Identification of Genetic Variation using Massively Paralleled Sequencing This year, we sequenced the exomes of rhesus macaque subjects that were selected based on variation in temperament. One big advantage of using this approach is that DNA extracted whole blood can be used without the need for extracting DNA from the tissue of interest. Whole Exome Sequencing allows for the capture of large insertion/deletions and SNPs, and is best for Mendelian traits where variation is highly penetrant and either non-synonomous or frameshift. Drawbacks to WES include the inability to interrogate sequence sites that are not currently recognized as genes, control/regulatory sequences, structural variants such as translocations and inversions, splice-site variation, and exonic repeats. However, due to exome enrichment and the resulting deep sequencing coverage, the enhanced coding-region interrogation WES provides will allow for the sensitive detection of variants at those sites that are targeted. While there are no commercially available reagents for performing whole exome sequencing in nonhuman primates, a human whole exome platform is available. Given that there would likely be more purifying selection in coding regions and, therefore, less interspecific variation, we proposed using a human exome capture and sequencing kit for performing exome sequencing in rhesus macaques that differed in impulsivity and aggression. This approach was successful, and we identified many snps, including 391 missense variants, 23 stop codon snps, and 4 frameshift mutations. Of these, 58 variants were in genes in which variation is proposed to moderate addiction risk in humans. Whether these variants predict individual differences in alcohol drinking and related behaviors in rhesus macaques is currently under investigation. In certain instances, genetic variants that are functionally similar or orthologous to those that increase risk for human psychiatric disorders are maintained across primate species. We have identified several examples of this phenomenon in performing studies in rhesus macaques in order to model how genetic variation moderates risk for developing psychopathology. Some of these studies have suggested there to be convergent evolution or allelic variants being maintained by selection in both species. We may use naturally occurring genetic variation to look at not only within-species differences in vulnerability to behavioral disorders, but also to examine how genetic variation may alter traits in closely related species. Macaque species, all of which exhibit the same core elements of social organization and demographic structure, differ in characteristic patterns of stress reactivity, temperament and social interaction. We are currently performing whole exome sequencing a higher number of primate species, including the greater and lesser apes, and Old World monkeys (with an enriched sampling of Macaca) and the New World monkeys. One other area of interest for the study of genetic factors that contribute to addiction vulnerability is the genetics underlying domestication in animals.At its most basic, domestication is a suite of heritable traits affecting behavior. There are intriguing phenotypic commonalities among domesticates. Most important among these traits, and the only one common to all domesticates, is the ability to coexist with humans. The systems that likely permitted early domestication range from those involving fear and impulse control to those involving reward and sociality. Genetics contributing to such traits are likely to be relevant to human psychiatric illness and, in particular, the addictions. We have sequenced the genomes of domestic and European Wildcats, two species of felids that differ only in their behavior. Whether genetic variation that might underlie behavioral differences in these species is present is currently under investigation. Neurogenetics of Stress Response and Reward Sensitivity We have been studying how spontaneous genetic variation that influences reward sensitivity, and which could be adaptive in certain contexts, may also increase risk for alcohol related problems. In both humans and rhesus macaques, there are polymorphisms in the mu-opioid receptor gene (OPRM1) that influence affinity of the receptor for its endogenous ligand, -Endorphin. We and others have shown that these polymorphisms predict increased alcohol-induced euphoria and decreased stress response, suggesting them to have gain-of-function roles. We also have demonstrated it to relate to individual differences in response to natural rewards, as shown by measures of both reward bias in humans (and social attachment in both human and macaque. Recently, we have been screening this gene for variation across species in order to look for signs of selection and to determine whether functional variation that predicts species might be present. Of interest, we found a variant that produces a non-neutral amino acid substitution in the OPRM1 receptors ligand-binding domain (Asp12>Lys12). Among the Old World Monkey species that were screened (N = 17) this fixed difference was observed in all 5 species in the Cercopthecini tribe (which includes Patas monkeys and vervets, to name a few), but this allele was not observed outside of this tribe. Field observations of Vervet and Patas suggest they are reward-sensitive and more susceptible to stress compared to other OWMs. Our data suggest that an OPRM1 variant arose and went to fixation after the divergence of Cercopthecini from Papionini. They further support data showing that polymorphism at OPRM1 contributes to behavioral variation in primates. Dopamine neurotransmission underlies many reward-dependent and reinforcing processes. Tandem repeats in the third exon of the dopamine receptor D4 gene (DRD4) exist across a variety of animal species (humans, vervets, dogs, dolphins, bears, raccoons, horses, and chimpanzees). Some species, including macaque species, exhibit variation in the number of repeats present, with various alleles differing in frequencies across species. In humans, the DRD4 7-repeat allele (7R) reduces efficacy of the receptor and is linked with traits such as sensation seeking. This has been replicated in vervet monkeys, in which a DRD4 length variant was also observed. Of interest, in the dog, which has been subject to intense artificial selection, a repeat polymorphism predicts social impulsivity and activity-impulsivity endophenotypes. We recently identified 4R, 5R, 6R, and 7R alleles in a colony of rhesus macaques. Compared to the ancestral allele (4R), the loss-of-function 7R allele predicted increased incidence of physical aggression toward an unknown age- and sex-matched conspecific. One system of potential relevance to the addictions via its effects on anxiety and reward is the oxytocin system. We screened the rhesus OXTR gene and identified 17 SNPs, including 4 non-synonymous SNPs in the first exon for the rhesus Oxytocin receptor gene (OXYR). This is a location at which there is high interspecific conservation yet high frequencies of non-synonymous SNPs in humans as well. Among these, some of them are conserved across primate taxa, suggesting that they have been maintained by selection. We also found there to be ancient polymorphisms maintained in humans and the existence of alternative haplotypes within this region indicating that human variation is being maintained by balancing selection. Oxytocin is a neuropeptide that produces affiliative, amnesic and anxiolytic affects. Given its roles in some of these processes, it has been proposed as a potential therapeutic agent for the treatment of anxiety and stress-related disorders, including the addictions.Functional variation that influences oxytocin system function may also be a good candidate for performing GxE studies.