Acquisition and retention of conditioned aversions to context and taste in laboratory mice.

We compared the rate of acquisition and strength of retention of conditioned context aversion (CCA) with conditioned taste aversion (CTA) using pigmented, genetically heterogeneous mice (derived from Large and Small strains). Extending previous findings, in Experiment 1, mice accustomed to drinking from large glass bottles in the colony room learned to avoid graduated tubes after a single conditioning trial when drinking from these novel tubes was paired with injections of LiCl. The results also showed that CCA could be developed even when there was a 30-minute delay between conditioned stimulus and unconditioned stimulus. Retention of the aversion lasted for 4 weeks in both Immediate and Delay groups. Studies of conditioned saccharin aversion were conducted in Experiment 2. CTA acquisition was very similar to that observed in CCA and duration of aversion retention was similar in the CCA and CTA Delay groups, although at least 2 weeks longer in the Immediate group. Thus, CCA acquisition and retention characteristics are closer to those seen for CTA than has previously been reported. In Experiment 3, we examined whether albino mice (which are known to have weaker visual abilities compared to pigmented mice) would develop CCA comparable to those of pigmented mice. The development of conditioned aversion and its duration of retention was similar in albinos and pigmented mice. Nonspecific aversion emerged as an important contributor to strength of aversion during retention trials in both CCA and CTA paradigms with pigmented (but not albino) mice and deserves additional scrutiny in this field of inquiry.

Conditioned context aversion learning in the laboratory mouse.

It is well known that pairing of large contextual changes with illness can cause conditioned context aversion in laboratory rats. The aim of present study was to develop a paradigm to study this phenomenon in laboratory mice, a species widely employed in neurobehavioral studies. Genetically heterogeneous mice, drinking from plastic bottles in the colony room, learned to avoid glass bottles after a single conditioning trial when drinking from these was paired with injections of lithium chloride. The aversion was independent of any difference in the taste of water in plastic vs. glass bottles. When the variation in the visual stimulus was less distinct, development of a strong aversion required two conditioning trials and was not retained as well. The results also showed that conditioned context aversion, just like conditioned taste aversion, could also be developed across a 30-minute CS-UCS delay. The fact that taste was not a factor in distinguishing drinking from glass and plastic water bottles raises the possibility that, contextual stimuli, not taste, may have been the CS when rats (in Garcia's original experiments) avoided drinking from plastic bottles that had been paired with radiation. The development of contextual aversion conditioning protocols for mice will enable the molecular resources available for this species to be exploited. Furthermore, representation of the CS by discrete rather than the multimodal CSs typically used in most studies on contextual conditioning offers more focus when considering its neuroanatomical basis.

A Chromosome 13 locus is associated with male-specific mortality in mice.

BACKGROUND AND AIM: Mortality is a highly complex trait influenced by a wide array of genetic factors. METHODS: We examined a population of 1200 mice that were F2 generation offspring of a 4-way reciprocal cross between C57BL6/J and DBA2/J strains. Animals were sacrificed at age 200, 500, or 800 days and genotyped at 96 markers. The 800 days old cohort, which were the survivors of a much larger breeding group, were examined for enriched frequency of alleles that benefit survival and depletion of alleles that reduce survival. RESULTS: Loci on Chr 13 in males and on Chr X in females were significantly distorted from Mendelian expectations, even after conservative correction for multiple testing. DBA2/J alleles between 35 and 80 Mb on Chr 13 were underrepresented in the age 800 male animals. D2 genotypes in this region were also associated with premature death during behavioral testing. Furthermore, confirmatory analysis showed BXD recombinant inbred strains carrying the D2 alleles in this region had shorter median survival. Exploration of available pathology data indicated that a syndrome involving dental malocclusions, pancreatic islet hypertrophy, and kidney lipidosis may have mediated the effects of DBA alleles on mortality specifically in male mice. The heterozygote advantage locus on the X Chr was not found to be associated with any pathology. CONCLUSIONS: These results suggest a novel locus influencing survival in the B6/D2 genetic background, perhaps via a metabolic disorder that emerges by 200 days of age in male animals.

The Defecation Index as a Measure of Emotionality: Questions Raised by HPA Axis and Prolactin Response to Stress in the Maudsley Model.

The Maudsley Reactive and Maudsley Non-Reactive strains have been selectively bred for differences in open-field defecation (OFD), a putative index of stress. We investigated whether variations in the hypothalamic-pituitary-adrenal (HPA) axis are correlated with strain differences in OFD in the Maudsley model. Exposure to the open-field test did not result in increases in ACTH in male rats of either strain and there were no strain differences in the large increases in ACTH and corticosteroid that occurred in response to intermittent footshock. Parallel studies of prolactin showed that Maudsley Reactive rats had greater response to the open-field and to footshock than Maudsley Non-Reactive rats. The lack of correlation between strain differences in OFD and reactivity of the HPA axis is consistent with the idea that HPA response to stress and OFD reflect the output of different neural systems and that individual differences in emotionality, as indexed by OFD do not influence other measures of stress-reactivity in a simple manner, if at all. The reactivity of the prolactin system to the open-field test and lack of response of ACTH to the same situation is consistent with the idea that the prolactin system is sensitive to lower levels of stress than the HPA axis, a finding at variance with the presumed extreme sensitivity of the latter system. Earlier comparisons of the HPA axis in these strains implicate local factors such as neuropeptide-Y peptide in the adrenal in attenuating the response of the adrenal cortex to ACTH and hints at the complexity of regulation of the HPA axis.

Genetic and genomic analyses of musculoskeletal differences between BEH and BEL strains.

Berlin high (BEH) and Berlin low (BEL) strains selected for divergent growth differ threefold in body weight. We aimed at examining muscle mass, which is a major contributor to body weight, by exploring morphological characteristics of the soleus muscle (fiber number and cross sectional area; CSA), by analyzing the transcriptome of the gastrocnemius and by initiating quantitative trait locus (QTL) mapping. BEH muscles were four to eight times larger than those of BEL. In substrain BEH+/+, mutant myostatin was replaced with a wild-type allele; however, BEH+/+muscles still were two to four times larger compared with BEL. BEH soleus muscle fibers were two times more numerous (P < 0.0001) and CSA was two times larger (P < 0.0001) compared with BEL. In addition, soleus femoral attachment anomaly (SFAA) was observed in all BEL mice. One significant (Chr 1) and four suggestive (Chr 3, 4, 6, and 9) muscle weight QTLs were mapped in a 21-day-old F2 intercross (n = 296) between BEH and BEL strains. The frequency of SFAA incidence in the F2 and in the backcross to BEL strain (BCL) suggested the presence of more than one causative gene. Two suggestive SFAA QTLs were mapped in BCL; however, their peak markers were not associated with the phenotype in F2. RNA-Seq analysis revealed 2,148 differentially expressed (P < 0.1) genes and 45,673 single nucleotide polymorphisms and >2,000 indels between BEH+/+ and BEL males. In conclusion, contrasting muscle traits and genomic and gene expression differences between BEH and BEL strains provide a promising model for the search for genes involved in muscle growth and musculoskeletal morphogenesis.

The genetics of neuroticism: Insights from the Maudsley rat model and human studies.

We examine some of the genetic features of neuroticism (N) taking as an animal model the Maudsley Reactive (MR) and Maudsley Nonreactive (MNR) rat strains which were selectively bred, respectively, for high and low open-field defecation (OFD) starting in the late 1950s. To draw analogies with human genetic studies, we explore the genetic correlation of N with irritable bowel syndrome (IBS). We review progress with the rat model and developments in the field of human complex trait genetics, including genetic association studies that relate to current understanding of the genetics of N. The widespread differences in the tone of the peripheral sympathetic nervous system that have been found between the Maudsley strains, particularly those observed in the colon, may underly the differences in OFD (MNR, higher sympathetic tone and zero defecation). In humans, a large genome-wide association study (GWAS) reported six genes contributing to IBS, four of which were implicated in mood and anxiety disorders or were expressed in the brain, with three of the four also expressed in the nerve fibers and ganglia of the gut. Heritability of N is estimated at around 50% in twin and family studies, and GWASs identified hundreds of loci, enabling estimation of genome-wide correlations (rg) with other traits. Significantly, the estimate for rg between risk of IBS, anxiety, N, and depression was >0.5 and suggested genetic pleiotropy without evidence for causal mechanisms. Findings on the adrenergic pharmacology of the colon, coupled with new understanding of the role of the locus ceruleus in modifying afferent information from this organ, generate hypotheses that challenge traditional cause/effect notions about the relationship of the central nervous system to peripheral events in response to stress, suggest specific targets for gene action in the Maudsley model and emphasize the value of reciprocal evaluation of genetic architecture underlying N in rodents and humans.

Resolving candidate genes of mouse skeletal muscle QTL via RNA-Seq and expression network analyses.

BACKGROUND: We have recently identified a number of Quantitative Trait Loci (QTL) contributing to the 2-fold muscle weight difference between the LG/J and SM/J mouse strains and refined their confidence intervals. To facilitate nomination of the candidate genes responsible for these differences we examined the transcriptome of the tibialis anterior (TA) muscle of each strain by RNA-Seq. RESULTS: 13,726 genes were expressed in mouse skeletal muscle. Intersection of a set of 1061 differentially expressed transcripts with a mouse muscle Bayesian Network identified a coherent set of differentially expressed genes that we term the LG/J and SM/J Regulatory Network (LSRN). The integration of the QTL, transcriptome and the network analyses identified eight key drivers of the LSRN (Kdr, Plbd1, Mgp, Fah, Prss23, 2310014F06Rik, Grtp1, Stk10) residing within five QTL regions, which were either polymorphic or differentially expressed between the two strains and are strong candidates for quantitative trait genes (QTGs) underlying muscle mass. The insight gained from network analysis including the ability to make testable predictions is illustrated by annotating the LSRN with knowledge-based signatures and showing that the SM/J state of the network corresponds to a more oxidative state. We validated this prediction by NADH tetrazolium reductase staining in the TA muscle revealing higher oxidative potential of the SM/J compared to the LG/J strain (p<0.03). CONCLUSION: Thus, integration of fine resolution QTL mapping, RNA-Seq transcriptome information and mouse muscle Bayesian Network analysis provides a novel and unbiased strategy for nomination of muscle QTGs.

Gustatory, trigeminal, and olfactory aspects of nicotine intake in three mouse strains.

Studies of nicotine consumption in rodents often intend to investigate nicotine's post-absorptive effects, yet little is known about the pre-absorptive sensory experience of nicotine drinking, including gustatory, trigeminal, and olfactory influences. We conditioned taste aversion (CTA) to nicotine in males of 3 inbred mouse strains: C57BL/6J, DBA/2J, and 129X1/SvJ by repeatedly pairing 150 µg/ml nicotine drinking with lithium chloride injections. Generalization to a variety of bitter, sour, sweet, salty, and irritant solutions and to nicotine odor was then examined. Nicotine CTA generalized to the bitter stimulus quinine hydrochloride and the chemosensory irritant spilanthol in all strains. It also showed strain specificity, generalizing to hydrogen peroxide (an activator of TRPA1) in C57BL/6J mice and to the olfactory cue of nicotine in DBA/2J mice. These behavioral assays demonstrate that the sensory properties of nicotine are complex and include multiple gustatory, irritant, and olfactory components. How these qualities combine at the level of perception remains to be assessed, but sensory factors clearly exert an important influence on nicotine ingestion and their contribution to net intake of nicotine should not be neglected in animal or human studies.

B6-MSM consomic mouse strains reveal multiple loci for genetic variation in sucrose octaacetate aversion.

Based on crosses among inbred strains derived principally from M. m. domesticus, sucrose octaacetate (SOA) aversion in laboratory mice has been thought for many years to be controlled by a single genetic locus (Soa) located on distal chromosome (Chr) 6. To expand knowledge of the genetic basis underlying SOA aversion, we have studied the M. m. molossinus derived strain (MSM) and MSM consomic strains on a M. m. domesticus (C57BL/6J: B6) host background. Using two-bottle preference procedures, MSM mice avoided 0.1 mM and 1 mM SOA while B6 mice were indifferent to 0.1 mM and exhibited slight aversion to 1 mM SOA. Preference tests of 16 available consomic strains implicated Chr 2, 4 and 15 in SOA aversion in addition to the prominent effect of the known Soa locus on Chr 6 (implicated by study of two congenic strains). The originally defined Soa locus is presumably associated with one or more members of the cluster of Tas2r genes on distal Chr 6 that code for bitter taste receptors. Our results point to the possible role of established Tas2r genes on Chr 2 and 15, as well as to genes not coding for bitter receptors (Chr 4), in SOA aversion. SOA aversion emerges from this consomic screen as being definitively under polygenic control. The genetic diversity captured by the MSM model system is shown to be a valuable tool to complement the limited genetic variation in the commonly used stocks derived from M m. domesticus.

Genomic analysis of variation in hindlimb musculature of mice from the C57BL/6J and DBA/2J lineage.

The precise locations of attachment points of muscle to bone-the origin and insertion sites-are crucial anatomical and functional characteristics that influence locomotor performance. Mechanisms that control the development of these interactions between muscle, tendon, and bone are currently not well understood. In a subset of BXD recombinant inbred (RI) strains derived from the C57BL/6J and DBA/2J strains, we observed a soleus femoral attachment anomaly (SFAA) that was rare in both parental strains (Lionikas, Glover et al. 2006). The aim of the present study was to assess suitability of SFAA as a model to study the genetic mechanisms underlying variation in musculoskeletal anatomy. We scored the incidence of SFAA in 55 BXD strains (n = 9 to 136, median = 26, phenotyped animals per strain, for a total number of 2367). Seven strains (BXD1, 12, 38, 43, 48, 54, and 56) exhibited a high incidence of unilateral SFAA (47-89%), whereas 23 strains scored 0%. Exploration of the mechanisms underlying SFAA in 2 high incidence strains, BXD1 and BXD38, indicated that SFAA-relevant genes are to be found in both C57BL/6J and DBA/2J regions of the BXD1 genome. However, not all alleles relevant for the expression of the phenotype were shared between the 2 high-incidence BXD strains. In conclusion, the anatomical origin of the soleus muscle in mouse is controlled by a polygenic system. A panel of BXD RI strains is a useful tool in exploring the genetic mechanisms underlying SFAA and improving our understanding of musculoskeletal development.

Blood pressure and heart rate QTL in mice of the B6/D2 lineage: sex differences and environmental influences.

A quantitative trait locus (QTL) approach was used to define the genetic architecture underlying variation in systolic blood pressure (SBP) and heart rate (HR), measured indirectly on seven occasions by the tail cuff procedure. The tests were conducted in 395 F(2) adult mice (197 males, 198 females) derived from a cross of the C57BL/6J (B6) and DBA/2J (D2) strains and in 22 BXD recombinant-inbred (RI) strains. Interval mapping of F(2) data for the first 5 days of measurement nominated one statistically significant and one suggestive QTL for SBP on chromosomes (Chr) 4 and 14, respectively, and two statistically significant QTL for HR on Chr 1 (which was specific to female mice) and Chr 5. New suggestive QTL emerged for SBP on Chr 3 (female-specific) and 8 and for HR on Chr 11 for measurements recorded several weeks after mice had undergone stressful blood sampling procedures. The two statistically significant HR QTL were confirmed by analyses of BXD RI strain means. Male and female F(2) mice did not differ in SBP or HR but RI strain analyses showed pronounced strain-by-sex interactions and a negative genetic correlation between the two measures in both sexes. Evidence for a role for mitochondrial DNA was found for both HR and SBP. QTL for HR and SBP may differ in males and females and may be sensitive to different environmental contexts.

QTL analyses of temporal and intensity components of home-cage activity in KJR and C57BL/6J strains.

BACKGROUND: A variety of mouse strains exhibit diversity in spontaneous activity consistent with an important genetic contribution. To date, many studies have defined spontaneous home-cage activity as total distance or total counts of activity within a test period. However, spontaneous activity is, in fact, a composite of elements of 'temporal' and 'intensity' that is similar to 'velocity'. Here, we report on quantitative trait loci for different components of spontaneous activity, an important step towards dissection of the underlying genetic mechanisms. RESULTS: In the analysis of total home-cage activity (THA) after habituation in female mice, KJR strain exhibit higher activity than C57BL/6J (B6). In this study, THA was partitioned into two components: active time (AT) was an index of the 'temporal element' of THA, average activity during active time (AA) was an index of 'intensity'. Correlation analysis using B6xKJR F2 female mice indicated that AA is a major component of THA, whereas AA and AT were associated to a lesser degree. To explore the genetic basis of the activity differences, we conducted quantitative trait loci (QTL) analysis on data of THA and its components, AT and AA. Three significant QTL affecting variation of different components of home cage activity were identified, two linked QTL Hylaq1 and Hylaq2 on Chr 2, and Hylaq3 on Chr 10. Chromosomal positions of these QTL were previously implicated in locomotor activity (Chr 2) or open-field ambulation (Chr 10). The results indicated that Hylaq1 influences AT, Hylaq2, AA, while Hylaq3 is associated with both AA and AT. CONCLUSION: Through this study, we found that variation in total home cage activity over a 3 day period is affected by variation in active time and intensity of activity. The latter two variables are distinct components of home cage activity with only partially overlapping genetic architecture.

Learning in the 2-bottle alcohol preference test.

BACKGROUND: The 2-bottle preference test is a popular protocol for characterizing a rodent's selection of a variety of solutions. Little attention has been paid, however, to the role of learning in this procedure. METHODS: We explored the role of learning in 2-bottle alcohol preference (AP) in mice by recording changes between days and periods (every 3 days the alcohol and water tubes were interchanged) throughout a 15-day standard exposure protocol in use in our laboratory. RESULTS: Male and female ethanol-naive mice of 2 BALB strains (cJ and cByJ), both characterized by low AP scores in the 2-bottle test, exhibited decreases in AP among days but the magnitude of the change depended on test period: relatively large reductions in AP between Day 1 and the subsequent 2 days of the first 3-day test period, smaller decreases between days during Period 2, while there were no significant differences between days during Periods 3 and 4. Thus, the ability of the mice to adapt to changes in tube position improved with increasing experience with the test until asymptote was reached. Study of mice from a C57BL/6JXBALB/cCrgl intercross with a wide range of AP scores showed that learning in the 2-bottle test was not restricted to inbred animals. In this genetically heterogeneous group, learning was shown to be flexible according to an animal's idiosyncratic pattern of alcohol intake: mice characterized by low AP scores on the basis of their 15-day mean AP index exhibited decreases across Days and Periods similar to those shown by the BALB mice (who also had low alcohol consumption) but F(2) mice characterized by high overall AP scores exhibited increases in AP across Periods. CONCLUSIONS: Two-bottle AP scores are known to be affected by genetic influences and environmental variation before test administration. The present data provide evidence of learning within the 2-bottle test situation and this phenomenon may help understand the biobehavioral mechanisms underlying preference behavior.

Replication and discovery of musculoskeletal QTLs in LG/J and SM/J advanced intercross lines.

The genetics underlying variation in health-related musculoskeletal phenotypes can be investigated in a mouse model. Quantitative trait loci (QTLs) affecting musculoskeletal traits in the LG/J and SM/J strain lineage remain to be refined and corroborated. The aim of this study was to map muscle and bone traits in males (n = 506) of the 50th filial generation of advanced intercross lines (LG/SM AIL) derived from the two strains. Genetic contribution to variation in all musculoskeletal traits was confirmed; the SNP heritability of muscle mass ranged between 0.46 and 0.56; and the SNP heritability of tibia length was 0.40. We used two analytical software, GEMMA and QTLRel, to map the underlying QTLs. GEMMA required substantially less computation and recovered all the QTLs identified by QTLRel. Seven significant QTLs were identified for muscle weight (Chr 1, 7, 11, 12, 13, 15, and 16), and two for tibia length, (Chr 1 and 13). Each QTL explained 4-5% of phenotypic variation. One muscle and both bone loci replicated previous findings; the remaining six were novel. Positional candidates for the replicated QTLs were prioritized based on in silico analyses and gene expression in muscle tissue. In summary, we replicated existing QTLs and identified novel QTLs affecting muscle weight, and replicated bone length QTLs in LG/SM AIL males. Heritability estimates substantially exceed the cumulative effect of the QTLs, hence a richer genetic architecture contributing to muscle and bone variability could be uncovered with a larger sample size.

QTL analysis of trabecular bone in BXD F2 and RI mice.

UNLABELLED: A sample of 693 mice was used to identify regions of the mouse genome associated with trabecular bone architecture as measured using microCT. QTLs for bone in the proximal tibial metaphysis were identified on several chromosomes indicating regions containing genes that regulate properties of trabecular bone. INTRODUCTION: Age-related osteoporosis is a condition of major concern because of the morbidity and mortality associated with osteoporotic fractures in humans. Osteoporosis is characterized by reduced bone density, strength, and altered trabecular architecture, all of which are quantitative traits resulting from the actions of many genes working in concert with each other and the environment over the lifespan. microCT gives accurate measures of trabecular bone architecture providing phenotypic data related to bone volume and trabecular morphology. The primary objective of this research was to identify chromosomal regions called quantitative trait loci (QTLs) that contain genes influencing trabecular architecture as measured by microCT. MATERIALS AND METHODS: The study used crosses between C57BL/6J (B6) and DBA/2J (D2) as progenitor strains of a second filial (F2) generation (n = 141 males and 148 females) and 23 BXD recombinant inbred (RI) strains (n approximately 9 of each sex per strain). The proximal tibial metaphyses of the 200-day-old mice were analyzed by microCT to assess phenotypic traits characterizing trabecular bone, including bone volume fraction, trabecular connectivity, and quantitative measures of trabecular orientation and anisotropy. Heritabilities were calculated and QTLs were identified using composite interval mapping. RESULTS: A number of phenotypes were found to be highly heritable. Heritability values for measured phenotypes using RI strains ranged from 0.15 for degree of anisotropy in females to 0.51 for connectivity density in females and total volume in males. Significant and confirmed QTLs, with LOD scores 4.3 in the F2 cohort and 1.5 in the corresponding RI cohort were found on chromosomes 1 (43 cM), 5 (44 cM), 6 (20 cM), and 8 (49 cM). Other QTLs with LOD scores ranging from 2.8 to 6.9 in the F2 analyses were found on chromosomes 1, 5, 6, 8, 9, and 12. QTLs were identified using data sets comprised of both male and female quantitative traits, suggesting similar genetic action in both sexes, whereas others seemed to be associated exclusively with one sex or the other, suggesting the possibility of sex-dependent effects. CONCLUSIONS: Identification of the genes underlying these QTLs may lead to improvements in recognizing individuals most at risk for developing osteoporosis and in the design of new therapeutic interventions.

Anomaly of anatomical origin of soleus muscle: a mouse model.

In the laboratory mouse, the soleus muscle arises at the head of the fibula and inserts via the Achilles tendon on the tuber calcanei together with the gastrocnemius muscle. During routine dissection of mice from the BXD recombinant inbred (RI) strains, we found that the soleus often originated from the lateral epicondyle of the femur instead of the head of the fibula. This soleus femoral attachment anomaly (SFAA) changes the soleus from being a single-joint to a two-joint muscle. The incidence of SFAA was 45% in the BXD38 RI strain. Bilateral inspection indicated that SFAA may be present unilaterally or bilaterally within an individual mouse. We explored the effect of SFAA on muscle weight in mice with unilateral expression. The weight of SFAA soleii was significantly less (P < 0.01) than that of the soleii with normal attachment by 6% (females) and 14% (males). Similar anatomical anomalies of the soleus muscle have been noted in humans. The mouse model will provide the means to explore the physiological consequences and genetic basis for such anomalies.

'Return to home cage' as a reward for maze learning in young and old genetically heterogeneous mice.

Recent studies have shown that 'return to home cage' can serve as a reward for maze learning in adult male mice. The present study examined whether the same reward is an effective motivator of learning in young and old mice and included females in the study design. We tested 25- and 65-d-old HS mice and 85- and 800-d-old B6D2F2 mice in a Lashley III maze. Return to home cage motivated maze acquisition in all groups. Compared with 65-d-old HS mice, 25-d-olds acquired the maze more slowly, took longer to achieve the test criterion, and showed increased latency to reach the goal box. There was no difference between 85- and 800-d-old B6D2F2 mice in rate of acquisition. This reward procedure may reduce the potentially confounding effects of deprivation or aversive stimuli on maze performance and may be suitable as a motivational procedure for a wide range of subject groups.

Genome-wide association study of behavioral, physiological and gene expression traits in outbred CFW mice.

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.

Genome-wide association of multiple complex traits in outbred mice by ultra-low-coverage sequencing.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.

Quantitative trait loci analysis of structural and material skeletal phenotypes in C57BL/6J and DBA/2 second-generation and recombinant inbred mice.

UNLABELLED: QTL analyses identified several chromosomal regions influencing skeletal phenotypes of the femur and tibia in BXD F2 and BXD RI populations of mice. QTLs for skeletal traits co-located with each other and with correlated traits such as body weight and length, adipose mass, and serum alkaline phosphatase. INTRODUCTION: Past research has shown substantial genetic influence on bone quality, and the impact of reduced bone mass on our aging population has heightened the interest in skeletal genetic research. MATERIALS AND METHODS: Quantitative trait loci (QTL) analyses were performed on morphologic measures and structural and material properties of the femur and tibia in 200-day-old C57BL/6J x DBA/2 (BXD) F2 (second filial generation; n = 400) and BXD recombinant inbred (RI; n = 23 strains) populations of mice. Body weight, body length, adipose mass, and serum alkaline phosphatase were correlated phenotypes included in the analyses. RESULTS: Skeletal QTLs for morphologic bone measures such as length, width, cortical thickness, and cross-sectional area mapped to nearly every chromosome. QTLs for both structural properties (ultimate load, yield load, or stiffness) and material properties (stress and straincharacteristics and elastic modulus) mapped to chromosomes 4, 6, 9, 12, 13, 15, and 18. QTLs that were specific to structural properties were identified on chromosomes 1, 2, 3, 7, 8, and 17, and QTLs that were specific to skeletal material properties were identified on chromosomes 5, 11, 16, and 19. QTLs for body size (body weight, body length, and adipose mass) often mapped to the same chromosomal regions as those identified for skeletal traits, suggesting that several QTLs identified as influencing bone could be mediated through body size. CONCLUSION: New QTLs, not previously reported in the literature, were identified for structural and material properties and morphological measures of the mouse femur and tibia. Body weight and length, adipose mass, and serum alkaline phosphatase were correlated phenotypes that mapped in close proximity of skeletal chromosomal loci. The more specific measures of bone quality included in this investigation enhance our understanding of the functional significance of previously identified QTLs.