DNA Markers to Discriminate Cannabis sativa L. ‘Cheungsam’ with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes

Abstract

Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. Therefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. Thus, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar “Cheungsam” (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars.

Origin and Evolution of the Cannabinoid Oxidocyclase Gene Family

“…Type 2/3 ( Onofri et al 2015 ) comprises accession which differs by two synonymous substitutions from type 2/1 and can therefore be considered functionally equivalent. Type 2/4 comprises sequences from Boseung province in Korea described by Doh et al (2019) that share three nonsynonymous substitutions. Type 2/5 comprises accession MN422091.1 from Jecheon province in Korea that has five nonsynonymous substitutions.…”

“…Type 2/5 comprises accession MN422091.1 from Jecheon province in Korea that has five nonsynonymous substitutions. Type 2/6 comprises sequences from low-THCA cultivar “Cheungsam” described by Doh et al (2019) and can be characterized by ten nonsynonymous substitutions. Other types remain ungrouped.…”

Origin and evolution of the cannabinoid oxidocyclase gene family

“…Type 2/5 comprises accession MN422091.1 from Jecheon province in Korea that has five nonsynonymous substitutions. Type 2/6 comprises sequences from low-THCA cultivar 'Cheungsam' described by (Doh et al 2019) and can be characterised by ten nonsynonymous substitutions. Other types remain ungrouped.…”

“…Level of sequence divergence between THCAS from different geographic areas in South Korea is relatively high (Table S3, Figure S1). Accessions from Boseung province share three unique aa substitutions, the accession from Jecheon province in Korea that has five unique aa substitutions, and sequences from Cheungsam share ten unique aa substitutions (Doh et al 2019). Moreover, the THCAS sequence from the putatively wild accession from Jilong, Tibet is also relatively different from canonical THCAS and placed as sister to all other clade A1 sequences (S. ).…”

“…In addition, we compiled homologous nucleotide sequences available from ncbi databases the majority of which came from published studies (Sirikantaramas et al 2004;Kojoma et al 2006;Taura, Sirikantaramas, Shoyama, Yoshikai, et al 2007;El Alaoui et al 2013;McKernan et al 2015;Onofri et al 2015;Weiblen et al 2015;Cascini et al 2019;Doh et al 2019). Based on preliminary analyses, some sequences described by (Cascini et al 2019) were found to have relatively high levels of ambiguous nucleotides, probably due to unspecific amplification of multiple genes or gene variants.…”