Cannabis 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 vigour 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 (>50x depth of coverage) to compare the variability within an individual Cannabis sativa cv. Honey Banana plant sampled at the bottom, middle and top. Overall, we called over 6 million sequence variants based on a published reference genome (SNPs, MNPs, and indels) and found that that the top had the most by a sizable amount. We compared the variants among the samples and uncovered that nearly 600K (34%) were unique to the top while the bottom only contained 148K (12%) and middle with 77K (9%) unique variants. Bioinformatics tools were used to identify high impact mutations in critical cannabinoid/terpene biosynthesis pathways. While none were identified, some contained more than double the average level of nucleotide diversity (π) in or near the gene, including OLS, CBDAS, HMGR2 and CsTPS9FN. The first two genes code for essential enzymes required for the cannabinoid pathway while the other two are involved in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, these data identified a measurable number of intra-plant genetic diversity that could impact the long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.
Accumulation of somatic mutations leads to genetic mosaicism in cannabis