Quantitative trait loci controlling agronomic and biochemical traits in Cannabis sativa

Understanding the genetic basis of complex traits is a fundamental goal of evolutionary genetics. Yet, the genetics controlling complex traits in many important species such as hemp (Cannabis sativa) remain poorly investigated. Because hemp’s change in legal status with the 2014 and 2018 U.S. Federal Farm Bills, interest in the genetics controlling its numerous agriculturally important traits has steadily increased. To better understand the genetics of agriculturally important traits in hemp, we developed an F₂ population by crossing two phenotypically distinct hemp cultivars (Carmagnola and USO31). Using whole-genome sequencing, we mapped quantitative trait loci (QTL) associated with variation in numerous agronomic and biochemical traits. A total of 69 loci associated with agronomic (34) and biochemical (35) trait variation were identified. We found that most QTL co-localized, suggesting that the phenotypic distinctions between Carmagnola and USO31 are largely controlled by a small number of loci. We identified TINY and olivetol synthase as candidate genes underlying co-localized QTL clusters for agronomic and biochemical traits, respectively. We functionally validated the olivetol synthase candidate by expressing the alleles in yeast. Gas chromatography-mass spectrometry assays of extracts from these yeast colonies suggest that the USO31 olivetol synthase is functionally less active and potentially explains why USO31 produces lower cannabinoids compared to Carmagnola. Overall, our results help modernize the genomic understanding of complex traits in hemp.