Distinguishing CPT gene family members and vetting the sequence structure of a putative rubber synthesizing variant in Hevea brasiliensis

Abstract

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze the cis-1,4-polymerization of isoprene units to generate isoprenoids with carbon skeletons varying from C10 (neryl pyrophosphate) to C > 10,000 (natural rubber). Though the previously reported CPTs in Hevea are designated based on sequence variations, their classification was done mostly by phylogenetic analysis using a mixture of partial as well as full length sequences often excluding the UTRs. In this context an attempt was made to reclassify the CPTs strictly based on their sequence similarity and distinguish the members putatively associated with rubber biosynthesis from the others. Extensive computational analysis was carried out on CPT sequences obtained from public resources and whole genome assemblies of Hevea. Based on the results from BLAST analysis, multiple sequence alignments of protein, nucleotide and untranslated regions, open reading frame analysis, gene prediction analysis and sequence length variations, we conclude that there exists mainly three CPTs namely RubCPT1, RubCPT2 and RubCPT3 putatively associated with rubber biosynthesis in Hevea brasiliensis. The rest were categorised as variants of dehydrodolichyl diphosphate synthase (DHDDS) involved in the synthesis of dolichols having short chain isoprenoids. Analysis of the sequence structure of the most highly expressed RubCPT1 in latex revealed the allele richness and diversity of this important variant prevailing in the popular rubber clones. Haplotypes consisting of SNPs with high degree of heterozygosity were also identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor towards the generation of allelic diversity rather than point mutations. Alternatively, gene expression analysis indicated the possibility of association between specific haplotypes and RubCPT1 expression in Hevea clones which may have downstream impact up to the level of rubber production. The conclusions from this study may pave way for the identification and better understanding of CPTs directly involved with natural rubber biosynthesis in Hevea and the SNP data generated may aid in the development of molecular markers putatively associated with yield in rubber.