Analysis of Morphological Traits, Cannabinoid Profiles, THCAS Gene Sequences, and Photosynthesis in Wide and Narrow Leaflet High-Cannabidiol Breeding Populations of Medical Cannabis

Abstract

Cannabis sativa L. is one of the oldest cultivated crops, used in medicine for millennia due to therapeutic characteristics of the phytocannabinoids it contains. Its medicinal properties are highly influenced by the chemotype, that is, the ratio of the two main cannabinoids cannabidiol (CBD) and Δ-9-tetrahydrocannabinol (THC). Based on published data, the chemotype should correlate with plant morphology, genetics, and photosynthetic properties. In this work, we investigated leaf morphology, plant growth characteristics, cannabinoid profiles, THCAS gene sequences, and plant photosynthetic traits in two breeding populations of medical cannabis (MX-CBD-11 and MX-CBD-707). The populations differed significantly in morphological traits. The MX-CBD-11 plants were taller, less branched, and their leaves had narrower leaflets than the bushier, wideleaved MX-CBD-707 plants, and there were significant differences between populations in the dry biomass of different plant parts. Based on these morphological differences, MX-CBD-11 was designated as a narrow leaflet drug type or vernacular “Sativa” type, while MX-CBD-707 was classified as wide leaflet drug type or “Indica” type. Chemical characterisation revealed a discrepancy between the expected chemotypes based on plant morphology; although both populations have high CBD, within each Type II (CBD/THC intermediate) and Type III (CBD dominant) plants were detected. The THCAS gene sequence analysis clustered the plants based on their chemotypes and showed high similarity to the THCAS sequences deposited in NCBI. In silico complementary analysis, using published molecular markers for chemotype determination, showed their low discrimination power in our two populations, demonstrating the genotype dependence of the molecular markers. Basic photosynthetic traits derived from light and CO2 response curves were similar in the populations. However, measurements of gas exchange under chamber conditions revealed higher stomatal conductivity and photosynthesis in MX-CBD-707 plants, which were also characterised by higher day respiration. The results of this study showed that based on visual appearance and some morphological measurements, it is not possible to determine a plant’s chemotype. Visually homogenous plants had different cannabinoid profiles and, vice versa, morphologically distinct plants contained similar CBD and THC content. The two chemotypes identified in our experimental plants therefore did not correlate with plant visual appearance, leaf morphometry, and photosynthetic properties of the populations studied. Correlation was only demonstrated with the respect to THCAS sequences, which showed great discrimination power between the chemotypes.

Intra-leaf modeling ofCannabisleaflet shape produces leaf models that predict genetic and developmental identities

“…(2021b) conducted a study on clones of 21 cultivars and found a strong negative correlation between the width and length ratios of central leaflets and CBD, and a positive correlation with THC; however, Vergara et al . (2021) and Murovec et al . (2022) were unable to confirm these findings.…”

“…Using this new method, any leaf can be projected onto the morphospace, and the resulting PC values used to provide a quantitative measure of the leaf shape would be useful for studies investigating correlation between shape and chemical profiles or sex of the plant. For example, Jin et al (2021a) conducted a study on clones of 21 cultivars and found a strong negative correlation between the width and length ratios of central leaflets and CBD, and a positive correlation with THC; however, Vergara et al (2021) and Murovec et al (2022) were unable to confirm these findings. All three studies used lowresolution morphometric approaches.…”

“…Extensive variability in leaf morphology has already been described by Quimby et al (1973) and later Anderson (1980), who was the first to quantify the width, length, and ratio of the central leaflet. This or similar methods were then commonly used in studies investigating the morphological characteristics of Cannabis species, subspecies, cultivars, biotypes and chemotypes de Meijer et al, 1992;de Meijer & Keizer, 1996;Hillig, 2005a;Clarke & Merlin, 2013;Lynch et al, 2016;Karlov et al, 2017;Parsons et al, 2019;McPartland & Small, 2020;Carlson et al, 2021;Islam et al, 2021;Jin et al, 2021a;Vergara et al, 2021;Buzna & Sala, 2022;Chen et al, 2022;Murovec et al, 2022), often with contradictory results. Leaf shape has therefore played an important and sometimes controversial role in Cannabis taxonomy.…”

“…Leaf shape has therefore played an important and sometimes controversial role in Cannabis taxonomy. While researchers in previous Cannabis studies were aware of enormous plasticity and the effect the environment has on leaf shape (Vergara et al ., 2021; Murovec et al ., 2022), they very rarely paid attention to the effects of developmental processes, even though heteroblastic changes (differences in leaf shape arising from juvenile-to-adult phase transitions in the meristem) profoundly affect the arrangement and shape of Cannabis leaves along the shoot. While some studies briefly mention the developmental changes of leaves (Hillig, 2005a; Carlson et al ., 2021; Jin et al ., 2021b; Spitzer-Rimon et al ., 2022), the only two studies focusing on heteroblastic phase changes in leaves along the plant axis were done by Heslop-Harrison and Heslop-Harrison (1958) and Hesami et al .…”

Examining population structure across multiple collections of Cannabis

“…Cannabis varietals are typically classified as high THC (Type I), containing a ratio of THC to CBD (Type II) or high CBD (Type III) plants [38] as well as the rarer types, with high CBG (Type IV) and complete absence of cannabinoids (Type V) [39]. Hemp varietals are typically classified as Type III, often presenting with a CBD enriched phenotype largely the result of a non-functional copy of the synthase THCAS [54]. However, this is not the case for all hemp varietals, with functional and non-functional THCAS genes known to be present in germplasm presenting challenges for farmers who are required to have less than 0.3% THC at harvest based on current regulations.…”