Genetic structure of Cannabis sativa var. indica cultivars based on genomic SSR (gSSR) markers: Implications for breeding and germplasm management

Abstract

Cannabis sativa L. is cultivated for its fiber or seeds (var. sativa; hemp), or for its high content in cannabinoids (var. indica; marijuana). Knowledge of the genetic structure of C. sativa var. indica is important for selection and breeding of cultivars with medicinal interest. We used six genomic SSRs (gSSRs) for genotyping 154 individual plants of 20 cultivars of C. sativa var. indica, plus two cultivars of C. sativa var. sativa. A very high polymorphism was observed, with an average of 17 alleles and 23.8 genotypes per locus. Expected (He) and observed (Ho) heterozygosities were high, with average values of 0.753 and 0.429, respectively. In some cultivars He and Ho presented similar values, while in others He was considerably higher than Ho suggesting that consanguinity and fixation had taken place during its development. In addition, some cultivars had a reduced number of alleles per locus (in some cases only two) indicating that a genetic bottleneck had taken place during its development. Gene flow (Nm) between both botanical varieties was high, with Nm=1.736. The molecular analysis of variance (AMOVA) revealed that only 31.94% of the molecular variation observed was caused by differences among cultivars, while the variation among plants of the same cultivar was of 37.11%, and within individual variation, due to heterozygosity, was of 30.96%. This indicates that a large variation exists within cultivars, which can be exploited for selection, but also complicates germplasm management and regeneration. The population structure analysis identified 14 genetic clusters, with most individuals of a single cultivar clustering together. This analysis, together with UPGMA cluster analysis shows that the two C. sativa var. sativa cultivars studied are differentiated from C. sativa var. indica, and that some cultivars of C. sativa var. indica seem to represent different selections from a common original cultivar. Our results represent the first comprehensive study of intra-3 varietal diversity in C. sativa var. indica and provide information of relevance for selection, breeding, and germplasm conservation, as well as for forensic studies in this crop.

Research grade marijuana supplied by the National Institute on Drug Abuse is genetically divergent from commercially availableCannabis

“…The first group contained a majority of hemp (88 - 100%, depending on analysis) and both NIDA samples (100%), while the second group contained a majority of drug-type samples (82 - 95%). These results contribute to the growing consensus that hemp and drug-type Cannabis can be consistently differentiated 7,10–12,48–51 . To our knowledge, this is the first genetic study to include research grade marijuana from NIDA, and its placement with hemp samples was unexpected.…”

“…Although Cannabis sativa is the only described species in the genus Cannabis (Cannabaceae), there are several commonly described subcategories of Cannabis that are widely recognized. There are two primary Cannabis usage groups, which are well supported by genetic analyses 7,10–12 : Hemp is defined by a lack of THC (< 0.3% THC in the U.S.), and marijuana or drug-types have moderate to high THC concentrations (> 0.3% THC in the U.S.). Hemp-type Cannabis tends to have higher concentrations of CBD than drug-types 13 .…”

Foliar Sprays of Silver Thiosulfate Produce Male Flowers on Female Hemp Plants

“…Increased interest in CBD has led to increased cultivation of hemp, but because hemp has been selected for fiber and seed production, it is generally low in CBD content (2% to 4%) (Mead, 2017). Therefore, the development of hemp cultivars with increased CBD content is a current breeding objective (Caplan et al, 2017;Soler et al, 2017).…”

“…Female plants are preferred for cannabinoid production because females accumulate, predominantly in the inflorescences, significantly greater cannabinoid content than male plants (Small, 2015). Rahn et al (2016) and Soler et al (2017) report that hemp growers are using hormones or chemicals like STS to make feminized seed, in which 100% of the seed is female. Mohan Ram and Sett (1982) , 1982).…”

Developing Robust Standardised Analytical Procedures for Cannabinoid Quantification: Laying the Foundations for an Emerging Cannabis-Based Pharmaceutical Industry

“…Production and quality control of Cannabis -based biological medicines derived from recombinant DNA technology are also subject to market authorisation guidelines (e.g., EMA European Guidelines 3AB1a), which require product purification to remove protein and nucleic acid contaminants derived from the host cell. Given the obligate outcrossing nature of Cannabis [34] as well as heterozygosity within the gene pool [3, 35], seed-grown Cannabis botanical raw material can be highly variable in content and composition of target and non-target active ingredients [31, 36]. Thus, clonal propagation using callus- [37] or nodal- (axillary buds) [38] mediated regeneration is favoured over seed propagation [22], with explants typically grown under environmentally controlled conditions in an effort to minimise genetic by environment interactions and to maximise product uniformity [31].…”