The emergence of a thriving hemp industry in the U.S. will depend on the breeding of high-yielding regionally adapted cultivars. Despite the latest research efforts, little is known regarding the genetic basis of important agronomic traits in hemp. The objective of this research was to identify and characterize genomic regions associated with seed morphology and quality traits. F 2 mapping populations were developed by crossing hemp germplasm bred or cultivated for cannabinoids (‘FL 58’ × ‘TJ’s CBD’), grain (GVA- H-20-1179 × ‘Picolo’), or fiber (‘Si-1’ × GVA-H-21-1003) market classes. These populations were investigated due to their variation in seed size and seed crude protein. The cannabinoid, grain, and fiber populations were grown and seed was harvested in 2021, 2022, and 2023, respectively. Harvested seeds were phenotyped for thousand seed weight (TSW) and crude protein content predicted by near-infrared (NIR) spectroscopy. The high-cannabinoid population was genotyped using an Illumina array, while the fiber and grain populations were genotyped using an Agilent SureSelect Custom Target Enrichment Probe Set. Marker-associated sequences were aligned to the CBDRx v.2.0 reference genome to align the physical and genetic maps. The TSW and protein content in the cannabinoid population ranged from 9.62 to 23.93 g and 19.25 to 31.89 %, respectively. In contrast, the TSW of the fiber and grain populations ranged from 7.34 to 45.17 g and 8.73 to 31.42 g, respectively. Numerous quantitative trait loci (QTL) of varying effect sizes were identified genome-wide. Notably, in the high- cannabinoid population, major and minor effect QTL for TSW were detected on Chr01 corresponding to 642 kb and 5.56 Mb genetic regions, respectively. Our results in the cannabinoid population highlight the importance of developing more than one F 2 mapping population in a given cross to capture the effect of more alleles due to high heterozygosity in hemp and evaluating distinct pedigrees to sample additional alleles in diverse genetic backgrounds. Narrowing the region around or identifying candidate genes will allow the development of high-throughput molecular markers for beneficial alleles across mapping pedigrees. These findings will accelerate hemp breeding programs through the implementation of marker-assisted selection for high-yielding and high-quality hemp cultivars for grain production.
