Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments

Cannabis sativa (2n = 2x = 20) is a popular species belonging to the Cannabaceae family. Despite its use for medical, recreational, and industrial purposes as well as its long history, the genetic research on this species is in its infancy due to the legal implications and the prohibition campaigns. The recent legalization of Cannabis in many countries along with the use of genomics boosted the approaches aimed at marker-assisted selection, germplasm management, genetic discrimination, and authentication of cultivars. Nonetheless, the exploitation of molecular markers for the development of commercial varieties through marker-assisted breeding schemes is still rare. The present study aimed to develop an informative panel of simple sequence repeat markers to be used for the genotyping of high breeding value C. sativa lines. Starting from 41 nuclear SSR designated by in silico analyses, we selected 20 highly polymorphic and discriminant loci that were tested in 104 individuals belonging to 11 distinct hemp varieties. The selected markers were successful in accessing homozygosity, genetic uniformity, and genetic variation within and among varieties. Population structure analysis identified eight genetic groups, clustering individuals based on sexual behaviors (dioecious and monoecious) and geographical origins. Overall, this study provides important tools for the genetic characterization, authentication, conservation of biodiversity, genetic improvement and traceability of this increasingly important plant species.