Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82E10 gene mutants reducing nicotine to nornicotine conversion in tobacco

Nornicotine accumulation in tobacco is of concern because nornicotine is a precursor of N-nitrosonornicotine (NNN), a tobacco constituent recognized as a carcinogen by the health community. Nornicotine is derived from nicotine through a demethylation process catalyzed by nicotine demethylase enzymes. Three genes (CYP82E4, CYP82E5v2, and CYP82E10) have currently been identified that encode for these enzymes. Ethyl methane sulfonate has been used to introduce mutations into each of these genes to prevent production of functional gene products. These mutants represent a valuable tool for reducing nornicotine and NNN levels in cured tobacco leaves and their derived products. Methods are currently needed to rapidly and efficiently develop new cultivars possessing these mutant alleles. The objective of this study was to develop efficient, user-friendly DNA markers to identify these mutations based on single nucleotide polymorphisms (SNPs). Four dCAPS (derived cleaved amplified polymorphic sequence) markers were designed for a truncation mutation in CYP82E4, and a single marker was developed for a similar mutation in CYP82E5v2. Two CAPS (cleaved amplified polymorphic sequence) markers were designed for a missense mutation in CYP82E10. Because of the co-dominant nature of the CAPS and dCAPS markers, heterozygous and homozygous plants can be easily differentiated. Genotypes determined by the CAPS and dCAPS marker methods were validated by DNA sequencing and phenotypic analysis of plants carrying various mutant combinations. These markers can be used in marker-assisted selection programs to quickly introgress the desired mutations into commercial varieties in order to reduce nornicotine and NNN levels in tobacco leaves.