Accumulation of somatic mutations leads to genetic mosaicism in Cannabis

Abstract

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) to compare the variability within an individual Cannabis sativa cv. "Honey Banana" plant sampled at the bottom, middle and top. We called over 6 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 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 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 ({pi}) 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 intra-plant genetic diversity was discovered that could impact long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.