Non Technical Summary
The promise of hemp, with thousands of possible uses, potential suitability to numerous environments, and emerging, fast-growing new product markets, could be a welcome alternative crop for many producers. However, despite significant developments in the U.S. hemp industry over the past several years, there remain substantial gaps in the knowledge needed to fully support this fledgling industry. A recent survey of over 1,100 hemp stakeholders found that over 75% think additional research in breeding and genetics to produce stable and uniform cultivars and regional adaptability is very or extremely important. This is directly related to seed characteristics and considerations such as germination rate, dormancy, shelf life, state certification, cost, and quality. Genetic improvement of cultivars will have a direct effect on yield and production cost and therefore the return on investment for stakeholders. In order for public and private breeders and scientists to develop regionally adapted hemp cultivars it will be necessary for them to have access to a broad range of germplasm. Initial characterization of hemp germplasm shows a high level of genetic diversity in naturalized hemp populations compared to cultivated Cannabis, likely due to selection pressures and genetic drift in different regional populations. The purpose of this research task is to build and characterize a genetically diverse compliant collection of feral hemp populations that are adapted to environmental conditions in the regions where they have been growing since escaping from agronomic cultivation. Through the engagement and use of citizen scientists and regional collaborators we will be able to cover more ground than any small group of individuals. This collection will serve as a public resource for the characterization of genes and mining of alleles for beneficial traits in breeding new cultivars.
Animal Health Component
Research Effort Categories
Goals / Objectives
Overall Goal: Our integrated research and extension goal is to collect a geographically distributed range of genetically diverse and THC-complaint hemp germplasm that will kick start research and breeding efforts, while collaborating with and educating citizen scientists and stakeholders about industrial hemp and its potential downstream uses.Specific ObjectivesObjective 1: Collect seed and passport data from feral hemp populations representing the diversity of ecosystems in the United StatesObjective 2: Characterize cannabinoid traits for regulatory compliance and seed bankingObjective 3: Distribute a core collection of compliant discovery populations to collaborators for trait identification and breeding efforts
Obj 1 Experimental approach:Population identification-We will enlist citizen scientists to identify, verify, and collect site specific data for geographically distributed feral hemp populations. Specifically, we will use the iNaturalist application, which is free, easy to use, and one of the most popular nature applications with over one million registered users.In addition to using the citizen scientist approach, we have identified key regional partners who will assist with regional documentation of feral populations and collections. These partners will work with their local governments, state agencies, farmer groups, and nature groups to identify additional populations.Population collection-The country will be divided into geographic regions of interest based on the National Health and Environmental Effects Research Laboratory U.S. Environmental Protection Agency Level III Ecoregions. These ecoregions are areas where ecosystems and the type, quality, and quantity of environmental resources are generally similar. Regional collaborators have been selected based on geographic distribution and will assist with collection efforts in the late summer of 2022 and 2023. We will target ~50 ecoregions with 5 populations per region for a total of 250 geographically dispersed populations. All collaborators are familiar with their state licensing policies and must have a license for each year they are involved with collections. We will provide a collection protocol and list of supplies for each collaborator. The protocol will consist of harvesting seed from up to 50 female plants per population.Analysis:Passport data collected via iNaturalist and from regional collaborators will use standard Multi-Crop Passport Descriptors (MCPD) which has been developed as a uniform, global format. Analysis of materials is described further in Objective 2 and 3.Obj 2 Experimental Approach:Genetic Testing-We propose to pre-characterize every seed source (maternal plant) individually and a large random sample of seed from hemp-type maternal plants for each population. Testing maternal parents excludes drug-type genetics from the maternal line (dam). Seedling tests can estimate the frequency of the drug-type allele in the population using the paternal line (sire). Taken together, the two levels of testing can identify the best candidate populations for seed banking as well as populations of special concern.A single leaf taken from each female plant for which seed is harvested, stored separately in a coin envelope and individually indexed to the seed lots will be genotyped. Genomic DNA from the leaf sample of each female plant will be isolated using a CTAB protocol adapted from (Doyle and Doyle 1987). A portion of the gDNA will be used to determine the CBDAS genotype of the maternal field plants using the low cost manual CAPS assay described and validated by Wenger et al. (2020). One individual, determined by the cannabinoid profiling to be CBD-type and verified by the CAPS assay to be homozygous for functional CBDAS, will be selected as seed source for further genetic characterization. Approximately 5 ug of the isolated gDNA from each of the field collected plants from each site will be shipped to PI Ellison at UW-Madison for GBS analyses (see Objective 3).From the selected CBD-type individual for each collection site, we aim to germinate 100 seeds and maintain seedlings for two weeks of vegetative growth. True leaves of each seedling will be harvested, dried in the lab at ambient conditions, and submitted to the University of Minnesota Genomics Center (UMGC) where gDNA will be isolated after bead-shake pulverization using a CTAB protocol adapted for high-throughput (HTP). From gDNA, the CBDAS genotype of each seedling will be scored at UMGC using Illumina-based targeted amplicon sequencing.Cannabinoid Testing-Cannabinoid screening of a subset of seed heads for each population will serve several purposes. First, cannabinoid profiles will maintain quality control of the genetic assay. Second, the CBD:THC ratio test is insufficient to measure overall cannabinoid content (TCC). Third, we will have comparative data on variation in cannabinoid levels across the geographic distribution of source populations. Lastly, field measures of THC are required for state and federal compliance while aiding local collaborators and officials in decision-making should non-compliant populations be identified as a public safety concern.From each location from which feral seed is collected, seeded inflorescences (colas) from a minimum of ten individual female plants (10 plants per site where N = 50, otherwise 20% of N) will be reserved as indicated in Objective 1. Seeded inflorescences will be dried by collectors and shipped to UMN for determination of cannabinoid content of six cannabinoid compounds (CBC, CBD, CBG, d8-THC, d9-THC, THCV) using GC-MS. All cannabinoid phenotyping is performed in the blind with respect to provenance and sample codes to minimize the potential for investigator bias. Seed from each individual plant inflorescence will be separated from maternal tissue and held individually to allow subsequent genetic analyses of seed informed by maternal cannabinoid phenotype (THC:CBD ratio and TCC).Data Analysis and Application-For plants collected in the field by regional collaborators, CBDAS locus genotypes will be called by direct inspection from CAPS assay gel experiments (Fig. 1B, Wenger et al. 2020) and chemotypes will be determined using % dry weight inflorescence fractions as x = log(%THC/%CBD) where values of x >= 1 are THC-type, x =< -1 are CBD-type, and 1 > x > -1 are intermediate-type. CBDAS genotypes of seedlings will be called by UMGC from Illumina-based targeted amplicon sequencing using a marker assay (primer set) validated against the Weiblen laboratory's THC-type x CBD-type F2 mapping population (Weiblen et al. 2015). CBDAS genotype and allele frequencies of seedlings will be calculated using JMP Pro 14.2.0 (SAS, Cary, NC, USA) and tests of departure from Hardy-Weinberg equilibrium will be performed using a likelihood ratio test implemented in ExactoHW 1.1 software (Engels 2009).Obj 3 Experimental approach:We will acquire gDNA from Objective 2 from three of the original collected female plants per population that proved compliant from both cannabinoid screening and CBDAS genotyping. The gDNA will be genotyped-by-Sequencing on an Illumina NovaSeq 6000 at the University of Wisconsin Biotechnology Center DNA Sequencing Facility. Single nucleotide polymorphisms will be called using the GBS TASSEL pipeline using the Cannabis sativa reference genome CBDRx assembly (project PRJEB29284) (Glaubitz et al. 2014, Grassa et al. 2021). SNPs will be filtered for quality, depth, and minor allele frequency.Analysis:To better understand genetic relatedness, a series of analyses will be conducted including population structure (Alexander and Lange 2011), phylogenetic relatedness (neighbor-joining tree in PHYLIP), Principal Component Analysis (SNPRelate), Nei's gene diversity, Shannon's information index, and polymorphism information content (PICcalc). The consensus number of subpopulations will be used for AMOVA and Nei's genetic distance, fixation index (Fst). In addition, genetic indices such as number of loci with private allele, number of different alleles (Na), number of effective alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho) and expected heterozygosity (He) will be calculated with the R package Poppr (Kamvar Z. N. 2014). Core collections will be made using Core Hunter 3 (http://www.corehunter.org/).The project will be evaluated based on the number of collaborators (20+), citizen scientists (+50), ecoregions coverage (+50), hemp samples obtained (+100), hemp samples distributed to researchers (+50), and hemp samples distributed to the hemp germplasm repository (+100).