Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection. © 2014 Mantello et al.97 Sakdapipanich, J.T., Structural characterization of natural rubber based on recent evidence from selective enzymatic treatments (2007) J Biosci Bioeng, 103, pp. 287-292. , doi:10.1263/jbb.103.287Cornish, K., Similarities and differences in rubber biochemistry among plant species (2001) Phytochemistry, 57, pp. 1123-1134Gronover, C.S., Wahler, D., Prüfer, D., (2005) Natural Rubber Biosynthesis and Physic- Chemical Studies on Plant Derived LatexSaha, T., Priyadarshan, P.M., (2012) Genomics of Tree Crops, , Schnell RJ, Priyadarshan PM, editors New York, NY: Springer New York. doi:10.1007/978-1-4614-0920-5Priyadarshan, P.M., Goncalves, P.D.S., (2003) Hevea Gene Pool for Breeding, pp. 101-114Leitch, A.R., Lim, K.Y., Leitch, I.J., Neill, M.O., Molecular cytogenetic studies in rubber (1998) Hevea, pp. 464-467Pires, J.M., Secco, R., Gomes, J.I., (2002) Taxonomia e Filogeografia Das Seringueiras (Hevea Spp) Belem: Embrapa Amazonia Oriental, p. 103Pushparajah, E., Natural rubber (2001) Tree Crop Ecosystems, , Last, F.T. (ed) Amsterdam, The Netherlands: Elsevier ScienceRaj, S., Das, G., Pothen, J., Dey, S.K., Relationship between latex yield of Hevea brasiliensis and antecedent environmental parameters (2005) International Journal of Biometeorology, 49 (3), pp. 189-196. , DOI 10.1007/s00484-004-0222-6Le Guen, V., Garcia, D., Doaré, F., Mattos, C.R.R., Condina, V., A rubber tree's durable resistance to Microcyclus ulei is conferred by a qualitative gene and a major quantitative resistance factor (2011) Tree Genet Genomes, 7, pp. 877-889. , doi:10.1007/s11295-011-0381-7Mantello, C.C., Suzuki, F.I., Souza, L.M., Gonçalves, P.S., Souza, A.P., Microsatellite marker development for the rubber tree (Hevea brasiliensis): Characterization and cross-amplification in wild Hevea species (2012) BMC Res Notes, 5, p. 329. , doi:10.1186/1756-0500-5-329Feng, S.P., Li, W.G., Huang, H.S., Wang, J.Y., Wu, Y.T., Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (Hevea brasiliensis) (2008) Mol Breed, 23, pp. 85-97. , doi:10.1007/s11032-008-9216-0Triwitayakorn, K., Chatkulkawin, P., Kanjanawattanawong, S., Sraphet, S., Yoocha, T., Transcriptome sequencing of Hevea brasiliensis for development of microsatellite markers and construction of a genetic linkage map (2011) DNA Res, 18, pp. 471-482. , doi:10.1093/dnares/dsr034Li, D., Deng, Z., Qin, B., Liu, X., Men, Z., De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.) (2012) BMC Genomics, 13, p. 192. , doi:10.1186/1471-2164-13-192Lespinasse, D., Rodier-Goud, M., Grivet, L., Leconte, A., Legnate, H., Seguin, M., A saturated genetic linkage map of rubber tree (Hevea spp.) based on RFLP, AFLP, microsatellite, and isozyme markers (2000) Theoretical and Applied Genetics, 100 (1), pp. 127-138Souza, L.M., Gazaffi, R., Mantello, C.C., Silva, C.C., Garcia, D., QTL mapping of growth-related traits in a full-sib family of rubber tree (Hevea brasiliensis) evaluated in a sub-tropical climate (2013) PLoS One, 8, pp. e61238. , doi:10.1371/journal.pone.0061238Chow, K.-S., Wan, K.-L., Isa, M.N.M., Bahari, A., Tan, S.-H., Harikrishna, K., Yeang, H.-Y., Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex (2007) Journal of Experimental Botany, 58 (10), pp. 2429-2440. , DOI 10.1093/jxb/erm093Chow, K.-S., Mat-Isa, M.-N., Bahari, A., Ghazali, A.-K., Alias, H., Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex (2012) J Exp Bot, 63, pp. 1863-1871. , doi:10.1093/jxb/err363Rahman, A.Y.A., Usharraj, A.O., Misra, B.B., Thottathil, G.P., Jayasekaran, K., Draft genome sequence of the rubber tree Hevea brasiliensis (2013) BMC Genomics, 14, p. 75. , doi:10.1186/1471-2164-14-75Wang, Z., Gerstein, M., Snyder, M., RNA-Seq: A revolutionary tool for transcriptomics (2009) Nat Rev Genet, 10, pp. 57-63Lister, R., O'Malley, R.C., Tonti-Filippini, J., Gregory, B.D., Berry, C.C., Highly integrated single-base resolution maps of the epigenome in Arabidopsis (2008) Cell, 133, pp. 523-536. , doi:10.1016/j.cell.2008.03.029Lu, T., Lu, G., Fan, D., Zhu, C., Li, W., (2010) Function Annotation of the Rice Transcriptome at Single-nucleotide Resolution by RNA-seq, pp. 1238-1249. , doi:10.1101/gr.106120.110Hansey, C.N., Vaillancourt, B., Sekhon, R.S., De Leon, N., Kaeppler, S.M., Maize (Zea mays L.) genome diversity as revealed by RNA-sequencing (2012) PLoS One, 7, pp. e33071. , doi:10.1371/journal.pone.0033071Egan, A.N., Schlueter, J., Spooner, D.M., Applications of next-generation sequencing in plant biology (2012) Am J Bot, 99, pp. 175-185. , doi:10.3732/ajb.1200020Xia, Z., Xu, H., Zhai, J., Li, D., Luo, H., RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis (2011) Plant Mol Biol, 77, pp. 299-308. , doi:10.1007/s11103-011-9811-zChang, S., Puryear, J., Cairney, J., A simple and efficient method for isolating RNA from pine trees (1993) Plant Mol Biol Report, 11, pp. 113-116. , doi:10.1007/BF02670468Doyle, J., Doyle, J., A rapid DNA isolation procedure for small quantities of fresh leaf tissue (1987) Phytochem Bull, 19, pp. 11-15Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research (2005) Bioinformatics, 21 (18), pp. 3674-3676. , DOI 10.1093/bioinformatics/bti610Ye, J., Fang, L., Zheng, H., Zhang, Y., Chen, J., Zhang, Z., Wang, J., Wang, J., WEGO: A web tool for plotting GO annotations (2006) Nucleic Acids Research, 34 (WEB. SERV. ISS.), pp. W293-W297. , DOI 10.1093/nar/gkl031Zdobnov, E.M., Apweiler, R., (2001) Signature-recognition Methods in InterPro., 17, pp. 847-848Silva, C.C., Mantello, C.C., Campos, T., Souza, L.M., Gonçalves, P.S., Leaf-, panel- and latex-expressed sequenced tags from the rubber tree (Hevea brasiliensis) under cold-stressed and suboptimal growing conditions: The development of gene-targeted functional markers for stress response (2014) Mol Breed, , doi:10.1007/s11032-014-0095-2Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R., Primer3Plus, an enhanced web interface to Primer3 (2007) Nucleic Acids Res, 35, pp. W71-W74. , doi:10.1093/nar/gkm306Miller, M., 27. Miller MP: Tools for population genetic analysis (TFPGA) 1.3: A Windows program for the analysis of allozyme and molecular population genetic data (1997) Computer Software, , distributed by the author 1997Tatusov, R.L., Fedorova, N.D., Jackson, J.D., Jacobs, A.R., Kiryutin, B., The COG database: An updated version includes eukaryotes (2003) BMC Bioinformatics, 4, p. 41. , doi:10.1186/1471-2105-4-41Stirnimann, C.U., Petsalaki, E., Russell, R.B., Müller, C.W., WD40 proteins propel cellular networks (2010) Trends Biochem Sci, 35, pp. 565-574. , doi:10.1016/j.tibs.2010.04.003Rushton, P.J., Somssich, I.E., Ringler, P., Shen, Q.J., WRKY transcription factors (2010) Trends Plant Sci, 15, pp. 247-258. , doi:10.1016/j.tplants.2010.02.006Cheng, Y., Zhou, Y., Yang, Y., Chi, Y.-J., Zhou, J., Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors (2012) Plant Physiol, 159, pp. 810-825. , doi:10.1104/pp.112.196816Kanehisa, M., Goto, S., KEGG: Kyoto Encyclopedia of Genes and Genomes (2000) Nucleic Acids Research, 28 (1), pp. 27-30Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., Katayama, T., Yamanishi, Y., KEGG for linking genomes to life and the environment (2008) Nucleic Acids Research, 36 (SUPPL. 1), pp. D480-D484. , DOI 10.1093/nar/gkm882Sando, T., Takeno, S., Watanabe, N., Okumoto, H., Kuzuyama, T., Cloning and Characterization of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) Pathway Genes of a Natural-Rubber Producing Plant, Hevea brasiliensis (2008) Biosci Biotechnol Biochem, 72, pp. 2903-2917. , doi:10.1271/bbb.80387Dao, T.T.H., Linthorst, H.J.M., Verpoorte, R., Chalcone synthase and its functions in plant resistance (2011) Phytochem Rev, 10, pp. 397-412. , doi:10.1007/s11101-011-9211-7Kim, J.Y., Kim, W.Y., Kwak, K.J., Oh, S.H., Han, Y.S., Glycine-rich RNA-binding proteins are functionally conserved in Arabidopsis thaliana and Oryza sativa during cold adaptation process (2010) J Exp Bot, 61, pp. 2317-2325. , doi:10.1093/jxb/erq058Caverzan, A., Passaia, G., Rosa, S.B., Ribeiro, C.W., Lazzarotto, F., Plant responses to stresses: Role of ascorbate peroxidase in the antioxidant protection (2012) Genet Mol Biol, 35, pp. 1011-1019Lam, K.C., Ibrahim, R.K., Behdad, B., Dayanandan, S., (2007) Structure, Function, and Evolution of Plant O -Methyltransferases, 1013, pp. 1001-1013. , doi:10.1139/G07-077Badger, M., The roles of carbonic anhydrases in photosynthetic CO(2) concentrating mechanisms (2003) Photosynth Res, 77, pp. 83-94. , doi:10.1023/A:1025821717773Tanaka, Y., Matsuoka, M., Yamanoto, N., Ohashi, Y., Kano-Murakami, Y., Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato (1989) Plant Physiol, 90, pp. 1403-1407Morgante, M., Hanafey, M., Powell, W., Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes (2002) Nat Genet, 30, pp. 194-200. , doi:10.1038/ng822Marconi, T.G., A, C.E., Miranda, H.R., Mancini, M.C., Cardoso-Silva, C.B., Functional markers for gene mapping and genetic diversity studies in sugarcane (2011) BMC Res Notes, 4, p. 264. , doi:10.1186/1756-0500-4-264La Rota, M., Kantety, R.V., Yu, J.-K., Sorrells, M.E., Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley (2005) BMC Genomics, 6, p. 23. , doi:10.1186/1471-2164-6-23Wang, Z., Fang, B., Chen, J., Zhang, X., Luo, Z., De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas) (2010) BMC Genomics, 11, p. 726. , doi:10.1186/1471-2164-11-726Chen, C., Zhou, P., A, C.Y., Huang, S., Gmitter, F.G., Mining and characterizing microsatellites from citrus ESTs (2006) Theor Appl Genet, 112, pp. 1248-1257. , doi:10.1007/s00122-006-0226-1Wei, W., Qi, X., Wang, L., Zhang, Y., Hua, W., Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers (2011) BMC Genomics, 12, p. 451. , doi:10.1186/1471-2164-12-451Fraser, L.G., Harvey, C.F., Crowhurst, R.N., De Silva, H.N., EST-derived microsatellites from Actinidia species and their potential for mapping (2004) Theoretical and Applied Genetics, 108 (6), pp. 1010-1016. , DOI 10.1007/s00122-003-1517-4Aggarwal, R.K., Hendre, P.S., Varshney, R.K., Bhat, P.R., Krishnakumar, V., Singh, L., Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species (2007) Theoretical and Applied Genetics, 114 (2), pp. 359-372. , DOI 10.1007/s00122-006-0440-xMeglécz, E., Nève, G., Biffin, E., Gardner, M.G., Breakdown of phylogenetic signal: A survey of microsatellite densities in 454 shotgun sequences from 154 non model eukaryote species (2012) PLoS One, 7, pp. e40861. , doi:10.1371/journal.pone. 0040861Toth, G., Gaspari, Z., Jurka, J., Microsatellites in different eukaryotic genomes: Surveys and analysis (2000) Genome Research, 10 (7), pp. 967-981. , DOI 10.1101/gr.10.7.967Kumpatla, S.P., Mukhopadhyay, S., (2005) Mining and Survey of Simple Sequence Repeats in Expressed Sequence Tags of Dicotyledonous Species, 998, pp. 985-998. , doi:10.1139/G05-060Splicing, P.R.N.A., Coleman, T.P., Roesser, J.R., Commonwealth, V., Uni, V., (1998) RNA Secondary Structure: An Important Cis -Element in Rat Calcitonin/CGRP, pp. 15941-15950Novaes, E., Drost, D.R., Farmerie Jr., W.G., P, G.J., Grattapaglia, D., (2008) High-throughput Gene and SNP Discovery in Eucalyptus Grandis, An Uncharacterized Genome, 14, pp. 1-14. , doi:10.1186/1471-2164-9-312Chagné, D., Crowhurst, R.N., Troggio, M., Davey, M.W., Gilmore, B., Genome-wide SNP detection, validation, and development of an 8K SNP array for apple (2012) PLoS One, 7, pp. e31745. , doi:10.1371/journal.pone.0031745Lijavetzky, D., Cabezas, J.A., Ibáñez, A., Rodríguez, V., Martínez-Zapater, J.M., High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology (2007) BMC Genomics, 8, p. 424. , doi:10.1186/1471-2164-8-424Pootakham, W., Chanprasert, J., Jomchai, N., Sangsrakru, D., Yoocha, T., Single nucleotide polymorphism marker development in the rubber tree, Hevea brasiliensis (Euphorbiaceae) (2011) Am J Bot, 98, pp. e337-e338. , doi:10.3732/ajb.1100228Salgado, L.R., Koop, D.M., Pinheiro, D.G., Rivallan, R., Le Guen, V., De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers (2014) BMC Genomics, 15, p. 236. , doi:10.1186/1471-2164-15-236Allegre, M., Argout, X., Boccara, M., Fouet, O., Roguet, Y., 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 (2012) DNA Res, 19, pp. 23-35. , doi:10.1093/dnares/dsr039Neale, D.B., Kremer, A., Forest tree genomics: Growing resources and applications (2011) Nat Rev Genet, 12, pp. 111-122. , doi:10.1038/nrg2931Souza, L.M., Mantello, C.C., Santos, M.O., Goncalves, P.S., Souza, A., Microsatellites from rubber tree (Hevea brasiliensis) for genetic diversity analysis and cross-amplification in six Hevea wild species (2009) Conserv Genet Resour, 1, pp. 75-79Le Guen, V., Gay, C., Xiong, T.C., Souza, L.M., Rodier-Goud, M., Seguin, M., Development and characterization of 296 new polymorphic microsatellite markers for rubber tree (Hevea brasiliensis) (2011) Plant Breed, 130, pp. 294-296Hirakawa, H., Shirasawa, K., Ohyama, A., Fukuoka, H., Aoki, K., Genome-wide SNP genotyping to infer the effects on gene functions in tomato (2013) DNA Res, 20, pp. 221-233. , doi:10.1093/dnares/dst005