Genotype screening of Cannabis sativa L. based on the specifics of minor cannabinoids manifestation

Cannabinoids belonging to the chemical class of aromatic compounds and accumulating predominantly in glandular hairs (trichomes), are specific substances in industrial hemp (Cannabis sativa L.) [1–3]. The most common and well-known are tetrahydrocannabinolic (THCA), cannabidiol (CBDA) and cannabigerolic (CBGA) acids. In the plant organism they are synthesized in acid form, bioactive forms of cannabinoids – tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabigerol (CBG), reUDC 633.522:631.52:577 https://doi.org/10.21498/2518-1017.17.3.2021.242949


Introduction
Cannabinoids belonging to the chemical class of aromatic compounds and accumulating predominantly in glandular hairs (trichomes), are specific substances in industrial hemp (Cannabis sativa L.) [1][2][3]. The most common and well-known are tetrahydrocannabinolic (THCA), cannabidiol (CBDA) and cannabigerolic (CBGA) acids. In the plant organism they are synthesized in acid form, bioactive forms of cannabinoids -tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabigerol (CBG), re-spectively -are formed as a result of decarboxylation reaction under the influence of external conditions [4]. In addition to the main ones, hemp contains many minor (rare) cannabinoidscompounds whose content is very low and usually does not exceed 0.5% of dry biomass. The pharmacological properties of minor cannabinoids have not yet been reliably confirmed in clinical studies or in vitro and in vivo rational analyzes. Due to the lack of such biological and pharmacological information, minor cannabinoids have some potential as possible drug candidates [5]. If there is now a significant interest from manufacturers and scientists in THC and CBD, it is expected that in the near future they will focus on the still unknown variety of secondary minor cannabinoids. At the same time, the extraction and purification of these compounds from hemp plant extracts is a rather difficult task due to the low yield of isolates and, during subsequent processing, due to the low stability of substances [6].
The pharmacological activity of minor cannabinoids is now being actively investigated, in particular, their effect on the CB1R and CB2R receptors is being studied [7]; CBG, cannabichromene (CBC), canabinol (CBN) and tetrahydrocannabivarin (THCV), for example, demonstrate anticonvulsant, anti-inflammatory, antidepressant and antibacterial properties, relieve pain and promote muscle relaxation. There is a possibility of their use as anticancer agents, but further research is needed to confirm the possibility of their medical use [8].
The biosynthesis of minor cannabinoids in cannabis has not been completely elucidated, it is unlikely that they are synthesized enzymatically. It was suggested that the chemical transformation in trichomes under the influence of light, temperature, and ultraviolet radiation is the main catalytic mode of their formation; it is these transformations that can explain the significant chemical diversity and low structural stability of these compounds [5,6].
The precursors of cannabinoid biosynthesis are formed in two different biosynthetic pathways: polyketide, which produces olivetholic acid, and plastid, which produces geranyldiphosphate. Of these, with the participation of prenyltransferases, CBGA, which is the main precursor of several different cannabinoids is synthesized [9]. In this case, specific synthases ferment a certain cannabinoid compound: THCA synthase converts CBGA into THCA, CBDA synthase -into CBDA and CBCA synthase -into CBCA [10]. A non-functional allelic variant of the THCA synthase gene was also found: the so-called "null" THCA synthase with a single nucleotide polymorphism (SNP), which makes the synthase unable to convert CBGA into THCA, which leads to a significant accumulation of the first compound [11]. Apart from the aforementioned enzymes (THCA, CBDA, and CBCA synthas es), no other genes or enzymes that control the biosynthesis of cannabinoids from CBGA have yet been found [5].
Most authors suggest the membrane localization of the enzyme CBGA synthase and the cytosolic localization of THCA synthase, but recently THCA synthase has been found outside the plasma membrane of the glandular cell and catalysis under non-aqueous conditions [12]. Therefore, trichome can play a significant role in the formation of secondary metabolites; it is a biosynthetic organ where photosynthetic reactions take place. The following arguments are made in favor of this statement: 1) almost ideal spherical shape in the lower micrometric range, very conducive to the refraction and focusing of light rays as in a lens filled with liquid; 2) raising the temperature to 50 °C under direct sunlight probably accelerates the formation of bonds between carbon atoms and other chemical reactions [6]. In general, the number of minor cannabinoids reaches at least 150 [13], their biological role and physiological functions in the plant are not known, many of them are artifacts of the processes following the synthesis of the main cannabinoids, and some acquire stability after acetylation, methylation or dimerization reactions. Chemical diversity of cannabinoids is still not fully understood, and the biosynthetic effect of enzymatic catalysis was overestimated, so photochemical transformations in a non-aqueous "tric home bioreactor" become an acceptable hypothesis for explaining the patterns of their occurrence [5]. So, in the end, we can state about two types of formation of cannabinoid compounds -enzymatic and non-enzymatic (photochemical).
It should be noted that collectible hemp samples of unrelated origin are characterized by variability in the characteristics of the main (common) cannabinoid compounds -CBD, THC and CBG. According to the results of studies conducted with the national collection of hemp, the content of CBD in dried inflorescences ranged from 0.0052 to 1.7251%, THC -from 0.0000 (absent) to 0.0775% (does not exceed the norm permitted by law), CBG -from 0.0000 (absent) to 0.8892%. The established range of variation makes it possible to select samples with an increased content of CBD and/or CBG in the absence of THC. The analyzed material was assigned to 3 hemp chemotypes: III (65.5%) is fibrous cannabis with a predominance of CBD and a high CBD : THC ratio, in which the THC content ranges from a small percentage to a complete absence; IV (1.8%) -hemp with a predominance of CBG, which is the main compound and low or no THC; V (32.7% of the total) -hemp with a complete absence of cannabinoid compounds, which are practically impossible to isolate within the sensitivity of the chromatograph. Furthermore, strong correlations between CBD and THC and, in fact, the absence of a relationship between CBD and other cannabinoid compounds were revealed; moreover, in the samples of chemotype III bonds are weaker compared to chemotype V [14]. The issue of characterizing cannabis genetic resources by the content of minor (rare) nonpsychotropic cannabinoids -CBC, cannabidivarin (CBDV) and CBN, assessing the correlation between them and common compounds, identifying sources and donors of valuable traits and selecting the initial breeding material on this basis remains unclear.

Materials and methods
The material for the research was 210 samples from Ukrainian National Collection of Flax and Hemp of the Institute of Bast Crops of the National Academy of Sciences of Ukraine, belonging to various ecological and geographical types (northern, Central European and southern), genetic origin (local and wild forms, self-pollinated lines, hybrids, varieties, synthetic populations, polyploids ), obtained from 10 countries (mainly from Ukraine, Russia, France, Germany and China) and for which the THC content is within the limits permitted by the current legislation, and does not exceed 0.08%.
Three-year field research (cultivation) was carried out in the northeastern part of Ukraine on the southern border of the mixed forest zone of the lowest part of the Ukrainian Polissia. Height above sea level was 166 m, geographic coordinates of the area: 51°39' north latitude and 33°59' east longitude. The soils in crop rotation were dark and light gray forest, slightly podzolized loams formed on moraine clay. Fertilizer application rate was N 120 P 90 K 90 . The weather conditions during the research years (2018-2020) were varied and characterized by deviations from the average annual air temperature, precipitation and relative humidity: 2018 and 2020 were hot (in September up to 3.9 °C above normal) and arid during the hemp growing season, and 2019 was characterized by excessive rainfall from May to July and almost absence of rainfall in August (9.9 mm). This enabled comprehensive evaluation of the performance of collection samples under various weather conditions.
In order to identify cannabinoid compounds during threshing of hemp plants grown in a valuation plant nursery with a feeding area of 30 × 5 cm (phase BBCH 89) [15], a combined sample of plant material was taken from each area of 1 m 2 from the inflorescences, dried and stored at a laboratory temperature. Before the analysis, the samples were dried to constant weight at a temperature of 105 °C in an oven, ground to a fine powder and thoroughly mixed; samples weighing 0.5 g were taken in two repetitions, and 5 ml of methanol was added (the ratio "plant type : extractant" -1 : 10). The extraction time was 24 h, after that the extract was filtered using a paper filter. In the obtained methanol extracts of samples, the quantitative content of cannabinoid compounds was determined by gas chromatography on an HP 6890 Series GC System chromatograph with detection. Chromatography conditions: -capillary column -Agilent Technologies 19091J-413 (HP-5), length -30 m, diameter -0.320 mm, phase -0.25 µm, SN: USN493366H, constant flow -1.5 ml/min, carrier gas -helium; -injector -auto-injector 7683, Split 20 : 1, evaporator temperature -T = 250 °C; oven -T initial = 100 °C, hold for 2 minutes, heating -15 °C/min, T final = 280 °C, hold for 11 minutes; -detector -flame ionization; -sample -1.0 µl. Compounds were identified by retention time. The concentration of cannabinoids was determined using an internal standard (stearic acid methyl ester at a concentration of 0.392% of the sample), for which, the chromatographic peak areas of the internal standard and the chromatographic peak areas for the studied compounds were compared using the Chemstation software. Statistical data processing was carried out according to the method of field experiment [16]. A sample with a high content of a certain cannabinoid is one with content higher than the median (Me).

Research results
The findings of the quantitative analysis of cannabinoid compounds showed that CBC in the inflorescences of collection hemp samples was in a small amount, namely, from absence to 0.6836%. The Cumulative graph of the frequency distribution of the values of the trait of a given compound content in all studied samples shows that their lion's share (88.1% of the total amount) is within the 0.0000-0.0683% class, a significantly smaller number of samples (5.2%) falls within the class 0.0684-0.1367%, the rest (from 0 to 4 samples, or up to 1.9%) belongs to other classes (Fig. 1).
CBC was isolated at the very beginning of modern biochemical studies of cannabis, but it is less studied in comparison with other phytocanabinoids in terms of biological profile and chemical activity [17]. Traditionally, together with CBD, THC, and CBG, it is considered the main phytocanabinoid and a constituent of the so-called "Big Four" components of cannabinoid compounds, even the second most abundant, but its concentration was significantly overestimated due to the difficulty of separating CBC and CBD in gas chromatography with subsequent assignment of the peak area exclusively to the CBC. In fact, the concentration of CBC is much lower, it rarely exceeds 0.2-0.3% of dry biomass; it was found that CBC accumulates in one-or two-digit concentrations [13]. Our research confirms this feature. Genetics CBDV was identified in even smaller amounts compared to CBC -from complete absence to 0.1719% of dry biomass. The class with a content of 0.0000-0.0171% includes 62.4% of all studied samples, with a content of 0.0172- Among the studied minor cannabinoids, CBN was identified in the smallest number of samples, its maximum content was 0.3274%. The Cumulative graph of the frequency distribution of the values of the trait clearly shows that this compound is absent in the vast majority of plants or present in insignificant quantities. Thus, 192 collection samples out of 210 analyzed, or 91.4%, contain 0.0000-0.0327% of this compound. At the same time, a negative kurtosis of the studied trait was revealed, since 7.6% of genotypes contained from 0.0328 to 0.0654% CBN, 0.5% of genotypes each contained from 0.2620 to 0.2947% and from 0.2948 to 0.3274% of this compound, respectively. No plants with CBN content from 0.0655 to 0.2619% were found (Fig. 3).
In general, the discovered biochemical features of collection hemp samples indicate the difficulty of using them in breeding as sour ces and donors of the trait of a high content of minor cannabinoids. In addition, the issue of establishing correlations between cannabinoid compounds becomes relevant, since the pre-sence of strong relationships can cause difficulties in breeding work, when it is necessary to increase the content of some non-psychotropic component, and reduce the content of another, for example THC.
Within the framework of the studied samples, it was found that between the traits of the content of CBC and CBD there is an average negative relationship (r = -0.53), CBC and CBDV -a weak negative relationship (-0.35), and between the traits of the content of CBC and THC, CBC and CBN -average positive relationship (0.57 and 0.59, respectively). A mean positive correlation was found between CBDV and CBD traits (0.57), and CBN had a strong positive relationship with THC (0.82). It should be noted that CBG almost does not correlate with the studied minor cannabinoids (Table 1).  Since the biosynthesis of various cannabinoid compounds is rather complex, and the expression of the signs of their content depends on many, both genetic and external factors, it makes sense to establish a partial and multiple linear correlation. Partial correlation coefficients indicate that CBD and THC, at a con-  Recent studies [18] show that while the high transcription level of THCA synthase and CBDA synthase clearly reflects the chemical phenotype of cannabis, the low but stable level of transcription of CBCA synthase in all genotypes suggests that these genes may contribute to the final amount of cannabinoids. It was hypothesized that CBCA synthases are not only enzymes for converting CBGA to CBCA, but can also participate in the formation of THCA in a material with a predominance of CBD; therefore, the reciprocal (mutual) influence of gene groups takes place in the quantitative expression of the chemotype [18].
Our screening of a large number of collection samples for traits of cannabinoid content and the establishment of relationships between them gives grounds to assert that the presence of a high CBC content in samples, which mainly belong to chemotype ²²² (with a high CBD : THC ratio), is interconnected with a high THC content. It can be assumed that the CBCA synthase gene is not located in an independent locus; in this case, a more complex genetic mechanism for determining the synthesis of cannabinoids opera tes, and CBCA synthase gene, for example, has several linked loci, in particular with CBDA synthase genes and, especially, THCA synthase.
CBN is the end product of THC biosynthesis -aromatized THC, an oxidation product that is usually identified if hemp plant material or isolated THC is exposed to ultraviolet radiation or sunlight for a long time [5,19], therefore, the features of the correlation between CBN and THC and CBC that we have established are logical. The more THC was synthesized, the more of it could turn into CBN, the more active CBCA synthase was, the more THC accumulated. Since CBDV is an n-propyl analogue of CBD [19], the ave rage correlation between these compounds is obvious.
As a result of studying the collection of genetic resources of this culture, it was possible to identify valuable collection samples (sources and donors of traits) for the practical breeding of industrial hemp varieties for medical use, namely, with a high content of minor cannabinoids and a content of psychotropic THC within the limits permitted by current legislation. A tight linear connection of minor cannabinoids with each other and with common components makes it possible to find the source material with the above characteristics. So, 5 samples with a high content of CBC were isolated (2.4% of the total amount analyzed), CBDV -7 samples (3.3%), CBN -2 sa mples (1.0%), finally with a combination of a high CBDV content and widespread CBD compounds -5 samples (2.4%) ( Table 3).

Conclusions
In the analyzed collection samples of hemp, the content of CBC did not exceed 0.6836%, CBDV -0.1719% and CBN -0.3270%, in the vast majority of them minor cannabinoids were not identified at all. The level of accumulation of separate minor cannabinoids depends Ãåíåòèêà on the influence of many genetic and external factors, a different nature of the correlations between them was established: CBDV is most associated with CBD, CBN is most associated with THC, the trait of CBC content affects the formation of CBD and, especially, THC. A tight linear connection of minor cannabinoids with each other and common components makes it possible to isolate valuable samples for breeding with an increased content of both one compound and several compounds with a simultaneous absence or low content of psychotropic THC.