Application of a Simple Genetic Assay to Discriminate Hemp from Drug-Type Cannabis

December 16, 2019
Abstract / Synopsis: 

The explosion in the hemp industry within the United States has created confusion for state regulators and hemp farmers. The passage of the 2018 Farm Bill provides for the widespread planting of hemp containing less than 0.3% tetrahydrocannabinol (THC). While some hemp is being grown for fiber and seed, most of the 2019 hemp harvest is intended for extraction of cannabidiol (CBD). Therein lies the anxiety, since resinous hemp is often above that 0.3% THC threshold. An alternative to relying on THC content alone is to distinguish the categories of fiber hemp, resin hemp, and THC cannabis based on genetics. Here we report on the findings from the initial field evaluation of one such molecular DNA-based assay. The assay ID markers correctly identified 420 out of 420 individual seed, leaf, and flower cannabis samples. Correlation with a subset of available cannabinoid data shows that while low in THC content, 72% of these resin hemp samples fall outside of the 2018 Farm Bill’s definition of hemp of less than 0.3% THC.

The Agriculture Improvement Act of 2018, otherwise known as the 2018 Farm Bill, was signed into law on December 20, 2018. The 2018 Farm Bill legalized the cultivation and sale of hemp at the federal level effective January 1, 2019 (1). The change in federal status had broad ramifications for the hemp and cannabidiol (CBD) industries in the United States. Until December 20, 2018, hemp was categorized as a Schedule I substance under the Controlled Substances Act. It was illegal at the federal level to cultivate, possess, or distribute the hemp plant or CBD derived from the hemp plant. Almost all CBD products were illegal at the federal level. Notwithstanding, the CBD industry has flourished across the United States operating in the gray zone created under the federal policy in state jurisdictions where cannabis business is legal and regulated to some degree.

Concomitant acreage of hemp has quickly grown to more than 500,000 acres in the U.S. alone with much more under licensure. There have been many challenges associated with this ramp up in hemp cultivation starting with the definition of hemp as Cannabis sativa L. with a Δ9-tetrahydrocannabinol (Δ9-THC) concentration of not more than 0.3%. Some might strictly interpret that 0.3% to mean the Δ9-THC content only, others might take additional steps to account for tetrahydrocannabinolic acid (THCA) content. This is hardly the only point of confusion. Is moisture content taken into account? What is the analytical method? How are extracts and infused products regulated? Who performs this analysis? Each of these questions add layers of confusion and slow the growth of the industry.

Let’s consider the specification of Δ9-THC, for example. Most of the THC in the plant is not Δ9-THC but THCA. Therefore, if only Δ9-THC is considered, 17% of THC cannabis from the tightly regulated medical and adult-use cannabis industry in Nevada could also be categorized as hemp no matter how much THCA is present. If THCA is accounted for, a significant percentage of the resin hemp being grown under the current hemp industry expansion may be in a gray zone, since the high end of total THC content variation in these plants seems to be around 1% or higher.

The 0.3% THC distinction is artificial and creates confusion, but there actually are three distinct phenotypes of Cannabis sativa L. (see Figure 1) with an unmet need for easy discrimination. The molecular assay described in this article was developed as a DNA-based alternative to differentiate fiber hemp, resin hemp, and THC cannabis. This simple evidence based genetic test provides a clear picture of what hemp farmers are growing and thereby provides an alternative to the 0.3% THC cutoff as proposed in the 2018 Farm Bill.

Figure 1

The assay uses two single nucleotide polymorphisms (SNPs) that were previously identified by Henry in 2018 (2). Based on data from 192 cannabis DNA samples, we proposed the use of a set of two SNPs found in two disparate regions of the cannabis genome; the mitochondria and the THCA synthase gene (Table I). These two molecular markers provide a rapid and cost-effective means to differentiate fiber hemp, resin hemp, and THC cannabis.

Table 1

Materials and Methods

Cannabis sativa L. Plant Samples

Hemp samples including seed, leaf, and flower were collected in Vermont, North Carolina, Colorado, and Nevada for DNA extraction and testing on the Tru-Hemp ID assay (GroSciences). Additional analysis on Tru-Hemp ID markers was performed on samples accessed from retention held at Digipath Labs in Las Vegas, Nevada under a state approved method development R&D project. Additional analysis on Tru-Hemp ID markers was also performed at VSSL in Kelowna, British Columbia. The Tru-Hemp ID data output is available in real time, and the Solas 8 instrument (MatMaCorp) can be linked to a remote computer, or cellphone. Eight samples can be tested in parallel, and the reaction takes approximately 2 h. The associated DNA prep method adds about 1 h. Representative graphic displays of the amplification of the two observed outcomes for each of the two SNPs is shown in Figure 3.

Figure 3

  1. Text - H.R.2 - 115th Congress (2017-2018): Agriculture Improvement Act of 2018, (2018), (last visited Dec 26, 2018).
  2. P. Henry, The Journal of Brief Ideas (2018).
  3. C.S. Orser, Cannabis Science and Technology 1(1), 22 (2018).
  4. C.S. Orser, S. Johnson, M. Speck, A. Hilyard, and I. Afia, Natl. Prod. Chem. Res. 5(8),. DOI: 10:4172/2329-6836. 1000304 (2017).
  5. E. Small and A. Cronquist, TAXON 25(4), 405–435 (1976).

About the Authors

A. Hilyard, S. Johnson, and C. Orser are with Digipath Inc., in Las Vegas, Nevada. S. Lewin is with MatMaCorp Inc., in Lincoln, Nebraska. P. Henry is with VSSL, in Kelowna, British Columbia, Canada. Direct correspondence to: [email protected]

How to Cite this Article

A. Hilyard, S. Lewin, S. Johnson, P. Henry, and C. Orser, Cannabis Science and Technology 2(6), 42-46 (2019).