinBesutMalaysia
  3. 3.Centre for Chemical BiologyUniversiti Sains MalaysiaBayan LepasMalaysia

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