Identification of SNPs in nicotine biosynthesis related genes by targeted re-sequencing of TILLING population and germplasm with varying nicotine levels in tobacco

Abstract

Nicotine is an important plant metabolite having high commercial value due to wide applications as insecticide and therapeutic agent apart from its parasympathomimetic properties. In addition, nicotine biosynthesis serves as model pathway to study the genetic regulation of secondary metabolites in plants. In this study, we report the identification of genetic variations in terms of SNPs in the nicotine biosynthesis related genes. Targeted re-sequencing had identified a total of 50 natural variations across 19 genes in the nicotine biosynthesis related genes. In addition, mutations (n = 32) were also identified in TILLING mutant population of tobacco. A non-synonymous SNP in PMT2 gene leads to conversion of H25Q in PMT2 protein. The change results in formation of a highly conserved N terminal repeat motif in PMT2 protein. Germplasm regression analysis revealed that this particular SNP contributes 3 % of total variation observed for nicotine content. It was also observed that all the germplasm lines with the minor allele had nicotine content higher than the commercial cultivar Kanchan (K326). Functional characterisation of the natural/induced variations identified in this study would be of use to understand the genetic regulation of secondary metabolites and for development of pre-breeding lines with varied content of nicotine.