Progress 09/01/23 to 08/31/24
Outputs
Target Audience:We have reached several dozen citizen scientists over iNaturalist to help identify and collect feral hemp populations. Citizen scientists were educated through emails and virtual meetings about the importance of making these collections to preserve germplasm and fill the new hemp seed bank to initiate research and breeding programs. We also presented our work to fellow scientists and plant breeders through conferences and symposia to describe the current state of collections and enlist new collectors for the upcoming year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral researcher working on this project participated in the Open Science Grid summer school in 2023, a high-throughput computing course, and the UW-Madison Biotechnology courses "Intro to Linux command line" and "Next-generation sequencing analysis". They were also able to present preliminary findings at conferences and field days, gaining skills in scientific communication for both scientific and general audiences. Undergraduate researchers in the lab were trained in molecular biology techniques, data collection and data analysis. Citizen scientists received one-on-one training from the funded postdoc to collect feral hemp accessions. Meetings usually occurred over Zoom and the collection protocol was discussed as well as emphasizing the importance of the work and their contribution to germplasm preservation. How have the results been disseminated to communities of interest?Our preliminary results were presented a several symposia and conferences including: G. Weiblen "A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana" Packard Fellowship in Science & Engineering Meeting, Colorado Springs, Colorado, September 2023. Oral Presentation A. Aina "Genotypic and Chemotypic Diversity of Feral Hemp Germplasm" Plant and Animal Genome Conference. San Diego, CA Jan 12-17, 2024. Oral Presentation G. Weiblen "Cannabis conundrum: science & politics of America's most controversial plant" Hort 240 Science of Cannabis, University of Wisconsin-Madison, February 2024. Guest lecturer. G. Weiblen "Cultivating academic hemp research with indigenous collaborators: reflections on the past seven years" Great Lakes Indigenous Farming Conference, Grand Rapids, Minnesota March 2024, Oral Presentation S. Ellison Emerging Crops Showcase "Exploring Hemp in Wisconsin: Breeding, Research, and Commercialization" April 2, 2024. Oral Presentation S. Ellison "American Feral Hemp GermplasmCollection and Characterization" Institute of Cannabis Research, CSU Pueblo, February 21, 2024, Oral Presenation S. Ellison "Feral Hemp Germplasm and Genetic Diversity", 2024 Cornell Hemp Webinar Series May 2024 Oral Presentation Aina A. "Population Structure and Chemotypic Diversity of American Feral Hemp Germplasm" Botany 2024 Conference, Grand Rapids, MI June 15-17th, 2024. Oral Presentation Abendroth J. "Cannabis sativa L. Germination Response to Various Seed Pre-Treatments" National Association of Plant Breeders St. Louis, MO July 21-15, 2024. Poster Presentation Weiblen G. "Marker Assisted Selection to Achieve Industrial Hemp Regulatory Compliance". Cannabis Research Conference - Denver, CO August 7-9th, 2024. Oral Presentation Ellison S. "Genetic Diversity and Population Structure of US Feral Hemp". Cannabis Research Conference - Denver, CO August 7-9th, 2024. Oral Presentation Wenger JP. "Variation in the THC:CBD Ratio Among U.S. Feral Cannabis Populations". Cannabis Research Conference - Denver, CO August 7-9th, 2024. Oral Presentation S. Ellison and A. Aina. UW Madison Hemp Research Field Day - Arlington, WI August 20th, 2024. Oral Presentation The project was also discussed during guest lectures at various cannabis courses for undergraduates at UW Madison, Gateway Technical College, and Delaware Valley University. Student learned about the importance of germplasm preservation and some even enlisted friends and family members to help make seed collections. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 - Complete Objective 2 - Dr. Weiblen's group will test the remaining 2023 samples for cannabinoid content using GC-MS and summerize compliancy data. Objective 3 - In the next year we will finish all population genomics analyses (e.g. ADMIXTURE, PCA, DAPC) to determine population substructure across all collected feral samples and use genetic diversity statistics (e.g. AMOVA, FST, Nei's genetic distance) to determine genetic distance and relatedness between feral hemp populations. Seeds will be considered "hemp" or "drug-type cannabis" and submitted to Dr. Stansell for inclusion into the USDA hemp germplasm collection and a core collection of genetically unique samples will be redistributed to collectors to initiate regional breeding efforts. A planned submission to Scientific Reports by Aina et al. "Population Structure, Genetic Diversity, Cannabinoid Compliancy, and Signatures of Selection in American Feral Hemp Germplasm" - is also anticipated to disseminate our research project protocol and findings to interested communities. We will also phenotype the entire feral collection at the Arlington Ag Research Station in Arlington, WI in the fall of 2024. This data will be shared with the USDA to be included in the GRIN database.
Impacts
What was accomplished under these goals?
The purpose of this research task is to build and characterize a genetically diverse collection of feral hemp populations that are adapted to environmental conditions in the regions where they have been growing since escaping from agronomic cultivation almost a century ago. We have already been able to successfully collect over one thousand five hundred feral hemp samples and find there is both significant genetic and phenotypic diversity across this collection. Additionally, we find most of these materials are THC compliant and they will be able to be immediately used by the hemp research community. These samples will serve as a resource for the characterization of genes and mining of alleles for beneficial traits in breeding new hemp cultivars. The genetic improvement of hemp cultivars will provide farmers with another option to diversify their current cropping system to improve their economic and environmental options and outcomes. Objective 1: Collect seed and passport data from feral hemp populations representing the diversity of ecosystems in the United States Collection and Sequencing An additional ~500 feral hemp accessions were collected from 8 states for a total collection of ~1,500 accessions from 12 states. Collections were made with the assistance of over 50 citizen scientists. These new materials were used for chemotyping and genotyping in objectives 2 and 3 below. Objective 2: Characterize cannabinoid traits for regulatory compliance and seed banking Characterization of Cannabinoid Traits Wild collected feral flower samples were chemotyped for cannabinoids using the PACE genotyping protocol (Toth et al., 2020). The majority of the sampled feral populations across the Midwest were compliant with CBD-type alleles while one population showed a fair amount of all three groups (CBD-type, Intermediate, and Drug-type). To date, 31 samples have been characterized as THC (Type I), 80 as Intermediate (Type II), and 505 as CBD (Type III). Every population collected to date contains samples that are Type III indicating they can be directly used or "cleaned" for redistribution through the USDA GRIN collection. In addition to chemotyping, 96 samples were screened for cannabinoid content using GC-MS. Chemotype and GC-MS results are congruent with Type I plants having the highest concentrations of THC and Type III plants having low to no THC content. The average amount of total cannabinoids, CBD, and THC for all samples is 1.16%, 0.87%, and 0.13%, respectively. Objective 3: Distribute a core collection of compliant discovery populations to collaborators for trait identification and breeding efforts Population Structure and Genetic Diversity Statistics A subset of 384 samples from the 2023 collection were submitted for Genotyping by Sequencing (GBS) to detect single nucleotide polymorphisms (SNPs). This dataset has been merged with the 2022 samples for a collective dataset of 768 individuals and over 150,000 markers. With a 0.05 minor allele frequency and a minimum call rate of 0.1 (i.e., no more than 10% of the genotyped feral accessions in the germplasm panel should have missing data) 80,000 SNPs were called on the entire dataset. These initial results shows significant genetic diversity was present in our collection of feral hemp samples and five major clusters were identified. Principal component analysis indicated that PC1 and PC2 explained 14.7% and 10.4% of the genetic variation among the accessions. Admixture analysis revealed five populations, one unique Indiana population, one unique New York population and three additional subpopulations. One of the subpopulations consisted mostly of WI, MN and IL feral hemp, another had mostly SD, ND, NE, and CO while the other had mostly KS, MO, and IA feral hemp. Discriminant analysis of principal component results corroborated Admixture results with a similar clustering patterns. Fixation indices values between populations (> 0.15) indicated that the populations were significantly different from one another.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Ford, T., Aina, A., Ellison, S., Gordon, T., & Stansell, Z. (2024). Utilizing digitized occurrence records of Midwestern feral Cannabis sativa to develop ecological niche models. Ecology and Evolution, 14, e11325. https://doi.org/10.1002/ece3.11325
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:We have reached several dozen citizen scientists over iNaturalist to help identify and collect feral hemp populations. Citizen scientists were educated through emails and virtual meetings about the importance of making these collections to preserve germplasm and fill the new hemp seed bank to initiate research and breeding programs. We also presented our work to fellow scientists through conferences and symposia to describe the current state of collections and enlist new collectors for the upcoming year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral researcher working on this project was able to receive collaborative research opportunities by working with another lab to gain exposure to the chemotyping assay. They then brought that back to our lab and were able to train others and gained skills in mentoring. They were also able to present preliminary findings a research symposium and field day, gaining skills in scientific communication for both scientific and general audiences. They will also be attending a week long bootcamp on high throughput computing in August 2023. Undergraduate researchers in the lab were trained in molecular biology techniques and data analysis. Citizen scientists received one-on-one training from the funded postdoc to collect feral hemp accessions. Meetings usually occurred over Zoom and the collection protocol was discussed as well as emphasizing the importance of the work and their contribution to germplasm preservation. How have the results been disseminated to communities of interest?Our preliminary results were presented a several symposia and conferences including the 2022 UW Madison Hemp Research Symposium and the Cannabis Research Conference - Denver, CO. The project will also be showcased at the UW Madison Hemp Research Field Day - Arlington, WI on August 17th, 2023 and UW Madison Plant and Agroecosystem Sciences and Soils Arlington Field Day - Arlington, WI on August 30th, 2023. The project was also discussed during guest lectures at various cannabis courses for undergraduates at UW Madison, Gateway Technical College, and Delaware Valley University. Student learned about the importance of germplasm preservation and some even enlisted friends and family members to help make seed collections. What do you plan to do during the next reporting period to accomplish the goals?During the late summer and fall of 2023 we will again enlist our collection collaborators to obtain feral hemp samples across the country. These samples will be sent to Dr. Weiblen's group where his team will test flower for cannabinoid content using HPLC and extract DNA from leaves. The DNA will be used to determine chemotype and the remaining DNA will be sent to Dr. Ellison's group for Genotype by Sequencing (GBS). Seeds will be considered "hemp" or "drug-type cannabis" and submitted to Dr. Zac Stansell for inclusion into the USDA hemp germplasm collection. After all feral hemp has been genotyped, samples will be analyzed using population genomics pipelines to determine genetic relatedness. A core collection of genetically unique samples will be redistributed to collectors to initiate regional breeding efforts. A planned submission to Advances in Conservation and Utilization of Plant Genetic Resources Special Edition - Frontier in Plant Science - is also anticipated to disseminate our research project protocol and findings to interested communities.
Impacts
What was accomplished under these goals?
The purpose of this research task is to build and characterize a genetically diverse collection of feral hemp populations that are adapted to environmental conditions in the regions where they have been growing since escaping from agronomic cultivation almost a century ago. We have already been able to successfully collect over a thousand feral hemp samples and find there is both significant genetic and phenotypic diversity across this collection. Additionally, we find most of these materials are THC compliant and they will be able to be immediately used by the hemp research community. These samples will serve as a resource for the characterization of genes and mining of alleles for beneficial traits in breeding new hemp cultivars. The genetic improvement of hemp cultivars will provide farmers with another option to diversify their current cropping system to improve their economic and environmental options and outcomes. Objective 1: Collect seed and passport data from feral hemp populations representing the diversity of ecosystems in the United States Collection and Sequencing One thousand and fifty (n = 1,050) feral accessions were collected from eight midwestern states in the late summer and early fall of 2022. Objective 2: Characterize cannabinoid traits for regulatory compliance and seed banking Characterization of Cannabinoid Traits Field-collected feral flower samples were chemotypedfor cannabinoids using PACE genotyping protocol (Toth et al., 2020). The majority of the sampled feral population across the Midwest were compliant with CBD-type alleles while one population show a fair amount of all three groups (CBD-type, Intermediate, and THC-type). Five to 10 flower samples from each collection were submitted to U. Miss for HPLC in May 2023. Objective 3: Distribute a core collection of compliant discovery populations to collaborators for trait identification and breeding efforts Population Structure and Genetic Diversity Statistics A subset (n = 453) of samples were used for DNA analysis. Genotyping by Sequencing (GBS) was employed for single nucleotide polymorphism (SNPs) marker development of a total of 98,298K SNPs. With a 0.05 minor allele frequency and a minimum call rate of 0.1 (i.e., no more than 10% of the genotyped feral accessions in the germplasm panel should have missing data), ~35K SNPs were used for further analysis. Data were analyzed using software ADMIXTURE (Alexander et al., 2009). Discriminant analysis of principal components and principal component analysis were performed using adegenet package in R (Jombart et al., 2010; Jombart and Ahmed, 2011). Analysis of molecular variance was also calculated with poppr package in R (Kamvar et al., 2014). Nei's genetic distances and Fst statistics were calculated using hierfstat package (Goudet 2005; Goudet and Jombart, 2015). These initial results shows significant genetic diversity was present in our first-year collection of feral hemp samples and three major clusters were identified. Principal component analysis indicated that PC1 and PC2 explained 15.7% and 9.21% of the genetic variation among the accessions. Admixture analysis revealed three populations, one unique Indiana population, and two additional subpopulations. One of the subpopulations consisted mostly of WI and IL feral hemp while the other had mostly NE and IA feral hemp. Discriminant analysis of principal component results corroborated Admixture results with a similar clustering patterns. Fixation indices values between populations (> 0.15) indicated that the populations were significantly different from one another. AMOVA results also indicated variation within samples, between populations, and between samples within a population.
Publications
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