Development of TILLING by sequencing platform towards enhanced leaf yield in tobacco

Tobacco being a non-food commercial crop, has gained attention for alternate uses apart from its use for making cigarettes. Leaf yield is a major economic produce in tobacco. In order to screen for mutants that enhance leaf yield and its associated metabolic traits, we generated an Ethyl Methane Sulphonate (EMS) mutant population also referred to as TILLING (Targeting Induced Local Lesions IN Genomes) population. DNA and seed libraries were prepared from 7800 M2 plants. High throughput sequencing technology combined with 3D pooling of amplicons from 3072 M2 plants was deployed for mutation screening. In total, seven induced mutations were identified in genes potentially linked with leaf biomass (Ntfw2.2 and NtLs) and secondary metabolism (NtMYB12 and NtGGR). Overall mutation density of one mutation per 1.423 million bases or ∼3162 mutations per genome was observed in the population. Out of the three candidate point mutations identified in Ntfw2.2, the mutation that resulted a premature stop codon was converted to a simple High Resolution Melting (HRM) DNA marker. Additionally, mutants with traits such as increase in number of leaves and broader leaves were selected from the population and advanced to further generations. This study provided evidence to suggest that mutants with agronomic superior phenotypes identified by forward and reverse genetic approaches (TILLING) could contribute potentially towards crop improvement in tobacco.

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► TILLING by sequencing platform in tobacco was established.
► Mutants with traits such as more leaves and broader leaves were selected from the population.
► Seven mutations in the selected genes (Ntfw2.2, NtLs, NtMYb12 and NtGGR) were identified.
► All the mutations identified in Ntfw2.2 gene inherited to M3 generation.
► High Resolution Melting (HRM) DNA marker developed for Ntfw2.2-1 allele.
► Mutants with beneficial phenotypes identified in this study are useful for crop improvement.