Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP
Introduction
Cannabis sativa ssp. sativa (hemp) is a traditional crop worldwide. Cannabis sativa ssp. indica (marijuana) is, on the other hand, subject of drug abuse. Both subspecies show no evident morphological distinctions, but they contain different levels of psychoactive substances, among them the most important Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp.1 The presence of psychoactive substances does not have any influence on the quality of plant fiber, but in certain circumstances one triggers the question whether the cultivated plant is harmless hemp, or potentially harmfull marijuana and whether the cultivation is acceptable. Possessing, growing and consuming marijuana is illegal in many world countries, while the production of industrial hemp is desirable. Cultivated variants of industrial hemp have higher disease and pest resistance, while at the same time they have psychoactive potential in acceptable boundaries.2 Research of Cannabis sp. in forensic studies usually has a goal of making the disctincion between the subspecies of Cannabis sativa, i.e. the discrimination of C. sativa ssp. indica and C. sativa ssp. sativa.
Many jurisdictions have an upper limit of 0,2% or 0,3% for Δ-9-tetrahydrocannabinol (THC) content in dried plants. In Serbia, growing and possessing of plants capable of producing THC concentrations higher than 0.3% (as in marijuana) is considered punishable by the law.
Standard methodology for determining the content of THC in a sample is gas-chromatography. The literature frequently cites few other methodologies based on High Performance Liquid Chromatography (HPLC). However, these methodologies have low sensitivity when separating different chemical compounds that are important in forensic analysis (such as THC, cannabinol (CBN), cannabidiol (CBD) etc.). Furthermore, when applying such methodologies, there is a risk of obtaining false negative results in the analysis due to different plant age, sampling from different parts of the plant or as a result of decomposition of THC in the sample over time.3
C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene and its activity. THCA synthase gene is coding for an enzyme that catalyzes oxidative cyclization of cannabigerolic acid (CBGA) into tetrahydrocannabinol acid (THCA), which is then decarboxylatedinto THC by heating.4 C. sativa ssp. sativa is homozigous for inactive variant of THCA synthase gene, which results in no production (or production on the very low level) of THC molecule in this plants. On the other hand, Cannabis sativa ssp. indica could be heterozygous for inactive/active or homozygous for active variant. The existence of at least one copy of the active gene enables production of THC in significant quantities.5
Recent reasearch is usually based on the analysis of THCA synthase gene.6, 7 There is a total of 63 nucleotide sequence variants (NSVs) that differentiate active and inactive copy of THCA synthase gene, which correspond to 37 amino acid substitutions in the THCA synthase.8 Rotherham and Harbison developed snap-shot multiplex assay in which they analyzed four variants in 399 bp long sequence of THCA synthase gene,5 while Sutipatanasomboon and Panvisavas developed duplex PCR test for C. sativa drug- and fiber-type detection and discrimination.9 We developed in house methods, based on allele specific PCR and on PCR and restriction digestion, to differentiate THCA synthase gene sequence in C. sativa subspecies.
The objective of this paper was to establish an algorithm for forensic DNA analysis in order to determine if a plant in question is capable of producing THC in concentration higher than 0.3%, i.e. if it belongs to marijuana or „drug-type“ of cannabis, by using fast, simple and inexpensive method for the analysis of THCA synthase gene.
Section snippets
Material and methods
As a starting material the leaves and dried parts of C. sativa were used. The leaves from oak (Quercus robur) and tulip (Tulipa sp.) were used as controll samples. The fresh oak and tulip samples were obtained from a botanical garden, and the same day DNA isolation was conducted.
C. sativa ssp. sativa leaves were two days old. Three different hemp varieties from three different countries (USO11 from Ukraine, Fedora 17 from France and Novosadska from Serbia) were used as to have varied and
Results and discussion
All samples analyzed, including oak and tulip, were positive for the presence of rbcL gene, confirming their plant origin.
400 bp long PCR fragment was amplified in both cannabis subspecies under the stringent conditions (Table 1). When using less stringent annealing temperature (50 °C), the inactive gene copy was preferentially amplified, probabily due to differences in nucleotide sequences that lie adjacent to primer binding sites. The other allele of the same gene (marijuana-specific fragment
Conclusion
An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing significant quantities of THC (concentrations higher than 0.3% in dried plants, as specified by Serbian legislative) and to overcome the problem that could arise from false – negative results due to different plant age, sampling from different parts of the plant or as a result of decomposition of THC in the sample.
Acknowledgements
C. sativa ssp. sativa specimens were kindly provided by Prof. d. Janos Bereni from the University of Novi Sad, Serbia.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest
The authors declare that they have no conflict of interest.
References (10)
- et al.
Variations of Δ9-THC content in single plants of hemp varieties
Ind Crops Prod
(2004) - et al.
Cannabidioloc-acid synthase, the chemotype-determining enzyme in the fiber-type
Cannabis sativa FEBS Lett
(2007) - et al.
Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay
Forensic Sci Int
(2011) - et al.
The gene controlling marijuana psychoactivity
J Biol Chem
(2004) - et al.
DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in ’drug-type’ and ’fiber-type’ Cannabis sativa L
Forensic Sci Int
(2006)
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Evaluation of tetrahydrocannabinolic acid (THCA) synthase polymorphisms for distinguishing between marijuana and hemp
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