Figure 2. Multiplexed co-selection and/or co-counterselection produces genotypically pure populations.

Heterozygous drug-resistant and/or drug-sensitive fly strains: Blast^R and G418^R (A); Blast^R and GCV^S (B); 5FC^S and Blast^R (C); and GCV^S and 5FC^S (D) were crossed together under four drug conditions. Fly crosses were tested on food with vehicle control (VC), drug A, drug B, or both drugs (A+B). Survival data matched expected frequencies, with selection or counterselection reducing normalized percent survival by 50% and dual-drug treatment by 75% versus vehicle control. For each cross, 24 flies from each of the four drug conditions were collected and individually genotyped. Genotyping produced expected genotypes for each drug condition in each individual cross. Selection with a single drug results in survival of only the corresponding resistant genotypes (A–C). Conversely, single-drug counterselection eliminates only the relevant sensitized strains (B–D). Finally, dual-drug co-selection produced only the dually resistant genotype (A), only Blast^R heterozygotes survived combination selection and counterselection (A–C), and co-counterselection resulted in only EGFP homozygotes, sensitive to neither drug, surviving treatment (D). See also Figures S4–S6.