Genetic analysis in the Collaborative Cross breeding population

Abstract

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations.

Figure 1.

Distribution of litter size across generations. The inbreeding generations lose the extreme litter size values observed in the G2:F1s, indicating that no recessive allele produces this value. When such a phenomenon is observed in inbreeding lines relative to a natural population, the result is attributable to overdominance as a mechanism of inbreeding depression.

Figure 2.

Comparison of phenotypic distributions among the CC and BXD mice. Shaded bars represent the phenotypic distribution of the BXD population. (A) Distribution of thermal nociception across the CC and BXD populations. (B) Distribution of distance traveled in the open field across the CC (during the first 3 min) and BXD populations (during the first 5 min). (C) Distribution of body lengths across the CC and BXD populations. Note that in all the three examples the phenotypic range of the BXD population is contained within one side of the distribution of CC phenotypes.

Figure 3.

Wildness variation among CC progenitors, outcrossed (G1 and G2) and inbreeding generations (G2:F1 to G2:F8). (A) Mean wildness scores among CC progenitors. Among the progenitors, behavioral wildness resembles a discrete trait with high wildness in WSB/EiJ and low wildness in the other lines. (B) Proportion of mice in CC generations with wildness scores of 1 (blue), 2 (red), and >2 (other colors). Among the outcrossed generations, wildness scores increase, and among the inbreeding generations, a greater proportion of intermediate values is observed. In general, while more mice have scores >1 in the CC lines, fewer extreme high scores were observed, suggesting a restoration of continuous variation of this phenotype.

Figure 4.

Parent–offspring similarity estimates for three phenotypes. Parent–offspring regression coefficients typically increase during inbreeding with a single generation drop in correlation. For behavioral wildness, parent–offspring similarity increases until the G2:F5 generation followed by a single generation of drop (A). Similar trends exist for body weight (B) and gonadal fat-pad weight (C).

Figure 5.

Strain-specific Minor Allele Frequencies (MAF) at the G2:F1 and G2:F7 generations. Strain-specific MAF in the G2:F1 (A) generation depicts less variation than the G2:F7 (B) generation, with some spread of allele frequencies evident in the G2:F7 generation. Allele frequency distributions become asymmetrical for some strains. The distribution becomes right skewed for PWK/PhJ, indicating that more loss than gain has occurred, and left skewed for WSB/EiJ.

Figure 6.

Assessment of allele loss during inbreeding. Comparison of the percent allele loss between (A) progenitors (G0) and final outcross generation (G2:F1). (B) G2:F1 and the seventh inbreeding generation (G2:F7). Positive values indicate SNPs with an increase in minor allele frequency, while negative values indicate a decrease (allele loss) in minor allele frequency. The y-axis indicates the percent change from G0 allele frequency.

Figure 7.

Genetic mapping of litter size. A genome-wide scan (A) reveals a suggestive QTL on chromosome 6 (B) for litter size. Within the confidence interval are several alleles that have decreasing frequency in the inbreeding CC lines and several genes associated with embryonic lethality (C).

Figure 8.

Significant genome-wide QTLs. (A) Red blood cell width distribution. (B) Periosteal circumference. (C) Peak activity time in hours from dark onset after sleep deprivation. (D) Average percentage of sleep time over dark cycles for all baseline days. (E) Average minimum distance of the center of the mouse from the absolute center of the open field (cm). (F) Thermal nociception. (G) Open field locomotion in the first 3 min. (H) Body length. Horizontal lines indicate genome-wide significance thresholds based on 1000 permutations. Dotted lines are genome-wide significant thresholds at p ≤ 0.05; dashed lines indicate genome-wide suggestive thresholds at p ≤ 0.10.