In recent years, Cannabis sativa has garnered attention for its myriad of uses beyond recreation. Its well-supported medicinal benefits were first documented in China more than 10,000 years ago.1 Since then, the plant has shown to be effective in the treatment of conditions like Alzheimer’s disease, epilepsy, and glaucoma, to name a few.2
Beyond medicine, C. sativa is part of a long history of domestication and selective breeding. Humans have produced hundreds of strains designed for a variety of practical uses like building materials and textiles.3,4 However, safeguarding the future of C. sativa depends on leveraging diverse cultivars that can overcome environmental threats like drought and disease.
To that end, researchers and breeders need tools to genetically assess C. sativa samples and make informed cultivation decisions. Next-generation sequencing (NGS) is the key to unlocking strategies that preserve plant samples for these therapeutic and industrial uses.
Complexities of Cannabis sativa
One of the challenges of investigating genetic diversity in C. sativa is how to classify strains. The plant is often divided into three species—C. sativa, C. indica, and C. ruderalis.5 Each can interbreed and produce hybrid varieties. As a result, there are more than 700 known strains. These strains differ in their ratio of cannabinoids, which, at the time of publishing, is a class of about 104 compounds.6 Moreover, factors like age, environment, harvesting time, and storage methods can alter the composition of C. sativa secondary metabolites.7
C. sativa is predominantly a dioecious plant, with some crops having female flowers and others having male flowers. To produce and propagate a specific C. sativa strain that has a desired trait, plants with the genetic variation associated with that trait must be genetically isolated and then selectively bred in isolation to reinforce and pass on that trait.8 Such conventional breeding practices are subject to difficulty in predicting phenotypes. Breeding requires monitoring offspring over several generations, which could lead to loss of genetic stability over time.
With the expansion of legalization of C. sativa for medicinal uses, maintaining and managing genetically diverse strains will be necessary to safeguarding environmental resilience. Strategies for preserving this genetic diversity include:
- Conserving landraces (locally adapted varieties that have evolved naturally in specific regions)
- Sharing of genetic repositories and resources
- Avoiding excessive inbreeding
- Leveraging advanced NGS methods for genetic studies to inform breeding decisions
Given that most C. sativa strains that exist today could not survive in the wild, humans must exercise responsible cultivation guided by genetic information.
NGS Methods for Optimized Research
DNA sequencing and molecular genotyping have proven to be powerful methods.9 Low-coverage whole genome sequencing (WGS) is a cost-effective genotyping approach that uses a relatively shallow depth of coverage while sequencing the entire genome. It provides genomic information on a large scale to aid in classification.10
The purePlex DNA Library Prep Kit supports truly multiplexed and highly scalable construction of library pools for low-pass WGS. This scientific poster demonstrates the effectiveness of purePlex in generating reproducible library characteristics of C. sativa despite sample plant tissue origins.11
Molecular genotyping is a tool that allows scientists to gain insight into the genetic makeup of crop plants. Innovations in this space are ushering in a new era of possibilities, from the rapid analysis of crops and livestock to the identification of traits via DNA sequencing.
For example, within the last twenty years, simple sequence repeats (SSRs), also called microsatellites, have been widely employed for plant genotyping.12 SSRs consist of highly polymorphic short repeated sequences of nucleotides that represent variation at one locus of a chromosome. Since SSRs show mutations at higher rates than sequences that do not repeat, this is a reliable and versatile tool for assessing genetic diversity. These markers enable researchers to analyze population structure, identify parentage, and map important traits or genes of interest.
NGS innovations like these make single nucleotide polymorphism (SNP) analysis (looking at variations in a single nucleotide) a more viable technique for higher resolution analysis of C. sativa genetics. In one study, researchers used genetic mapping with nearly 10,000 SNPs to uncover a gene related to resistance of powdery mildew, one of the most common fungal diseases affecting C. sativa.13
Another NGS technique that can boost the genetic diversity of C. sativa is CRISPR/Cas9 gene editing. This method can be used to alter concentrations of specific cannabinoids or other compounds and produce new varieties for pharmaceutical development, improve productivity, and enhance disease and climate resistance.
With transposase-based solutions like seqWell’s Tagify UMI reagents, researchers can create QC benchmarks that enable safer and more effective gene editing.
Investing in the Future of Cannabis sativa
From 2000 to 2018, cannabis research funding in the United States increased from roughly $30 million to $143 million.14 However, consistency across research is needed to improve the reliability and meaningfulness of subsequent findings.15
C. sativa has proven to have a long tenure as a cornerstone crop for a wide range of uses. As legality continues to expand, collaboration between research institutions, healthcare providers, policymakers, and regulatory bodies is crucial to conserve this plant’s genetic diversity for future generations.
In turn, demands for producing accessible and accurate methods that bolster research and strategic breeding decisions will only continue to grow.
NGS technologies, like seqWell’s purePlex and Tagify tools, enable higher resolution studies, which can uncover traits of interest, guide breeding decisions, and enable efficient and comprehensive DNA sequencing and genotyping. These insights have the potential to boost the genetic diversity and, thus, the potential of C. sativa.
- Genetic diversity and future prospects