By testing a control congenic strain in parallel, we have demonstrated that strain-specific polymorphisms from the introgressed D2 background do not produce or influence any of the phenotypes observed here. This possibility is often ignored [31] but is an important concern due to the quantitative nature of several of the phenotypes tested. Although we have ruled out strain-specific polymorphisms, we cannot formally rule out the possibility that an additional ENU-induced mutation in our congenic strain might influence some of the phenotypes we have observed. The mutation rate of ENU depends on many factors, but hovers around 1×10−6 [25]–[27]; therefore, it is unlikely that more than one or two point mutations were induced within this 1.4 Mb congenic region, and furthermore, the likelihood of mutating a coding exon is very rare [28]. We have sequenced the majority of coding exons in this congenic interval without finding any additional mutations other than Lightweight. Complete resequencing of the whole 1.4 Mb interval would confirm the absence of other confounding mutations.
