Genome-wide association mapping of ethanol sensitivity in the Diversity Outbred mouse population.

BACKGROUND: A strong predictor for the development of alcohol use disorder (AUD) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled in part by genetic factors. Mice offer a powerful tool to elucidate the genetic basis of behavioral and physiological traits relevant to AUD, but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations make it possible to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification. METHODS: We have taken advantage of the Diversity Outbred (DO) mouse population to identify and precisely map quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 798 male J:DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high-density MegaMUGA and GigaMUGA to obtain genotypes ranging from 77,808 to 143,259 SNPs. We also performed RNA sequencing in striatum to map expression QTLs and identify gene expression-trait correlations. We then applied a systems genetic strategy to identify narrow QTLs and construct the network of correlations that exists between DNA sequence, gene expression values, and ethanol-related phenotypes to prioritize our list of positional candidate genes. RESULTS: We observed large amounts of phenotypic variation with the DO population and identified suggestive and significant QTLs associated with ethanol sensitivity on chromosomes 1, 2, and 16. The implicated regions were narrow (4.5-6.9 Mb in size) and each QTL explained ~4-5% of the variance. CONCLUSIONS: Our results can be used to identify alleles that contribute to AUD in humans, elucidate causative biological mechanisms, or assist in the development of novel therapeutic interventions.

Analysis of independent cohorts of outbred CFW mice reveals novel loci for behavioral and physiological traits and identifies factors determining reproducibility.

Combining samples for genetic association is standard practice in human genetic analysis of complex traits, but is rarely undertaken in rodent genetics. Here, using 23 phenotypes and genotypes from two independent laboratories, we obtained a sample size of 3076 commercially available outbred mice and identified 70 loci, more than double the number of loci identified in the component studies. Fine-mapping in the combined sample reduced the number of likely causal variants, with a median reduction in set size of 51%, and indicated novel gene associations, including Pnpo, Ttll6, and GM11545 with bone mineral density, and Psmb9 with weight. However, replication at a nominal threshold of 0.05 between the two component studies was low, with less than one-third of loci identified in one study replicated in the second. In addition to overestimates in the effect size in the discovery sample (Winner's Curse), we also found that heterogeneity between studies explained the poor replication, but the contribution of these two factors varied among traits. Leveraging these observations, we integrated information about replication rates, study-specific heterogeneity, and Winner's Curse corrected estimates of power to assign variants to one of four confidence levels. Our approach addresses concerns about reproducibility and demonstrates how to obtain robust results from mapping complex traits in any genome-wide association study.

Integration of evidence across human and model organism studies: A meeting report.

The National Institute on Drug Abuse and Joint Institute for Biological Sciences at the Oak Ridge National Laboratory hosted a meeting attended by a diverse group of scientists with expertise in substance use disorders (SUDs), computational biology, and FAIR (Findability, Accessibility, Interoperability, and Reusability) data sharing. The meeting's objective was to discuss and evaluate better strategies to integrate genetic, epigenetic, and 'omics data across human and model organisms to achieve deeper mechanistic insight into SUDs. Specific topics were to (a) evaluate the current state of substance use genetics and genomics research and fundamental gaps, (b) identify opportunities and challenges of integration and sharing across species and data types, (c) identify current tools and resources for integration of genetic, epigenetic, and phenotypic data, (d) discuss steps and impediment related to data integration, and (e) outline future steps to support more effective collaboration-particularly between animal model research communities and human genetics and clinical research teams. This review summarizes key facets of this catalytic discussion with a focus on new opportunities and gaps in resources and knowledge on SUDs.

Alcohol Sensitivity as an Endophenotype of Alcohol Use Disorder: Exploring Its Translational Utility between Rodents and Humans.

Alcohol use disorder (AUD) is a complex, chronic, relapsing disorder with multiple interacting genetic and environmental influences. Numerous studies have verified the influence of genetics on AUD, yet the underlying biological pathways remain unknown. One strategy to interrogate complex diseases is the use of endophenotypes, which deconstruct current diagnostic categories into component traits that may be more amenable to genetic research. In this review, we explore how an endophenotype such as sensitivity to alcohol can be used in conjunction with rodent models to provide mechanistic insights into AUD. We evaluate three alcohol sensitivity endophenotypes (stimulation, intoxication, and aversion) for their translatability across human and rodent research by examining the underlying neurobiology and its relationship to consumption and AUD. We show examples in which results gleaned from rodents are successfully integrated with information from human studies to gain insight in the genetic underpinnings of AUD and AUD-related endophenotypes. Finally, we identify areas for future translational research that could greatly expand our knowledge of the biological and molecular aspects of the transition to AUD with the broad hope of finding better ways to treat this devastating disorder.

Genome-wide Associations Reveal Human-Mouse Genetic Convergence and Modifiers of Myogenesis, CPNE1 and STC2.

Muscle bulk in adult healthy humans is highly variable even after height, age, and sex are accounted for. Low muscle mass, due to fewer and/or smaller constituent muscle fibers, would exacerbate the impact of muscle loss occurring in aging or disease. Genetic variability substantially influences muscle mass differences, but causative genes remain largely unknown. In a genome-wide association study (GWAS) on appendicular lean mass (ALM) in a population of 85,750 middle-aged (aged 38-49 years) individuals from the UK Biobank (UKB), we found 182 loci associated with ALM (p < 5 × 10-8). We replicated associations for 78% of these loci (p < 5 × 10-8) with ALM in a population of 181,862 elderly (aged 60-74 years) individuals from UKB. We also conducted a GWAS on hindlimb skeletal muscle mass of 1,867 mice from an advanced intercross between two inbred strains (LG/J and SM/J); this GWAS identified 23 quantitative trait loci. Thirty-eight positional candidates distributed across five loci overlapped between the two species. In vitro studies of positional candidates confirmed CPNE1 and STC2 as modifiers of myogenesis. Collectively, these findings shed light on the genetics of muscle mass variability in humans and identify targets for the development of interventions for treatment of muscle loss. The overlapping results between humans and the mouse model GWAS point to shared genetic mechanisms across species.

Genome-wide association for testis weight in the diversity outbred mouse population.

Testis weight is a genetically mediated trait associated with reproductive efficiency across numerous species. We sought to evaluate the genetically diverse, highly recombinant Diversity Outbred (DO) mouse population as a tool to identify and map quantitative trait loci (QTLs) associated with testis weight. Testis weights were recorded for 502 male DO mice and the mice were genotyped on the GIGAMuga array at ~ 143,000 SNPs. We performed a genome-wide association analysis and identified one significant and two suggestive QTLs associated with testis weight. Using bioinformatic approaches, we developed a list of candidate genes and identified those with known roles in testicular size and development. Candidates of particular interest include the RNA demethylase gene Alkbh5, the cyclin-dependent kinase inhibitor gene Cdkn2c, the dynein axonemal heavy chain gene Dnah11, the phospholipase D gene Pld6, the trans-acting transcription factor gene Sp4, and the spermatogenesis-associated gene Spata6, each of which has a human ortholog. Our results demonstrate the utility of DO mice in high-resolution genetic mapping of complex traits, enabling us to identify developmentally important genes in adult mice. Understanding how genetic variation in these genes influence testis weight could aid in the understanding of mechanisms of mammalian reproductive function.

Systems Genetic Analysis in GeneNetwork.org.

Genome-wide association studies (GWAS) have emerged as a powerful tool to identify alleles and molecular pathways that influence susceptibility to psychiatric disorders and other diseases. Forward genetics using mouse mapping populations allows for a complementary approach that provides rigorous genetic and environmental control. In this unit, we describe techniques and tools that reduce the technical burden traditionally associated with genetic mapping in mice and enhance their translational utility to human psychiatric disorders. We provide guidance on choosing the appropriate mapping population, discuss the importance of phenotype, and offer detailed instructions on using the Web-based resource GeneNetwork to aid neuroscientists in better understanding the mechanisms through which genes influence behavior. We believe that the continued development of mouse mapping populations, genetic tools, bioinformatics resources, and statistical methodologies should remain a parallel strategy by which to investigate the genetic and environmental underpinnings of psychiatric disorders and other diseases in humans. © 2017 by John Wiley & Sons, Inc.

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.

A dendritic organization of lateral amygdala neurons in fear susceptible and resistant mice.

Subtle differences in neuronal microanatomy may be coded in individuals with genetic susceptibility for neuropsychiatric disorders. Genetic susceptibility is a significant risk factor in the development of anxiety disorders, including post-traumatic stress disorder (PTSD). Pavlovian fear conditioning has been proposed to model key aspects of PTSD. According to this theory, PTSD begins with the formation of a traumatic memory which connects relevant environmental stimuli to significant threats to life. The lateral amygdala (LA) is considered to be a key network hub for the establishment of Pavlovian fear conditioning. Substantial research has also linked the LA to PTSD. Here we used a genetic mouse model of fear susceptibility (F-S) and resistance (F-R) to investigate the dendritic and spine structure of principal neurons located in the LA. F-S and F-R lines were bi-directionally selected based on divergent levels of contextual and cued conditioned freezing in response to fear-evoking footshocks. We examined LA principal neuron dendritic and spine morphology in the offspring of experimentally naive F-S and F-R mice. We found differences in the spatial distribution of dendritic branch points across the length of the dendrite tree, with a significant increase in branch points at more distal locations in the F-S compared with F-R line. These results suggest a genetic predisposition toward differences in fear memory strength associated with a dendritic branch point organization of principal neurons in the LA. These micro-anatomical differences in neuron structure in a genetic mouse model of fear susceptibility and resistance provide important insights into the cellular mechanisms of pathophysiology underlying genetic predispositions to anxiety and PTSD.

Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders.

Mapping of Craniofacial Traits in Outbred Mice Identifies Major Developmental Genes Involved in Shape Determination.

The vertebrate cranium is a prime example of the high evolvability of complex traits. While evidence of genes and developmental pathways underlying craniofacial shape determination is accumulating, we are still far from understanding how such variation at the genetic level is translated into craniofacial shape variation. Here we used 3D geometric morphometrics to map genes involved in shape determination in a population of outbred mice (Carworth Farms White, or CFW). We defined shape traits via principal component analysis of 3D skull and mandible measurements. We mapped genetic loci associated with shape traits at ~80,000 candidate single nucleotide polymorphisms in ~700 male mice. We found that craniofacial shape and size are highly heritable, polygenic traits. Despite the polygenic nature of the traits, we identified 17 loci that explain variation in skull shape, and 8 loci associated with variation in mandible shape. Together, the associated variants account for 11.4% of skull and 4.4% of mandible shape variation, however, the total additive genetic variance associated with phenotypic variation was estimated in ~45%. Candidate genes within the associated loci have known roles in craniofacial development; this includes 6 transcription factors and several regulators of bone developmental pathways. One gene, Mn1, has an unusually large effect on shape variation in our study. A knockout of this gene was previously shown to affect negatively the development of membranous bones of the cranial skeleton, and evolutionary analysis shows that the gene has arisen at the base of the bony vertebrates (Eutelostomi), where the ossified head first appeared. Therefore, Mn1 emerges as a key gene for both skull formation and within-population shape variation. Our study shows that it is possible to identify important developmental genes through genome-wide mapping of high-dimensional shape features in an outbred population.

High-resolution genetic mapping of complex traits from a combined analysis of F2 and advanced intercross mice.

Genetic influences on anxiety disorders are well documented; however, the specific genes underlying these disorders remain largely unknown. To identify quantitative trait loci (QTL) for conditioned fear and open field behavior, we used an F2 intercross (n = 490) and a 34th-generation advanced intercross line (AIL) (n = 687) from the LG/J and SM/J inbred mouse strains. The F2 provided strong support for several QTL, but within wide chromosomal regions. The AIL yielded much narrower QTL, but the results were less statistically significant, despite the larger number of mice. Simultaneous analysis of the F2 and AIL provided strong support for QTL and within much narrower regions. We used a linear mixed-model approach, implemented in the program QTLRel, to correct for possible confounding due to familial relatedness. Because we recorded the full pedigree, we were able to empirically compare two ways of accounting for relatedness: using the pedigree to estimate kinship coefficients and using genetic marker estimates of "realized relatedness." QTL mapping using the marker-based estimates yielded more support for QTL, but only when we excluded the chromosome being scanned from the marker-based relatedness estimates. We used a forward model selection procedure to assess evidence for multiple QTL on the same chromosome. Overall, we identified 12 significant loci for behaviors in the open field and 12 significant loci for conditioned fear behaviors. Our approach implements multiple advances to integrated analysis of F2 and AILs that provide both power and precision, while maintaining the advantages of using only two inbred strains to map QTL.

Mice selectively bred for High and Low fear behavior show differences in the number of pMAPK (p44/42 ERK) expressing neurons in lateral amygdala following Pavlovian fear conditioning.

Individual variability in the acquisition, consolidation and extinction of conditioned fear potentially contributes to the development of fear pathology including posttraumatic stress disorder (PTSD). Pavlovian fear conditioning is a key tool for the study of fundamental aspects of fear learning. Here, we used a selected mouse line of High and Low Pavlovian conditioned fear created from an advanced intercrossed line (AIL) in order to begin to identify the cellular basis of phenotypic divergence in Pavlovian fear conditioning. We investigated whether phosphorylated MAPK (p44/42 ERK/MAPK), a protein kinase required in the amygdala for the acquisition and consolidation of Pavlovian fear memory, is differentially expressed following Pavlovian fear learning in the High and Low fear lines. We found that following Pavlovian auditory fear conditioning, High and Low line mice differ in the number of pMAPK-expressing neurons in the dorsal sub nucleus of the lateral amygdala (LAd). In contrast, this difference was not detected in the ventral medial (LAvm) or ventral lateral (LAvl) amygdala sub nuclei or in control animals. We propose that this apparent increase in plasticity at a known locus of fear memory acquisition and consolidation relates to intrinsic differences between the two fear phenotypes. These data provide important insights into the micronetwork mechanisms encoding phenotypic differences in fear. Understanding the circuit level cellular and molecular mechanisms that underlie individual variability in fear learning is critical for the development of effective treatment of fear-related illnesses such as PTSD.

Rats are the smart choice: Rationale for a renewed focus on rats in behavioral genetics.

Due in part to their rich behavioral repertoire rats have been widely used in behavioral studies of drug abuse-related traits for decades. However, the mouse became the model of choice for researchers exploring the genetic underpinnings of addiction after the first mouse study was published demonstrating the capability of engineering the mouse genome through embryonic stem cell technology. The sequencing of the mouse genome and more recent re-sequencing of numerous inbred mouse strains have further cemented the status of mice as the premier mammalian organism for genetic studies. As a result, many of the behavioral paradigms initially developed and optimized for rats have been adapted to mice. However, numerous complex and interesting drug abuse-related behaviors that can be studied in rats are very difficult or impossible to adapt for use in mice, impeding the genetic dissection of those traits. Now, technological advances have removed many of the historical limitations of genetic studies in rats. For instance, the rat genome has been sequenced and many inbred rat strains are now being re-sequenced and outbred rat stocks are being used to fine-map QTLs. In addition, it is now possible to create "knockout" rats using zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and related techniques. Thus, rats can now be used to perform quantitative genetic studies of sophisticated behaviors that have been difficult or impossible to study in mice. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Variation in the form of Pavlovian conditioned approach behavior among outbred male Sprague-Dawley rats from different vendors and colonies: sign-tracking vs. goal-tracking.

Even when trained under exactly the same conditions outbred male Sprague-Dawley (SD) rats vary in the form of the Pavlovian conditioned approach response (CR) they acquire. The form of the CR (i.e. sign-tracking vs. goal-tracking) predicts to what degree individuals attribute incentive salience to cues associated with food or drugs. However, we have noticed variation in the incidence of these two phenotypes in rats obtained from different vendors. In this study, we quantified sign- and goal-tracking behavior in a reasonably large sample of SD rats obtained from two vendors (Harlan or Charles River), as well as from individual colonies operated by both vendors. Our sample of rats acquired from Harlan had, on average, more sign-trackers than goal-trackers, and vice versa for our sample of rats acquired from Charles River. Furthermore, there were significant differences among colonies of the same vendor. Although it is impossible to rule out environmental variables, SD rats at different vendors and barriers may have reduced phenotypic heterogeneity as a result of genetic variables, such as random genetic drift or population bottlenecks. Consistent with this hypothesis, we identified marked population structure among colonies from Harlan. Therefore, despite sharing the same name, investigators should be aware that important genetic and phenotypic differences exist among SD rats from different vendors or even from different colonies of the same vendor. If used judiciously this can be an asset to experimental design, but it can also be a pitfall for those unaware of the issue.

Practical considerations regarding the use of genotype and pedigree data to model relatedness in the context of genome-wide association studies.

Genome-wide association studies of complex traits often are complicated by relatedness among individuals. Ignoring or inappropriately accounting for relatedness often results in inflated type I error rates. Either genotype or pedigree data can be used to estimate relatedness for use in mixed-models when undertaking quantitative trait locus mapping. We performed simulations to investigate methods for controlling type I error and optimizing power considering both full and partial pedigrees and, similarly, both sparse and dense marker coverage; we also examined real data sets. (1) When marker density was low, estimating relatedness by genotype data alone failed to control the type I error rate; (2) this was resolved by combining both genotype and pedigree data. (3) When sufficiently dense marker data were used to estimate relatedness, type I error was well controlled and power increased; however, (4) this was only true when the relatedness was estimated using genotype data that excluded genotypes on the chromosome currently being scanned for a quantitative trait locus.

Traits of fear resistance and susceptibility in an advanced intercross line.

Genetic variability in the strength and precision of fear memory is hypothesised to contribute to the etiology of anxiety disorders, including post-traumatic stress disorder. We generated fear-susceptible (F-S) or fear-resistant (F-R) phenotypes from an F8 advanced intercross line (AIL) of C57BL/6J and DBA/2J inbred mice by selective breeding. We identified specific traits underlying individual variability in Pavlovian conditioned fear learning and memory. Offspring of selected lines differed in the acquisition of conditioned fear. Furthermore, F-S mice showed greater cued fear memory and generalised fear in response to a novel context than F-R mice. F-S mice showed greater basal corticosterone levels and hypothalamic corticotrophin-releasing hormone (CRH) mRNA levels than F-R mice, consistent with higher hypothalamic-pituitary-adrenal (HPA) axis drive. Hypothalamic mineralocorticoid receptor and CRH receptor 1 mRNA levels were decreased in F-S mice as compared with F-R mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was used to investigate basal levels of brain activity. MEMRI identified a pattern of increased brain activity in F-S mice that was driven primarily by the hippocampus and amygdala, indicating excessive limbic circuit activity in F-S mice as compared with F-R mice. Thus, selection pressure applied to the AIL population leads to the accumulation of heritable trait-relevant characteristics within each line, whereas non-behaviorally relevant traits remain distributed. Selected lines therefore minimise false-positive associations between behavioral phenotypes and physiology. We demonstrate that intrinsic differences in HPA axis function and limbic excitability contribute to phenotypic differences in the acquisition and consolidation of associative fear memory. Identification of system-wide traits predisposing to variability in fear memory may help in the direction of more targeted and efficacious treatments for fear-related pathology.

Expression pattern of the cannabinoid receptor genes in the frontal cortex of mood disorder patients and mice selectively bred for high and low fear.

Although the endocannabinoid system (ECS) has been implicated in brain development and various psychiatric disorders, precise mechanisms of the ECS on mood and anxiety disorders remain unclear. Here, we have investigated developmental and disease-related expression pattern of the cannabinoid receptor 1 (CB1) and the cannabinoid receptor 2 (CB2) genes in the dorsolateral prefrontal cortex (PFC) of humans. Using mice selectively bred for high and low fear, we further investigated potential association between fear memory and the cannabinoid receptor expression in the brain. The CB1, not the CB2, mRNA levels in the PFC gradually decrease during postnatal development ranging in age from birth to 50 years (r2 > 0.6 & adj. p < 0.05). The CB1 levels in the PFC of major depression patients were higher when compared to the age-matched controls (adj. p < 0.05). In mice, the CB1, not the CB2, levels in the PFC were positively correlated with freezing behavior in classical fear conditioning (p < 0.05). These results suggest that the CB1 in the PFC may play a significant role in regulating mood and anxiety symptoms. Our study demonstrates the advantage of utilizing data from postmortem brain tissue and a mouse model of fear to enhance our understanding of the role of the cannabinoid receptors in mood and anxiety disorders.

QTLs for murine red blood cell parameters in LG/J and SM/J F(2) and advanced intercross lines.

Red blood cells are essential for oxygen transport and other physiologic processes. Red cell characteristics are typically determined by complete blood counts which measure parameters such as hemoglobin levels and mean corpuscular volumes; these parameters reflect the quality and quantity of red cells in the circulation at any particular moment. To identify the genetic determinants of red cell parameters, we performed genome-wide association analysis on LG/J×SM/J F2 and F34 advanced intercross lines using single nucleotide polymorphism genotyping and a novel algorithm for mapping in the combined populations. We identified significant quantitative trait loci for red cell parameters on chromosomes 6, 7, 8, 10, 12, and 17; our use of advanced intercross lines reduced the quantitative trait loci interval width from 1.6- to 9.4-fold. Using the genomic sequences of LG/J and SM/J mice, we identified nonsynonymous coding single nucleotide polymorphisms in candidate genes residing within quantitative trait loci and performed sequence alignments and molecular modeling to gauge the potential impact of amino acid substitutions. These results should aid in the identification of genes critical for red cell physiology and metabolism and demonstrate the utility of advanced intercross lines in uncovering genetic determinants of inherited traits.