Mining of trait specific gene candidates through mRNA sequencing emphasizing on expression study of terpenoid biosynthesis genes in betelvine cash crop

Abstract

Betelvine (Piper betle L., Family: Piperaceae) is one of the important cultivated cash crop and medicinal plant of India, lacking sufficient molecular resources in various databases for exploration of biosynthetic pathways involved in terpenoid synthesis. Molecular markers like SSRs (Simple Sequence Repeats) and SNPs (Single Nucleotide Polymorphisms) are generally used for genetic diversity study. However, limited number of nucleotides and absence of SSRs as well as SNPs restrict molecular study of betelvine landraces. These problems also minimise molecular characterization of novel genes that are involved in terpenoid biosynthesis. To elucidate the biochemical pathways of terpenoid biosynthesis, transcriptome profiling of RNA sample was performed from leaves of betelvine. The pair end transcriptome profiling generated 6.7 G bases (4.4Gb) of data containing 33,235,667 raw reads for each end of RNA Seq. After removing contaminants, 30,104,811 clean reads were subjected to de novo transcriptome assembling, which subsequently produced 46,656 clean transcripts. The clean transcripts were annotated and their functions were identified using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG), PlantCyc, Swissprot, and TrEMBL database. These annotations resulted mining of various trait specific genes including important terpenoid biosynthesis genes. Presence of 43 terpenoid compounds in essential oil of four betelvine landraces has also been confirmed through GCMS. Further, gene expression analysis of five terpenoid biosynthesis genes were carried out by real time polymerase chain reaction (RT PCR). The expression of monoterpene synthase genes (limonene synthase, linalool synthase, and α-terpeneol synthase) and sesquiterpene synthase genes (germacrene synthase and sesquiterpene) were validated using four landraces of betelvine. Landraces specific high quality 4181 SSRs and 350,676 SNPs were identified for further genetic diversity study. In this study, terpenoid biosynthesis specific transcription factors and 22 miRNAs having 33 target sites were also predicted. Overall, this study will enrich the molecular repository of betelvine for further improvement of the taxa and to develop new genotype of betelvine crop with quality traits.