A Study in Purple: An Investigation into the Genetics of Anthocyanin Accumulation in Cannabis sativa

Abstract

Cannabis sativa L. is an emerging crop with a variety of uses for its nutritious grains, long bast fibers, and significant public interest in its secondary metabolites. Anthocyanins are a class of secondary metabolites which provide visible purple and pink pigments in plants, driving consumer preferences in the floral cannabis market and enhancing abiotic stress tolerance. Identifying and characterizing the candidate gene(s) responsible for anthocyanin accumulation in C. sativa will create new opportunities for developing cultivars with heightened levels of these compounds. Prior mapping studies implicated a region on chromosome 6 putatively influencing leaf and flower pigmentation traits in two independent populations (S. Kim, Unpublished). To help identify potential candidate genes responsible for anthocyanin biosynthesis in leaf, flower, and stem tissues, and validate previous marker associations in C. sativa, an F1 population replicated from prior studies was used to identify significant SNP marker clusters associated with each trait using a genome wide association study (GWAS). This analysis validated the presence of a significant marker cluster on chromosome 6 from 66.75Mb-75.77Mb associated with flower anthocyanin, and containing the putative subgroup 6 R2R3-MYB transcription factor CsMYB78. A new significant marker cluster on chromosome 1associated with stem and leaf anthocyanin was also revealed from 85.08Mb-87.92Mb. Following this, CsMYB78 and CsMYB33, previously reported candidate genes from Kundan et al. (2022) were transformed into C. sativa plants at the Wisconsin Crop Innovation Center (WCIC). Initial results of generated T0 plants show a counterintuitive pattern of pigmentation with CsMYB78 failing to induce strong anthocyanin accumulation and CsMYB33 inducing strong accumulation across tissues. The results of these experiments will be invaluable in future breeding efforts focused on creating and maintaining purple cannabis cultivars, as well as in advancing our understanding of transcriptional regulation mechanisms of cannabis.