There was no statistical support for genetic variation within selected or control lines in flies whose dams were two generations removed from selection (Table 3), but this is likely more reflective of the relatively large and significant line (line type) * treatment * block term in the denominator of the F test rather than reflective of the biology.
After selection of resistant lines, cells were removed from selection 72 h before the induction of RNAi by the addition of 1 µg ml−1 tetracycline (Sigma-Aldrich) to normal growth medium.
Studies were conducted on flies zero, one, or two generations removed from selection.
For one generation removed from selection, the dams of the daughters for ovariole number determination were taken from lines in which selection had been relaxed for one generation.
For two generations removed from selection, the dams of the daughters for ovariole number determination were taken from lines in which selection had been relaxed for two generations.
For zero generations removed from selection, females used as dams in the experiments were taken directly from the starvation lines without intervening generations of rearing under control line conditions.
SNPs rs360718, rs360719, rs1946518, rs12721559 and rs360721 were removed from selection, as these SNPs were less than 100 bp apart and therefore could not be pooled together for the customized Illumina chip.
There was evidence for genetic variation within selected and control lines line [line type]) for ovariole number in flies whose dams were zero or one generation removed from selection (Tables 1 and 2, line (line type), P < 0.0001).
Following death of all mock-infected cells (approx. 1 to 2 weeks), cells were removed from selection, and plated at limiting dilution in 96-well plates to generate single-cell clones.
The motivation for using flies variable numbers of generations removed from selection was to be able to determine if the response to the starvation as a phenotypic manipulation, or the magnitude of the ovary number indirect response to selection, was related to generational proximity to lengthy starvation as the selective agent in the selection experiment.
