Although mice are the most widely used mammalian model organism, genetic studies have suffered from limited mapping resolution due to extensive linkage disequilibrium (LD) that is characteristic of crosses among inbred strains. Carworth Farms White (CFW) mice are a commercially available outbred mouse population that exhibit rapid LD decay in comparison to other available mouse populations. We performed a genome-wide association study (GWAS) of behavioral, physiological and gene expression phenotypes using 1,200 male CFW mice. We used genotyping by sequencing (GBS) to obtain genotypes at 92,734 SNPs. We also measured gene expression using RNA sequencing in three brain regions. Our study identified numerous behavioral, physiological and expression quantitative trait loci (QTLs). We integrated the behavioral QTL and eQTL results to implicate specific genes, including Azi2 in sensitivity to methamphetamine and Zmynd11 in anxiety-like behavior. The combination of CFW mice, GBS and RNA sequencing constitutes a powerful approach to GWAS in mice.
At a glance
- A HapMap harvest of insights into the genetics of common disease. J. Clin. Invest. 118, 1590–1605 (2008). , &
- Bringing genome-wide association findings into clinical use. Nat. Rev. Genet. 14, 549–558 (2013).
- Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature 511, 421–427 (2014).
- The role of regulatory variation in complex traits and disease. Nat. Rev. Genet. 16, 197–212 (2015). &
- Dissecting quantitative traits in mice. Annu. Rev. Genomics Hum. Genet. 14, 421–439 (2013). &
- Dark matter: are mice the solution to missing heritability? Front. Genet. 2, 32 (2011). &
- The so-called Swiss mouse. Lab. Anim. Care 19, 214–220 (1969).
- Genetic variance of laboratory outbred Swiss mice. Nature 283, 157–161 (1980). &
- Commercially available outbred mice for genome-wide association studies. PLoS Genet. 6, e1001085 (2010). et al.
- The origins and uses of mouse outbred stocks. Nat. Genet. 37, 1181–1186 (2005). , , &
- Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases. Am. J. Hum. Genet. 95, 535–552 (2014). et al.
- Quantitative trait locus mapping methods for diversity outbred mice. G3 (Bethesda) 4, 1623–1633 (2014). et al.
- The Mouse Universal Genotyping Array: from substrains to subspecies. G3 (Bethesda) 6, 263–279 (2015). et al.
- A customized and versatile high-density genotyping array for the mouse. Nat. Methods 6, 663–666 (2009). et al.
- A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6, e19379 (2011). et al.
- Linkage disequilibrium in humans: models and data. Am. J. Hum. Genet. 69, 1–14 (2001). &
- Linkage disequilibrium in wild mice. PLoS Genet. 3, e144 (2007). et al.
- Out of the bottleneck: the Diversity Outcross and Collaborative Cross mouse populations in behavioral genetics research. Mamm. Genome 25, 3–11 (2014).
- The Diversity Outbred mouse population. Mamm. Genome 23, 713–718 (2012). , , &
- Collaborative Cross Consortium. The genome architecture of the Collaborative Cross mouse genetic reference population. Genetics 190, 389–401 (2012).
- Estimation of SNP heritability from dense genotype data. Am. J. Hum. Genet. 93, 1151–1155 (2013). et al.
- Pitfalls of predicting complex traits from SNPs. Nat. Rev. Genet. 14, 507–515 (2013). et al.
- A simulation study of permutation, bootstrap, and gene dropping for assessing statistical significance in the case of unequal relatedness. Genetics 193, 1015–1018 (2013). &
- Empirical threshold values for quantitative trait mapping. Genetics 138, 963–971 (1994). &
- Activin A balances Sertoli and germ cell proliferation in the fetal mouse testis. Biol. Reprod. 84, 379–391 (2011). , , &
- Activin bioactivity affects germ cell differentiation in the postnatal mouse testis in vivo. Biol. Reprod. 82, 980–990 (2010). et al.
- Essential roles of inhibin βA in mouse epididymal coiling. Proc. Natl. Acad. Sci. USA 104, 11322–11327 (2007). , , &
- Quadrupling muscle mass in mice by targeting TGF-β signaling pathways. PLoS One 2, e789 (2007).
- Regulation of muscle mass by follistatin and activins. Mol. Endocrinol. 24, 1998–2008 (2010). et al.
- Resolving candidate genes of mouse skeletal muscle QTL via RNA-Seq and expression network analyses. BMC Genomics 13, 592 (2012). et al.
- Autophagy-regulating TP53INP2 mediates muscle wasting and is repressed in diabetes. J. Clin. Invest. 124, 1914–1927 (2014). et al.
- Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture. Nat. Genet. 44, 491–501 (2012). et al.
- Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture. Nature 526, 112–117 (2015). et al.
- SLC4A2-mediated Cl−/HCO3− exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts. Proc. Natl. Acad. Sci. USA 110, 2163–2168 (2013). et al.
- A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. BMC Genomics 11, 337 (2010). et al.
- Genetic heterogeneity in osteogenesis imperfecta. J. Med. Genet. 16, 101–116 (1979). , &
- Osteogenesis imperfecta is linked to both type I collagen structural genes. Lancet 2, 69–72 (1986). , , , &
- Association between COL1A1 gene polymorphisms and bone size in Caucasians. Eur. J. Hum. Genet. 12, 383–388 (2004). et al.
- A statistical framework for joint eQTL analysis in multiple tissues. PLoS Genet. 9, e1003486 (2013). , , &
- Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression. PLoS Genet. 4, e1000006 (2008). et al.
- Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature 464, 768–772 (2010). et al.
- Refining analyses of copy number variation identifies specific genes associated with developmental delay. Nat. Genet. 46, 1063–1071 (2014). et al.
- Genome-wide association studies and the problem of relatedness among advanced intercross lines and other highly recombinant populations. Genetics 185, 1033–1044 (2010). et al.
- Fine mapping of QTL for prepulse inhibition in LG/J and SM/J mice using F2 and advanced intercross lines. Genes Brain Behav. 9, 759–767 (2010). , , , &
- Fine-mapping alleles for body weight in LG/J × SM/J F and F34 advanced intercross lines. Mamm. Genome 22, 563–571 (2011). et al.
- High-resolution genetic mapping of complex traits from a combined analysis of F2 and advanced intercross mice. Genetics 198, 103–116 (2014). et al.
- High-resolution mapping of quantitative trait loci in outbred mice. Nat. Genet. 21, 305–308 (1999). et al.
- Further characterization and high-resolution mapping of quantitative trait loci for ethanol-induced locomotor activity. Behav. Genet. 31, 79–91 (2001). , , , &
- Genome-wide genetic association of complex traits in heterogeneous stock mice. Nat. Genet. 38, 879–887 (2006). et al.
- High-resolution mapping of gene expression using association in an outbred mouse stock. PLoS Genet. 4, e1000149 (2008). et al.
- Unraveling inflammatory responses using systems genetics and gene–environment interactions in macrophages. Cell 151, 658–670 (2012). et al.
- Integration of genome-wide association and extant brain expression QTL identifies candidate genes influencing prepulse inhibition in inbred F1 mice. Genes Brain Behav. 15, 260–270 (2016). , , , &
- High-resolution genetic mapping using the Mouse Diversity outbred population. Genetics 190, 437–447 (2012). et al.
- Genetic dissection of a behavioral quantitative trait locus shows that Rgs2 modulates anxiety in mice. Nat. Genet. 36, 1197–1202 (2004). et al.
- Allele frequency matching between SNPs reveals an excess of linkage disequilibrium in genic regions of the human genome. PLoS Genet. 2, e142 (2006). , , &
- Novel measures of linkage disequilibrium that correct the bias due to population structure and relatedness. Heredity 108, 285–291 (2012). et al.
- Subspecific origin and haplotype diversity in the laboratory mouse. Nat. Genet. 43, 648–655 (2011). et al.
- CONVERGE Consortium. Sparse whole-genome sequencing identifies two loci for major depressive disorder. Nature 523, 588–591 (2015).
- SNP detection and genotyping from low-coverage sequencing data on multiple diploid samples. Genome Res. 21, 952–960 (2011). &
- Low-coverage sequencing: implications for design of complex trait association studies. Genome Res. 21, 940–951 (2011). , , , &
- The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010). et al.
- A flexible and accurate genotype imputation method for the next generation of genome-wide association studies. PLoS Genet. 5, e1000529 (2009). , &
- Genome-wide association for fear conditioning in an advanced intercross mouse line. Behav. Genet. 42, 437–448 (2012). , , &
- Genome-wide association for methamphetamine sensitivity in an advanced intercross mouse line. Genes Brain Behav. 11, 52–61 (2012). , , &
- Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature 507, 371–375 (2014). et al.
- Genetics of gene expression surveyed in maize, mouse and man. Nature 422, 297–302 (2003). et al.
- WebQTL: rapid exploratory analysis of gene expression and genetic networks for brain and behavior. Nat. Neurosci. 7, 485–486 (2004). , , , &
- Complex trait analysis of gene expression uncovers polygenic and pleiotropic networks that modulate nervous system function. Nat. Genet. 37, 233–242 (2005). et al.
- Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'. Nat. Genet. 37, 225–232 (2005). et al.
- Gene expression differences in mice divergently selected for methamphetamine sensitivity. Mamm. Genome 16, 291–305 (2005). et al.
- High resolution mapping of expression QTLs in heterogeneous stock mice in multiple tissues. Genome Res. 19, 1133–1140 (2009). et al.
- Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet. 7, e1002038 (2011). et al.
- Systems genetic analysis of osteoblast-lineage cells. PLoS Genet. 8, e1003150 (2012). et al.
- Alternative mRNA transcription, processing, and translation: insights from RNA sequencing. Trends Genet. 31, 128–139 (2015). &
- Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008). , , , &
- Transcriptome sequencing of the Microarray Quality Control (MAQC) RNA reference samples using next generation sequencing. BMC Genomics 10, 264 (2009). et al.
- mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382 (2009). et al.
- TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009). , &
- Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012). et al.
- High throughput sequencing in mice: a platform comparison identifies a preponderance of cryptic SNPs. BMC Genomics 10, 379 (2009). et al.
- Combined sequence-based and genetic mapping analysis of complex traits in outbred rats. Nat. Genet. 45, 767–775 (2013). et al.
- CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014). &
- A high-resolution association mapping panel for the dissection of complex traits in mice. Genome Res. 20, 281–290 (2010). et al.
- The study of eQTL variations by RNA-seq: from SNPs to phenotypes. Trends Genet. 27, 72–79 (2011). &
- RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63 (2009). , &
- Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). , , &
- Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27, 2325–2329 (2011). , , &
- Population genomic variation reveals roles of history, adaptation and ploidy in switchgrass. Mol. Ecol. 23, 4059–4073 (2014). , , &
- Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009). &
- From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics 11, 11.10.1–11.10.33 (2013). et al.
- Mouse genomic variation and its effect on phenotypes and gene regulation. Nature 477, 289–294 (2011). et al.
- Inference of population splits and mixtures from genome-wide allele frequency data. PLoS Genet. 8, e1002967 (2012). &
- Genome-wide efficient mixed-model analysis for association studies. Nat. Genet. 44, 821–824 (2012). &
- Increased accuracy of artificial selection by using the realized relationship matrix. Genet. Res. (Camb.) 91, 47–60 (2009). , &
- Improved linear mixed models for genome-wide association studies. Nat. Methods 9, 525–526 (2012). et al.
- Polygenic modeling with Bayesian sparse linear mixed models. PLoS Genet. 9, e1003264 (2013). , &
- Permutation testing in the presence of polygenic variation. Genet. Epidemiol. 39, 249–258 (2015).
- Common SNPs explain a large proportion of the heritability for human height. Nat. Genet. 42, 565–569 (2010). et al.
- Improved heritability estimation from genome-wide SNPs. Am. J. Hum. Genet. 91, 1011–1021 (2012). , , &
- Supplementary Text and Figures (8,706 KB)
Supplementary Note and Supplementary Figures 1–29.
- Supplementary Table 1 (16,353 KB)
Summary of phenotypes.
- Supplementary Table 2 (18,098 KB)
- Supplementary Table 3 (10,779 KB)
The number of expression QTLs (eQTLs) found in the three brain tissues assayed.
- Supplementary Table 4 (10,512 KB)
Discordance rates when comparing GBS and MegaMUGA genotypes using 24 subjects genotyped using both platforms.