Phenotypic instability between the near isogenic substrains BALB/cJ and BALB/cByJ

Closely related substrains of inbred mice often show phenotypic differences that are presumed to be caused by recent mutations. The substrains BALB/cJ and BALB/cByJ, which were separated in 1935, have been reported to show numerous highly significant behavioral and morphological differences. In an effort to identify some of the causal mutations, we phenotyped BALB/cJ and BALB/cByJ mice as well as their F1, F2, and N2 progeny for behavioral and morphological phenotypes. We also generated whole-genome sequence data for both inbred strains (~3.5× coverage) with the intention of identifying polymorphic markers to be used for linkage analysis. We observed significant differences in body weight, the weight of the heart, liver, spleen and brain, and corpus callosum length between the two substrains. We also observed that BALB/cJ animals showed greater anxiety-like behavior in the open field test, less depression-like behavior in the tail suspension test, and reduced aggression compared to BALB/cByJ mice. Some but not all of these physiological and behavioral results were inconsistent with prior publications. These inconsistencies led us to suspect that the differences were due to, or modified by, non-genetic factors. Thus, we did not perform linkage analysis. We provide a comprehensive summary of the prior literature about phenotypic differences between these substrains as well as our current findings. We conclude that many differences between these strains are unstable and therefore ill-suited to linkage analysis; the source of this instability is unclear. We discuss the broader implications of these observations for the design of future studies.