Scanning Regions of

Cannabinoid
Synthases to
Identify Relevant
SNPs
Christopher S. Pauli
 Cannabinoid Biosynthesis Background

• Cannabinoid synthase enzymes were identified

over 20 years ago
• (Shoyama, 1998)
• Previously thought to be a single-locus gene
• (de Meijer et. al., 2003)
• Gene duplications have been discovered –
suggesting multiple paralogs and psuedogenes
• (Weiblen et. al., 2015; Van Bakel et. al., 2018)
• We’ve identified over 30 copies of cannabinoid
synthase-like enzymes that are being classified
and studied.
Mandolino, G., & Carboni, A. (2004). Potential of
marker-assisted selection in hemp genetic
improvement. Euphytica, 140(1-2), 107-120.

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 The Current Cannabinoid Synthase-Like Family

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 The Structure of THCA Synthase
• 3-D Crystalized Structure Identifies
• Substrate Active Site Residues : CBGA
• Co-Factor Active Site Residues: FAD
• Signal Peptide Identification to Predict Tissue Specific Expression

https://www.rcsb.org/pdb/protein/Q8GTB6

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 Building From Previous Work
• Bp706 = G -> C
• AA236 = Glutamic Acid -> Glutamine
• Significant Reduction of THCA Bioaccumulation
• Shows active/inactive variation pattern

Onofri, C., de Meijer, E.P.M., and Mandolino, G. (2015). Sequence heterogeneity of CBDAS and THCAS in
Cannabis sativa L. and its relationship with chemical phenotype. Phytochemistry 116, 57–68

• Bp1426 = C -> T
• AA476 = Proline -> Serine
• Significant Reduction of CBDA
Bioaccumulation
• Greater overall production variation
with CBDAS modifications

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 Designing & Refining Molecular Markers

• Re-Iterative Primer Design Process

• Using Sequencing to Observe Variation in Amplicons
• Consistent Chemical Analyses Data Allows for Correlation
Studies

Sequence
• In-Silico PCR Analyses
• PCR on Various
• Compare Chemotype &
Representative Cultivars
Genotype
• Sequence Amplicons
• Identify Specific SNPs or
SNP Patterns
Initial Design
Chemical
• Target the Informative Validation
SNPs or SNP Patterns
• Correlate Haplogroups to
• Test Primers on Our
Chemical Content
Validation Set
• Identify Nearby Regions
as Potential Secondary
Markers
Re-Design

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 Identifying SNPs in the Paralogs

• Targeting unique regions of each synthase

paralog allowed us to identify relevant SNPs in
each gene copy.
• TASSEL 5.0 phenotype-genotype correlation
• Able to identify the SNPs that are most indicative of
cannabinoid content.
• Allows for better haplogroup discrimination of
our primers by selectively targeting more
informative regions.
• As we target more paralogs, we can better
determine specific SNPs in certain paralogs
that can be rapidly tested in large populations.

Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y., and Buckler, E.S. (2007).
TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23,
2633–2635.
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 Applying These Concepts
#1 #2

#3 #4

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 Summary of Our Marker Application

Strain Fingerprinting Marker Assisted Breeding

• Profiles the cannabinoid synthase genes • Guided approach to stabilizing

cannabinoid production and ratios
• Creates a unique barcode identifier
• Select for:
• Identify clones from siblings
• More & less active synthases
• Can determine if a plant and a flower are • Minor cannabinoid synthases
the same • Certain parental synthases
• Tell which offspring more closely • Developing the ability to help breeders
resembles each parent select the cannabinoid and terpene
profiles of future Cannabis strains.

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 Future Directions

• Extremely complex secondary

metabolite profile
• 480 identified secondary metabolites
(Verpoote, 2008)
• Target and classify upstream
biosynthetic enzymes
• Track and predict segregation of
cannabinoid synthases
• Create a database of functional
genetic markers
• Focused on medically or agriculturally
important traits

Andre, C. M., Hausman, J., & Guerriero, G. (2016). Cannabis sativa: The Plant of
the Thousand and One Molecules. Frontiers in Plant Science, 7.
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 What About Terpene Synthases?

• My colleague will speak on our work in the terpene biosynthetic pathway

• Dr. Keith Allen’s Talk
• Get a chance to see the most complete set of terpene synthases identified in Cannabis

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 Acknowledgements

Steep Hill Genetics Team: Collaborators:

• Dr. Reginald Gaudino • Dr. Nolan Kane & Lab Members
• Dr. Keith Allen • Agricultural Genomics Foundation
• Dr. Daniela Vergara • Cannabis Genomic Research
Initiative
• Dr. Robert Givens
• Dr. Thomas Blank
• Anthony Torres
• Christian Cizek
• Kimberlee Neubauer

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 Thank you for Attending

Contact Information:
Email: Christopher.Pauli@SteepHill.com
LinkedIn: https://www.linkedin.com/in/c-pauli/
CannabisChristopher.weebly.com

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 References

• Shoyama, 1998
• https://sci-hub.tw/https://doi.org/10.1016/S0031-9422(98)00278-7
• Verpoorte, 2008
• https://link.springer.com/article/10.1007/s11101-008-9094-4
• Guerriero, 2016
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740396/
• Gene Model of THCAS
• https://www.rcsb.org/pdb/protein/Q8GTB6

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