Accumulation of somatic mutations leads to genetic mosaicism in cannabis

Abstract

Cannabis (Cannabis sativa L.) is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigor and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50×) to compare the variability within an individual cannabis cultivar Honey Banana plant sampled at the bottom, middle, and top. We called over six million sequence variants based on a reference genome and found that the top had the most by a sizable amount. Comparing the variants among the samples uncovered that nearly 600,000 (34%) were unique to the top while the bottom only contained 148,000 (12%), and middle with 77,000 (9%) unique variants. Bioinformatics tools were used to identify mutations in critical cannabinoid–terpene biosynthesis pathways. While none were identified as high impact, four genes contained more than double the average level of nucleotide diversity (π) in or near the gene. Two genes code for essential enzymes required for the cannabinoid pathway while the other two are in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, a measurable number of intraplant genetic diversity was discovered that could impact long‐term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigor and cannabinoid content.

Impact of Harvest Time and Pruning Technique on Total CBD Concentration and Yield of Medicinal Cannabis

“…Worldwide cannabis cultivation has increased due to the recent changes in legalization, regulation and marketization of Cannabis sativa L. for industrial, medicinal and recreational use [ 1 , 2 ]. To date, 177 phytocannabinoids have been identified in cannabis plants [ 3 ], among which the two most abundant are the psychoactive compound Δ 9 -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) [ 4 ]—whose medical properties have accumulated evidence for decades [ 5 , 6 ]. Both compounds have shown pharmacological effects for several medical treatments [ 7 ].…”

Effect of Explant Source on Phenotypic Changes of In Vitro Grown Cannabis Plantlets over Multiple Subcultures

“…Conventional cutting methods used for cannabis are time-consuming and associated with a wide range of diseases (e.g., viruses, bacteria, and fungi) that hamper the phytosanitary quality of the plants obtained [ 7 ]. While vegetative propagation through cuttings theoretically produces plants that are phenotypically and genetically identical to their mother plants, cannabis producers have noted that cuttings taken from mother plants change over time and generally become less vigorous with lower levels of cannabinoids [ 8 ].…”

“…Indeed, based on the explant source, in vitro plant rejuvenation is accompanied by specific changes in gene expression patterns and epigenetic regulation, which finally lead to different phenotypical changes such as phyllotaxy, the shape and size of leaves, as well as the capacity for multiplication [ 26 ]. In relation to cannabis, Adamek et al [ 8 ] previously showed that the accumulation of somatic mutations in top-part-derived cuttings was significantly higher than in bottom-part-derived cuttings from the cannabis mother plant, showing the importance of explant source. The physical position and age of the explant largely impact the composition of internal phytohormones, which can influence tissue responsiveness [ 27 ].…”

Characterization of CBD oils, seized on the Belgian market, using infrared spectroscopy: Matrix identification and CBD determination, a proof of concept

“…These compounds accumulate mainly in the glandular trichomes of the female plant 2 . To date, 177 phytocannabinoids have been identified among which the two well‐known cannabinoids, to which medical properties or health claims are assigned: the psychoactive substance Δ 9 ‐tetrahydrocannabinol (THC) and cannabidiol (CBD) 3 . They are synthetized as carboxylic acids and decarboxylated to the neutral form during exposure to light, heat, or prolonged storage; Δ 9 ‐tetrahydrocannabinolic acid (THCA) is decarboxylated to THC, and cannabidiolic acid (CBDA) is decarboxylated to CBD 4 …”