The study of natural variation advances our understanding of plant trait evolution. However, the analysis of variation in metabolism has been biased and limited to well-characterized metabolites, rarely utilizing the abundant natural variation in complex, specialized metabolite profiles (Li et al. 2015). The wild tobacco N. attenuata has evolved adaptive phenotypic plasticity including specific induced defense mechanisms against the attack by herbivores. In order to understand the molecular basis of the ecological interactions of N. attenuata with its biotic and abiotic environment, we have identified ecologically relevant genes, manipulated them using reverse genetic approaches (gene silencing and overexpression) and studied their ecological function in the field in their native environment. 

One of our forward genetics approaches is the creation of a Multi Parent Advanced Generation InterCross (MAGIC) population. For this, in 2012 we phenotyped individuals from 230 accessions with respect to morphological, biochemical and ecological traits. 26 individuals from different accessions with the most extreme phenotypes including the sequenced UT30x line were chosen as founder plants and their genomes were low pass sequenced (Ray et al., 2019).

The establishment of the MAGIC population started with a complete intercross of all 26 founder plants. The resulting 325 lines of generation ‘A’ were systematically crossed over 4 generations (‘B’ to ‘E’) to maximize recombination and include all 26 founder plants as ancestors of all 325 lines of generation ‘E’. Six Recombinant Inbred Lines (RILs) were created through seven selfing steps from each line of generation ‘E’. The resulting 1950 RILs (generation ‘M’) show >99% of homozygosity. Two sets of RILs (650 plants) of the ‘L’ generation were phenotyped in the greenhouse and low-pass sequenced. The resulting genetic map will enable us to perform QTL (Quantitative Trait Locus) mapping and to identify the genomic positions of new ecologically relevant genes.