Reference
The Diversity Outbred heterogeneous stock (J:DO) is a developing mouse population derived from progenitor lines of the Collaborative Cross (CC). The CC is a panel of recombinant inbred (RI) mouse strains that combines the genomes of eight genetically diverse founder strains - A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ - to capture nearly 90% of the known variation present in laboratory mice (Churchill et al. 2004). Animals from 160 incipient CC lines at early stages of inbreeding were used to establish the DO population, which is maintained by a randomized outbreeding strategy that avoids brother-sister matings. The DO and CC populations thus capture the same set of natural allelic variants derived from a common set of eight founder strains, with DO mice being outbred and the CC population being inbred.
CTC (2004), Churchill, G. A., et al.. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet. 36, 1133-7.
Experiment
The authors describe analytical methods for genetic mapping using the DO mapping resource and demonstrate the power and high mapping resolution achieved with this population by mapping a serum cholesterol trait to a 2-Mbp region on chromosome 3 containing only 11 genes. Analysis of the estimated allele effects in conjunction with complete genome sequence data of the founder strains reduced the pool of candidate polymorphisms to seven SNPs, five of which are located in an intergenic region upstream of the Foxo1 gene.
A pilot study was conducted using 150 early generation DO animals (G4: n = 100, 50 females, 50 males; G5: n = 50, 25 females, 25 males).
Blood was obtained from the retro-orbital sinus after administration of a topical anesthetic (tetracaine HCl) using a heparin-coated microcapillary tube and collected into a 1.5 mL eppendorf tube. To measure plasma components, approximately 150 mL whole blood was collected into a tube containing 2 mL of 10% sodium heparin, and plasma was separated by centrifugation at 10,000 rpm for 10 min at 4 degrees and removed into a clean eppendorf tube. Plasma component (total cholesterol, HDL cholesterol, glucose, triglycerides) were measured using the Beckman Synchron DXC600Pro Clinical chemistry analyzer. For whole-blood analysis approximately 200 mL of blood was collected into a tube containing 2 mL of 10% sodium EDTA. Hematological parameters were measured using the Siemens Advia 2120 haematology analyzer.
Body composition was assessed when mice were 12 weeks of age by dual energy X-ray absorptiometry (DEXA) using a Lunar PIXImus densitometer (GE Medical Systems) after mice were anesthetized intraperitoneally with tribromoethanol (0.2 mL 2% solution/10 g body weight). The skull is omitted from the DEXA analysis because it is so bone-dense.
DNA was prepared by standard methods from tail biopsies and genotyping was outsourced to GeneSeek (http://www. neogen.com/GeneSeek) for analysis using a Mouse Universal Genotyping Array (MUGA), a 7,851 SNP array built on the Illumina Infinium platform.
Haplotype reconstruction was based on Illumina's normalized MUGA intensity values rather than genotype calls.
The genotyping HMM (hidden Markov model) produced a matrix of 36 genotype state probabilities for each sample at each SNP. This matrix was condensed to produce an eight-founder state probability matrix by summing the probabilities contributed by each founder at each SNP. The eight-founder probabilities were used to fit a linear model, with sex and diet as additive covariates and variance components that account for genetic relatedness using QTLRel.
The authors performed QTL analysis by pooling the available animals across sexes and two dietary conditions. Sex, diet, and a random polygenic effect were included as covariates in the QTL analysis. The authors generated 113 phenotypes, some of which were calculated change between values measured at two time points in the phenotyping protocol. The number of animals with complete information varied across traits from 87 to 141. They identified significant QTL (genome-wide adjusted p < 0.05) for 11 traits, including 7 for hematological parameters, 2 for body composition, 1 for cardiac function, and 1 for plasma chemistries. These results represent an interim analysis of an ongoing study with a target sample size of 600 DO animals.
As a proof of principle, the authors mapped two coat color traits, albino and black. They coded each as binary traits and analyzed using linear regression as described above. They did not use any prior knowledge about the phenotypic mode of inheritance. Two QTL were identified:
QTL Albc2 (albino coat color QTL 2) maps to Chr7:88-96 Mbp and the underlying Tyr gene is at 94.6 Mb. The effect plot (Figure S1B) shows that the two albino founders, A/J and NOD/ShiLtJ, have positive coefficients.
QTL Agoc2 (agouti coat color QTL 2) Black trait mapped to Chr2:152-160 Mbp. The a gene lies at 154.7 Mb. The effect plot (Figure S1E) shows that A/J and C57BL/6J both have positive coefficients.
Among the clinical traits mapped in this study, the authors selected change in total plasma cholesterol from age 8 to 19 weeks to illustrate the potential precision of QTL mapping with the DO. A total of 91 DO animals had complete data for this trait. The genome-wide LOD profile identified one significant QTL:
QTL Tpcq1 (total plasma cholesterol QTL 1) maps to Chr3:50.3-52.3 Mbp (p <= 0.014) with a peak LOD score of 4.8 at 51 Mbp (interpreted from Figure 6A). The allele effects plot (Figure 6B) indicates that founder haplotypes from strains 129S1/SvImJ, WSB/EiJ, and NZO/HlLtJ are associated with a decrease in total plasma cholesterol over time.
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