Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges—along with potential solutions—behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F₁ hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs.

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.

Cannabis sativa L. (2n = 2x = 20) is a widely recognized species within the Cannabaceae family. Despite its utilization for medicinal, recreational, and industrial purposes, alongside its extensive historical background, the number of genetic and biotechnological studies of this plant species has decreased due to legal ramifications and prohibition campaigns associated with its use and cultivation. For many years, the development of novel varieties has been pursued solely by cultivators, as domestic growers have transitioned their work from cultivation to breeding Cannabis lineages. Recently, the application of genomics has facilitated a surge in methodologies aimed at marker-assisted selection, germplasm management, genetic differentiation, authentication of cultivated varieties or cultivars, and forensic applications such as safeguarding intellectual property rights. Nevertheless, the utilization of molecular markers for the advancement of commercial varieties through marker-assisted breeding (MAB) frameworks remains rare. This investigation was designed to evaluate a previously established informative microsatellite (SSR) array for the genotyping of drug-type Cannabis sativa cultivars derived from seeds of European origin. A total of 171 samples from 20 varieties were collected from European distributors and analyzed for genetic uniformity and population structure. The results were then compared with previously analyzed hemp samples and drug-type samples of Canadian origin, revealing the identification capabilities of our SSR genotyping method.