Addiction to smoked tobacco and its many comorbid diseases – from cancer to COPD – have a massive global impact. Although we have deep insights into brain circuits that influence addiction, we understand much less about the genetic variants responsible for the high level of individual variability in the risk for addiction to smoked tobacco and its principal psychoactive agent, nicotine. A large fraction of the vulnerability to initially smoke cigarettes in adolescence and to relapse in adults is due to heritable genetic differences. In response to NIDA PAR-21-244, this U01 is focused on discovering and understanding these complex genetic and molecular factors, using a powerful new resource – the hybrid rat diversity panel (HRDP). The HRDP is unique in that it integrates: 1. A very high level of genetic diversity similar to that of admixed human populations; 2. Ways to control and change drug exposures and to systematically study gene-by-environment and gene-by-drug inter- actions; 3. Ways to integrate a full quantitative "addictome" of genetic, genomic, and molecular data that can be used to promote the mechanistic development of Precision Medicine. The HRDP consists of 91 different, fully isogenic rats, both inbred and F1 hybrids (used for diallel crosses): all are "open access" and available to study facets of addiction. We have 3 aims: Aim 1. Beginning in adolescence, we will collect behavioral data from the HRDP using our established limited access operant model of nicotine intravenous self-administration (IVSA), which will be followed by an extended access model. By juxtaposing these two models, we emulate the initial phase of limited, episodic smoking during adolescence, and the subsequent persistent, habitual smoking, which leads to nicotine dependence. Our experimental design is sex-balanced and interleaved to minimize batch effects; we quantify nicotine intake, motivation for nicotine, withdrawal severity, prolongation of seeking despite lack of reinforcement, and cue-induced reinstatement of drug seeking; Aim 2. We generate telomere-to-telomere (T2T) genome assemblies of key strains of the HRDP and construct a rat pangenome. We use the sequencing methods established for the human T2T assembly, based on the highly accurate Pacific Biosciences (PacBio) HiFi long-read (20kb) and Oxford Nanopore (ONT) ultralong (>100 kb) data; Aim 3. We use the pangenome for improved genotyping and QTL mapping. Human genomic studies have shown that pangenomes reduce genomic "dark matter" – greatly improving genotyping. We will map gene loci for each nicotine-dependent behavior and associated phenome networks and evaluate the translational relevance of candidate genes by comparison to hu- man GWAS. Combining rat models of human smoking, advanced long read genome sequencing, T2T assemblies and pangenomics, we will accurately define high impact genetic variants and molecular networks associated with nicotine motivated behavior that models the key stages of human smoking.

Risk of smoking cigarettes depends on interactions between environmental factors, age, and genetics. This project uses advanced long-read genome sequencing and unbiased pangenomic analysis to fully identify genetic variants in the hybrid rat diversity panel. Combined with nicotine self-administration data from this panel, our ad- vanced systems genetics approach will define causal linkages between DNA variants and key nicotine-dependent behaviors including initial acquisition and motivation to take nicotine, withdrawal from nicotine dependence, and nicotine seeking under conditions that model aspects of human relapse to smoking.