QTL Mapping of Skeletal Traits in Diversity Outbred Mice

The cohorts used for the earlier QTL mapping of ML consisted of 577 Diversity Outbred mice from breeding generations G10 and G11^{62 (link)}. G10 cohort mice consisted of both males and females fed a defined synthetic diet (D10001, Research Diets, New Brunswick, NJ), and were euthanized and analyzed at 12–15 weeks of age. G11 cohort mice were all females fed a defined synthetic diet (D10001, Research Diets, New Brunswick, NJ) until 6 weeks of age, and were then subsequently fed either a high-fat, cholesterol-containing (HFC) diet (20% fat, 1.25% cholesterol, and 0.5% cholic acid) or a low-fat, high protein diet (5% fat and 20.3% protein) (D12109C and D12083101, respectively, Research Diets, New Brunswick, NJ), and were euthanized and analyzed at 24–25 weeks of age. Mice were weighed and then euthanized by CO₂ asphyxiation followed by cervical dislocation. Carcasses were frozen at −80 °C. Subsequently, the femur was dissected and length, AP width, and ML width were measured two independent times to 0.01 mm using digital calipers. Mice were genotyped using the MegaMUGA SNP array (GeneSeek; Lincoln, NE) designed with 77,800 SNP markers, and QTL mapping was performed as above, but with the inclusion of sex, diet, age, and weight at sacrifice as additive covariates.