Forward genetic screening of mutants of the wild diploid ancestor <i>Nicotiana sylvestris</i> identifies <i>aspartate oxidase 2</i> as a key gene for nicotine biosynthesis in cultivated allotetraploid tobacco ( <i>Nicotiana tabacum</i> )

SUMMARY Tobacco ( Nicotiana tabacum ) is one of the most important commercial crops. It is an allotetraploid with two subgenomes derived from two wild diploid species: the maternal ancestor Nicotiana sylvestris and the paternal ancestor Nicotiana tomentosiformis . The functional duplication of homologous genes derived from both subgenomes often hinders phenotypic screening because a recessive mutation on one homeolog is complemented by the other. In this study, to establish a forward genetics platform for the genus Nicotiana , we constructed an ethyl methanesulfonate‐induced N. sylvestris mutant library with high mutation frequency and generated an improved‐quality reference genome of this species. We phenotypically screened the mutants with reduced nicotine content and selected two non‐allelic mutants from the library. MutMap analysis using the reference genome, and subsequent molecular analyses, identified causal mutations in aspartate oxidase 2 ( AO2 ) and ethylene responsible factor 199 genes. We genotypically screened N. tabacum mutants for AO2 and confirmed that nicotine biosynthesis was significantly affected in N. tabacum mutants with nonsense mutations in AO2 s in both subgenomes, as observed in N. sylvestris . Thus, screening the N. sylvestris mutant library and gene identification using the reference genome are promising forward genetic approaches for gene discovery. This would allow researchers to improve N. tabacum by using reverse genetics and to elucidate the molecular mechanisms underlying traits. This study opens new avenues in Nicotiana functional genomics.