In this study we sought to identify allelic variants responsible for mediating paraquat (PQ) resistance between C.elegans strains N2 and MY16. We observed that MY16 is two to three-fold more resistant then N2 to oxidative stress from PQ exposure. To identify causative alleles, we developed an experimental evolution approach which relies on multiple rounds of strong selection on standing genetic variation. Genetic variation is created by making strains obligate outcrossing (by introducing a
fog-2 mutation) and forcing random mating for twelve generations to create starting pools (n=3). A similar approach was taken by Burga et al. 2019. In our method, which we call Evolutionary Segregant analysis (ESA), we start with a pool of 30k worms from the F12 generation. We performed a strong selection by recovering only ~ 0.3% of the starting population resistant to PQ. This protocol was performed for five rounds with appropriate controls. All samples were sequenced using RAD-seq. The mapping data pointed to a ~150kb region on LGV. To identify causative alleles, we focused on candidate genes with a role in oxidative stress. From this group,
gad-3 seemed like a solid candidate. Gad-3 (B0222.9) is an evolutionarily conserved aldehyde oxidase downstream of the sirtuin (Sir-2.1) pathway and has a role in oxidative stress response and longevity (Park et al., 2009). GAD-3 dependent production of hydrogen peroxide is responsible for longevity increases from sirtuin (
sir-2.1) pathway over-expression (Schmeisser et al., 2013). GAD-3 protein differs by three mutations between strains. We focused our efforts on testing a mutation in a widely conserved Iron-sulfur cluster domain of GAD-3. Using CRISPR, we performed congenic allele swaps for both MY16 and N2 strains. In both cases, the MY16 allele conferred increased resistance to PQ exposure. In addition, we found that hydrogen peroxide production is reduced with the
gad-3 N2 allele in an MY16 background. Ongoing experiments are testing the reverse allele swap. Our results suggest that GAD-3 dependent production of hydrogen peroxide is mainly responsible for oxidative stress resistance between these natural isolates.