Evaluation of rubber tree transcriptome and discovery of SNP and SSR from candidate genes involved in cellulose and lignin biosynthesis

Hevea brasiliensis (the rubber tree) is a well-known species with high economic value, and it is the primary source of natural rubber globally. Increasing demand for furniture and related industries has made rubberwood production as important as latex production. Molecular markers such as Single Nucleotide Polymorphisms (SNPs) and Simple Sequence Repeats (SSRs) are widely used for Marker Assisted Selection (MAS) which can be detected in large quantity by transcriptome sequencing. MAS is thought to be a useful method for the development of new rubberwood clones for its shorter breeding cycle compared to a conventional breeding procedure. In this study we performed RNA sequencing (RNA-seq) on four H. brasiliensis clones (RRIM 712, RRIM 2025, RRIM 3001 and PB 314) from three tissues including bark, latex and leaf samples to identify SSRs and SNPs associated with wood-formation related genes. The RNA sequencing using the Illumina NextSeq 500 v2 platform, generated 1,697,491,922 raw reads. A total of 101,269 transcripts over 400 bp in size were obtained and similarity search of the non-redundant (nr) protein database returned 83,748 (83%) positive BLASTx hits. The transcriptome analysis was annotated using the NCBI NR (National Center for Biotechnology Information Non-Redundant), UniProtKB/Swiss-Prot, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Differential expression analysis between later-timber rubber clone and non-later-timber rubber clone on wood-formation related genes, showed genes encoding phenylalanine ammonia-lyase (PAL), caffeic acid O-methyltransferase (COMT) and cinnamoyl-CoA reductase (CCR) were highly up-regulated in a latex-timber rubber clone. In total, about 3,210,629 SNPs and 14,956 SSRs were detected with 1,786 SNPs and 31 SSRs were found for wood-formation biosynthesis of H. brasilensis from 11 lignin and cellulose gene toolboxes. After filtering and primer selection, 103 SNPs and 18 SSR markers were successfully amplified and could be useful as molecular tool for marker assisted breeding to produce new timber rubber clones.