F18 Poolseq for D. americana x D. novamexicana cross

To identify single nucleotide polymorphisms (SNPs) associated with eye size variation between D. americana and D. novamexicana, we performed a genotype-phenotype association study using F18 individuals resulting from brother sister mating for 17 generations starting from crosses between D. novamexicana (15010-1031.00) males and D. americana (SF12) females. We phenotyped a total of 157 females, which represented the entire F18 female progeny of two independent vials, for eye and wing areas, as well as tibiae lengths of one of each pair of legs. Wings and legs were randomly dissected from left or right sides. Missing values for individuals ID=125 and 150 showing highly damaged wings were estimated based on the equation of the multiple linear regression between wing area and tibiae length. The residuals of the multiple regression between eye area and both tibiae lengths and wing areas were used to remove the variation in eye area that could be explained by variation in total body size. The grand mean was summed to the residuals and these new values were sorted in ascending order to divide the females into four quartiles