De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways.

Camila Campos Mantello,Anete Pereira De Souza,Livia Moura De Souza,Paulo De Souza Gonçalves,Erivaldo José Scaloppi Junior,Carla Cristina Da Silva,Claudio Benicio Cardoso-Silva,Renato Vicentini,Minami Matsui

doi:10.1371/journal.pone.0102665

Abstract

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.

Highlights

Natural rubber is one of the most important polymers that is produced by plants
Considering the annotations of all the key genes that are involved in the MVA and methyl-D-erythritol 4-phosphate (MEP) pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we observed that the genes encoding hydroxymethylglutaryl-CoA reductase (NADPH) in the MVA pathway and (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase and 4-hydroxy-3methylbut-2-enyl diphosphate reductase in the MEP pathway, were enhanced in the GT1 genotype, providing a strong evidence that these genes are differentially expressed in these two clones
Because contigs corresponding with genes that are involved in the MVA and MEP pathways were identified in the KEGG annotations, we searched for single nucleotide polymorphisms (SNPs) in these sequences

Summary

Introduction

Rubber is composed of 94% cis-1,4polyisoprene and 6% proteins and fatty acids [1] and exhibits unique properties including flexibility, impermeability to liquids and abrasion resistance. 2,500 plant species are known to synthesize natural rubber, but only a few plants, such as Hevea brasiliensis (rubber tree), Parthenium argentatum (guayule) and Taraxacum koksaghyz (Russian dandelion), can produce highquality natural rubber with molecular weights of greater than 1 million Daltons [3]. H. brasiliensis (Willd. ex Adr. de Juss.) Muell. –Arg., which is commonly referred to as the rubber tree, is the major source of natural rubber [2] and is planted on a large scale in fields encompassing approximately 11.33 million hectares [4]

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Results

Conclusion