Genotype and chemotype insights of high-THC medicinal Cannabis sativa L.: the role of SSR markers in the identification of cultivars

Standardized methods for distinguishing high-THC Cannabis sativa L. genotype versus chemotype of cultivars remain limited, posing challenges for quality control and regulatory compliance in the medicinal cannabis industry. This study addresses the integration of genetic and chemical profiling for the standardization and quality control of medicinal High-THC Cannabis sativa L. clonal cultivars, focusing on the potential use of SSR markers combined with high-performance liquid chromatography (HPLC) for cultivar characterization. DNA was extracted from C. sativa L. leaf using a modified Mini-CTAB protocol, followed by PCR amplification based on 12 SSR primers, and capillary electrophoresis for genetic analysis. HPLC-PDA was used to quantify cannabinoid content, while non-cannabinoid components, including phenolics, flavonoids, chlorophyll, and waxes, were investigated using specific methods. Using eight individual specimens of the plants, the study evaluated three distinct cultivars (identified as A, B, and C), representing key genetic variations. Genetic analysis revealed that the eight SSR markers amplified allele sizes ranging from 147 to 326 bp, depending on the locus. ANUCS303, ANNUCS304, and C11CANN1 amplified respectively two, three, and three alleles and were valuable tools for cultivar fingerprinting. Further validation based on a broader range of cultivars and primers is needed to enhance the reliability of SSR-based cultivar identification. Chemical analysis showed similar cannabinoid profiles across cultivars, with a high THC [82–84% (w/w)] and a low CBD [0.5–0.8% (w/w)]. Non-cannabinoids, including waxes and chlorophylls, were quantified and efficiently removed during process purification. The extraction and purification processes significantly increased ∆9-THC content, with total cannabinoid content reaching 97% in purified extracts while preserving cannabinoid profiles, with minor differences across the cultivars’ chemical fingerprints. The study highlights the importance of combining genetic and chemical profiling for standardizing C. sativa L. cultivars. SSR markers can effectively aid in cultivar identification and quality control, and the combined “identity fingerprint” is important to ensure therapeutic equivalence across batches of clonal lines. These efforts should be designed to enhance the safety, reliability, and efficacy of vegetatively propagated cannabis-based therapeutics.