Hemp (Cannabis sativa L.), which can be legally grown in the U.S. since 2018, is amulti-use crop producing fiber, seed, oil, and/or essential oils. A better understanding of the
genetic basis of major traits will enable plant breeders to improve the crop more effectively. One
trait of importance to hemp producers is crop maturity. Hemp is naturally a short-day plant,
flowering in autumn when photoperiod declines below 12-13 hours. Day neutral types of hemp
will flower at roughly the same time regardless of photoperiod, making this trait desirable for
many production reasons. In this experiment, we hybridized day neutral and day sensitive hemp
germplasm to locate the day neutral trait in the hemp genome. We conducted a multi-year, multi-
environment experiment in which we scored individual plants on a ‘0’ to ‘5’ scale based on plant
maturity and computed the average growing degree days at which each plant scored ‘3,’ the
beginning of flowering. We also obtained the day length at this time. In parallel, we obtained
over 50,000 single nucleotide polymorphism (SNP) from genome sequencing data on 1507
individual plants. We used the phenotype and genotype data to conduct a genome wide
association study using a mixed linear model. From our analysis we identified 54 SNPs were
associated with our two traits, which shared the five most significant SNPs on Chromosome 2.
Within a 21Mb region of our shared significant SNP hits we identified a gene: flowering locus K
homology domain (FLK), a known down regulator of CONSTANS. Most plants we evaluated
derived from paired crosses between divergent germplasm, resulting in large disequilibrium which resulted in a broad chromosomal region in which the day neutral flowering regulatory gene likely lies. While more work is needed, this experiment identifies the likely region, which controls day neutral flowering in hemp, a first step toward developing genetic markers to facilitate the incorporation of this trait into other germplasm.