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

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-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.