Progress 01/15/23 to 01/14/24

Outputs
Target Audience:The target audience for this project includes research scientists in the field of hop and hemp research, as well as members of the hop and hemp industry. We have created a public database of existing data on terpene levels for different cultivars of hop and hemp found in the literature. We have data from at least 6 articles related to terpene production in hop and hemp. We have created an interactive website for users to select different terpenes of interest, as well as different cultivars of interest. One of the challenges in the field is that almost all the experiments done have used different cultivars. The website is still in draft form, but is available here:https://cannabase.cqls.oregonstate.edu/ We have also collected flower samples from hemp and hop plants and are growing hop samples currently in greenhouses. This tissue collection is the basis of the proposed experiments that have been conducted. Samples have been taken, and going forward they will be analyzed and shared on our interactive website. More detail on the collected samples will be provided in the other parts of this report. Changes/Problems:Things are going well for the project. However, I just had a baby born in December that has kept my hands full. So I apologize that this is late! What opportunities for training and professional development has the project provided?Training graduate students in RNA extraction, bioinformatics, and presenting posters and seminars. Graduate students attended PAG this past year, and presented posters. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to analyze the data collected and integrate the GCMS and gene expression data from RNA-seq. We will identify the most diverging cultivars from hop and hemp in terms of expression of different compounds, with particular focus on compounds that are important for the hop and hemp industries and to their flavor and aroma. A crucial part of this work is in labeling the different compounds. We will also use the orthologous genes between hop and hemp related to terpene synthesis that we have already identified in this analysis to identify commonalities between hop and hemp.

Impacts
What was accomplished under these goals? Data Analysis We have collected data from published GCMS and LCMS experiments on hop and hemp for the creation of our web-accessible database and for further analysis. We will compare levels from prior papers to our collected data when conditions are appropriate and similar extraction techniques are used. Furthermore, we have identified orthologs between hop and hemp for the terpene synthase genes, which will enable comparative analysis of these genes. Hemp Collection We have collected paired female flower samples from diverse hemp accessions for parallel analysis of cannabinoid/terpene profiles and gene expression. They were immediately flash frozen on dry ice, then stored at -80C. To determine terpene-related gene expression patterns, total RNA was extracted from female flowers of 22 hemp accessions (Table 1). Almost all accessions are CBD cannabinoid producers. The RNA samples have been submitted for Illumina sequencing on the NextSeq2000 at the OSU Center for Quantitative Life Sciences. They will be barcoded and pooled prior to sequencing to a transcriptome coverage depth of at least 30x. Table 1. Hemp accessions submitted for transcriptome sequencing. Cultivar Charlottes Angel CBD Nurse Lily CBD Clinical White SDSD 32 B2B2-001-031 Spectrum EC 11 WICW-041 TRTR-009 CTCT 38 ABAB 032 CBG #1 SGCW-005 Green Mango CBD CBCW 8 771-B-000-104 CBCB-006 WICW-061 BaOx NCBD 019 CHCW 012 CFCF 43 Erdpurt Hop Collection Cultivar Fuggle N Cascade Mt Rainier Teamaker Hallertauer Tradition Saazer Galena Tardif de Bourgeage Horizon Sorachi Ace Aurora Brewers Gold Centennial Pride of Kent Wye Target Comet Kitamidori Whitsbred Golding Triumph Chinook Hallertauer Mittelfrue Northern Brewer Nugget Backa Wye Viking Perle A set of of 25 hop cultivars were selected and cloned and then planted into 5-gallon pots for producing hop flowers under field conditions at the USDA-ARS Hop Breeding Farm located outside of Corvallis, OR. Plants were arranged in a randomized complete block design on a low-trellis (8-ft) production system using drip irrigation. Filled capacity in the soil water within pots was maintained at all times. Plants were trained up string to the top of the trellis and then the terminal buds were cut to initiate and stimulate side-arm production for increased flowering. Flowers were collected from lower, mid and upper canopy during mid-September. Flower samples were placed in aluminum foil and immediately flash frozen in liquid nitrogen. After flash-freezing samples they were then placed in a cooler filled with dry ice with temperature maintained at approximately -20 deg C. All flower samples were collected during the peak temperature of the day (10-2 PM) for highest expression of floral volatiles. Once flowers were collected they were transferred to a -80 deg C freezer for storage prior to GC/MS and transcriptome assays. GCMS Data Collection 50 samples consisting of approximately 5 grams each of mature floral tissue was collected in duplicate from 25 unique hemp cultivars grown in the greenhouse. Samples from 25 unique cultivars of hop were collected in triplicate in approximately 10-gram quantities from plants grown in an outdoor plot. Samples were quickly frozen in liquid nitrogen and stored in a -80oC freezer until processing. A common protocol was developed for tissue processing and extraction of hemp and hop samples by experimentally evaluating various factors including homogenization method, solvent type, ratio of solvent to tissue, duration and temperature of extraction, sonication length, internal standard used, and sample stoarge. The efficiency of these different treatments was evaluated by running the resulting extracts on GC-MS using identical run parameters and comparing the relative composition of the volatile constituents (obtained by integration values of identified major chromatographic peaks) to those reported in the literature from the same cultivar. This was primarily done using the hop cultivar 'Cascade' for these tests. GC-MS run parameters were optimized by testing three different protocols to obtain sufficient sensitivity and resolution to identify chromatographic peaks for even minor constituents of hemp and hop previously reported in the literature. The protocols tested had total run times of 25, 39, and 86 minutes, respectively. The optimal protocol used a slightly modified version of the GC parameters recommended for evaluating hop volatile oils by the American Society of Brewing Chemists. Samples were run on a GC outfitted with a DB5-MS column paired with a single-quadrupole MS. The run protocol consisted of a splitless injection of 1 µL sample volume with a starting oven temperature of 50°C, a ramp rate of 3°C/min up to 260°C, followed by a 15 min hold. The carrier gas (helium) flow rate used was 0.6 mL/min and the total run time was 86 minutes. The MS scan range was set to 20-500 m/z. To confirm that our data in this study will be comparable to results from previous reports that have used hydro-distillation to obtain essential oils from hop for analysis, we ran a comparison between essential oil and solvent extracts both derived from the hop cultivar 'Comet.' Analysis showed that the relative levels of key compounds were similar with the most notable differences attributable to the presence of higher boiling-point compounds in the extract which were absent from the distillate. ?Solvent extracts from all 25 hemp cultivars have been processed and run on GC-MS and the majority of hop samples have also been analyzed. Tissue samples were processed and prepared in batches of 12 at a time. A first GC-MS run consisting of 12 sample injections and four blank injections to monitor sample carry over (an approximately 24-hour run) was started on the same day the extracts were transferred to vials. Duplicate vials for each extract sample were stored at -20oC until the first run was completed after which they were initiated in a second 24-hour run to obtain technical replicates for each sample. The sampling order for these runs were randomized within each group to account for differences due time spent in the autosampler prior to injection. Agilent Masshunter Qualitative Analysis (B.06.00) software was used to analyze chromatograms and obtain quantitative data for each sample. The ChemStation integrator method was used with default settings except that the Peak Filter parameter was set to a minimum absolute height of 1,000 counts and m/z peaks of approximately 28 and 32 (representing nitrogen and oxygen in air, respectively) were excluded both from peak integration and mass spectrum extraction. Only peaks eluting from the column in the range of 6-35 minutes were integrated to filter out compounds that would be absent from hydro-distilled essential oils. The raw datafiles were converted to mzML file format using msConvert and the resulting files were analyzed using mzMine (ver. 2.5.3). Features were picked, combined, and aligned using standard parameters. Principal component analysis (PCA) was subsequently performed. As expected, hop and hemp samples clustered together, while hemp was separated into two groups. Given the raw GC-MS data files, this was the expected result indicating our PCA was performing as expected. The second observation is that the biological and technical replicates from a given cultivar clustered together as expected. In the sample we can immediately identify two hemp cultivars that are significantly different from the majority of hemp samples. These samples will be submitted for RNAseq analysis. The remaining hemp samples, when analyzed separately have differences that can be observed (data not shown) and this has lead to the selection of other hemp samples for RNAseq analysis.

Publications

Progress 01/15/22 to 01/14/23

Outputs
Target Audience:The target audience for this project includes research scientists in the field of hop and hemp research, as well as members of the hop and hemp industry. Because this is a three-year project that has effectively two-years of funds, and because of some challenges with growing plants in the first year, we have moved the bulk of the work to years 2 and 3 of the project. This will also make it more feasible to maintain salaries of trainees employed by this project without a "gap year". For this reason, there is nothing to report regarding our audience. That said, we have initiated work on a public database of existing data on terpene levels for different cultivars of hop and hemp found in the literature. We have collected 13 articles with at least 13 associated data tables, one from each paper. The challenge with this is that the publications typically embed these tables in the publication PDF file, and they aren't always easily collected and require some manual typing of values into a unified format for collection and for presenting in our database. We have also collected paired samples from hemp plants, and are growing hop samples currently in greenhouses. This tissue collection is the basis of the proposed experiements that we will conduct this year. More detail on the collected samples will be provided in the other textboxes of this report. Changes/Problems:As stated previously, we sought to keep things uniform for hop and hemp, we wanted to grow them both under the same conditions. However, we could not get approval to grow hemp outdoors, leading us to grow both hop and hemp in greenhouses. This proved to be more challenging than anticipated for hop cone (flower) collection and these plants are still growing. That said, we expect that we will be on track with hop samples collected and analyzed by the next report date. However, these challenges only minimally affect our progress. The project is a three-year project, but with 2 years of funding to carry out the work. Therefore, it is beneficial for us to move the work to years 2 and 3 for the project, which will avoid any "gap years" for personnel or trainees. We are in the fourth month of year 2, and therefore are making good progress. What opportunities for training and professional development has the project provided?The work has several opportunities for professional development. This includes, plant husbandry, growing plants in the greenhouse, sample collection, data analysis, and data curation. Because this is a three-year project with two years of funding, we have put the work of the project in years 2 and 3, to avoid a "gap year" for paying salaries etc. At the moment, we are beginning the work of the proposed project, and therefore have little to report, but expect this to change in next year's progress report. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We expect to have a manuscript submitted, or at least in preparation for the next reporting period. The sample and data collection will happen over this summer and fall, and we expect to have data collected by Fall term. We will then work towards publications and disseminating our results.

Impacts
What was accomplished under these goals? Overview of Sample Collection We have collected flower samples from hemp, but are still working towards collecting flower samples from hops. Because we sought to keep things uniform for hop and hemp, we wanted to grow them both under the same conditions. However, we could not get approval to grow hemp outdoors, leading us to grow both hop and hemp in greenhouses. This proved to be more challenging than anticipated for hop cone (flower) collection and these plants are still growing. That said, we expect that we will be on track with hop samples collected and analyzed by the next report date. Hemp Collection We are collecting paired female flower samples from diverse hemp accessions for parallel analysis of cannabinoid/terpene profiles and gene expression. The first collection was done March 30, 2023, from fourteen hemp accessions. They were immediately flash frozen on dry ice, then stored at -80C. Table 1: hemp accessions that have been collected. Accession Arab-32 Berry Blossom Carmagnola x Arcadia Carmagnola x Santiam Carmagnola x Umpqua CBDV CBCW-8 HD Cherry Laos Mango Biche Nurse Lilly Solodiol CBD Special Sauce Temperanillo Hop Sample Collection Rhizomes from 25 genetically diverse hop cultivars (Table 2 below) were dug and transplanted into 3.8-liter pots during February 2023. Three replications of each cultivar were potted up and then placed in a greenhouse with 16-h light, 8-h dark period with a constant temperature of 24 Celsius. All plants were subsequently re-potted into 18.9-liter pots during April, 2023. Plants were trained up string inside of the GH and allowed to grow to a height of 3-m. Excess growth was re-trained onto the same string. Plants were watered daily and fertilized bi-weekly with "Miracle Grow" (https://miraclegro.com/en-us/shop/plant-food/miracle-gro-water-soluble-all-purpose-plant-food/miracle-gro-water-soluble-all-purpose-plant-food.html). At no point in time were the plants allowed to be stressed for water or temperature. Finally, all plant diseases and insect pests were controlled as needed using chemicals labeled for use in hops. Of the 25 cultivars grown for this study, only six (6) have formed cones. Of the six that have formed cones only one cultivar is near ready for harvesting cones. These cones will be harvested on May 15th. The other five (5) lines with cones present will be harvested during the week of May 22-26, 2023. The remaining 19 cultivars could potentially form cones. However, the current state of these genotypes is that they have not formed axillary branches that would have flowering nodes on them. There is one other issue noted from this study using greenhouse grown hop plants. We have noticed in all cases where cones have formed that lupulin gland production is significantly reduced in greenhouse grown plants versus plants grown out in field conditions. Whether or not this has an impact upon this study is not known at this time. All attempts will be made to collect cones from hop plants present in the greenhouse. The contingency for this study is to harvest these cultivars from outdoor plots. The original design for this experiment was to grow plants in an outdoor hop garden to maximize cone production and have lupulin production optimized. These cultivars are already planted in a hop garden and will be harvested during the Fall, 2023 at their optimum maturity date (historically recorded and estimated precisely by dry matter content). Table 2. List of cultivars and status of cone formation May 10, 2023. Cultivar Flower Status Cone Status Northern Brewer Short, no flowers Triumph No flowers Tea maker No flowers Aurora No flowers Cascade Flowers Cones Mt Rainier Flowers Cones Galena No flowers Giggle N No flowers Whitsbred Golding No flowers Hallertauer Tradition No flowers Hallertauer Mittelfrue Short, no flowers Horizon Some flowers Some cones Kitamidori Flowers Cones Centennial No flowers Pride of Kent Short, no flowers Saazer No flowers Sorachi Ace Flowers Cones Comet No flowers Target No flowers Brewers Gold Short, no flowers Chinook Flowers Cones Perle No flowers Eye Viking Short, no flowers Nugget No flowers Backa No flowers

Publications