Source: OREGON STATE UNIVERSITY submitted to
THE ESSENCE OF CANNABACEAE: COMPARATIVE GENOMICS AND METABOLOMICS TO UNRAVEL THE COMPLEXITIES OF AROMA AND FLAVOR
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1028026
Grant No.
2022-67013-36393
Project No.
ORE01015
Proposal No.
2021-07849
Multistate No.
(N/A)
Program Code
A1103
Project Start Date
Jan 15, 2022
Project End Date
Jan 14, 2025
Grant Year
2022
Project Director
Hendrix, D. A.
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Biochemistry & Biophysics
Non Technical Summary
The Cannabaceae family, which includes Cannabis sativa (hemp) and Humulus lupulus (hop), constitutes a multi-billion-dollar industry in the US, with over one billion dollars in sales for Oregon alone. Hemp and hop are high-value ingredients, prized for their production of specialized metabolites and pharmacologically relevant effects. The flowers of both plants are valuable for flavor and aroma, there is a need for research on alleles that control the biosynthesis of terpenes and other volatile organic compounds (VOCs). There is a need for comparative approaches for studying hop and hemp to reveal how they evolved, diverged, and what genotypes may regulate production of flavor and aroma compounds.Objective 1: Build a web-accessible infrastructure to study the comparative genomics and metabolomics of the Cannabaceae. We will create a web-accessible framework for comparative genomics in hop and hemp, and for organizing available data on VOC abundance. We will identify orthologous gene groups, and build a comparative genomics infrastructure for hop and hemp.Objective 2: Characterize the conservation and variability of VOC pathways in hop and hemp. We will quantify VOC levels using GC-MS, and gene expression using RNA-seq. We will compare co-expression networks between hop and hemp, and characterize the conservation of VOC pathways.Objective 3: Characterize the functional properties of metabolites and variants. We will build co-expression networks for both transcript and metabolite expression. We will identify genomic variants associated with metabolite expression. We will validate the role of these alleles in the activity of enzymes in an in vitro context.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012230100050%
2011730100050%
Goals / Objectives
The goal of our proposal is to enhance flavor and aroma in hop and hemp by identifying alleles that are responsible for the production of specific volatile organic compounds (VOCs). Hop and hemp are part of the Cannabaceae family, and they share common metabolic pathways. More work is needed to bridge the gap in knowledge about commonalities between these two closely-related species, thereby informing and improving research efforts for both species. First, we will create a web-accessible infrastructure and resource for Cannabaceae genomics, transcriptomics, and we will organize what is known about the biosynthesis of compounds that enhance flavor and aroma. Second, we will use genomics, metabolomics, and data science approaches to elucidate the allelic architecture that enhances the biosynthesis of VOCs, including terpenes, by using populations of hop and hemp, and third, we will validate these experimentally by heterologous expression.
Project Methods
For objective 1, we propose build a web-accessible meta-database of all available gas chromatography-mass spectrometry (GC-MS) data for hop and hemp, and create a unifying resource to serve as the basis of future studies.Available data from literature for a given cultivar will be mined for relationships between enzymes and associated compounds.We will to use JBrowse 2, including the built-in synteny browser for visualizing conserved genomic loci. We will also identify orthologous genes in the Cannabaceae family, which will allow us to study the evolution and conservation of genes relevant for the biosynthesis of terpenes and other volatile compounds. We will align reference proteomes for the species under investigation, including hemp and hop and other members of the Cannabaceae family.We will identify orthologous gene groups (OGGs) from the above set of species using OrthoFinder.We will investigate OGGs with genes only from members of the Cannabaceae, as well as OGGs with all species present, to assess sequence evolution among more highly-conserved groups, well as a comparison to genes from our pathways of interest.Objective 2 will begin by selecting 25 hop and 25 hemp cultivars for GC-MS. These cultivars will be selected based on consultation with our stakeholders (John I. Haas and OregonCBD) and based on diverse flavor and aroma characteristics. After performing GC-MS and measuring their metabolic profiles, we will identify a diverse subset of 8 hop and 8 hemp cultivars for RNA-sequencing. The RNA-sequencing will identify both gene expression and allelic variation in the different cultivars for the genes of interest, as well as other genes that could be relevant.As part of this objective, we will perform an untargeted metabolomics approach using GC-MS to identify and quantify the metabolite composition across different cultivars within hemp and hop. Additionally, we will investigate the levels of monoterpenes and sesquiterpenes following a GC-MS approach described previously.Briefly, the plant material will be harvested and immediately frozen in liquid nitrogen. The plant material will be ground to a fine powder in a liquid nitrogen cooled mortar and pestle, and the powder divided into two equal portions. The first portion will be placed in RNAlater for RNA isolation and sequencing, while the remaining material will be weighed and extracted with hexane (10 mL per 1 g of tissue) containing 0.03% (vol:vol) of toluene as an internal standard in a sealed glass vial. The tissue will be extracted for a total of two hours at room temperature, and then the plant material pelleted via centrifugation. The cleared extract will be transferred to a new glass vial and stored at -20 °C until GC-MS analysis. Volatile compounds separation will be achieved using a Shimadzu GC-MS equipped with 60-m DB-5MS column and a quadrupole mass spectrometry detector with electron ionization source. Known metabolite quantification will be accomplished by comparison to standard curves of commercially available or synthetic compounds. While we are specifically interested in terpenes and other volatile compounds for our proposed work, the strength of the untargeted GC-MS approach will allow us to characterize a broad range of metabolites beyond terpenes. The data collected will be a starting point for future projects related to other volatile compounds, with the goal of mapping compounds that contribute most to flavor. However, for this study our focus will be on metabolites that are common between hop and hemp, where insight into flavoring will be gained from a comparative approach. Industry consultants have recommended a list of compounds as critical targets for this study.We propose to select 8 hop and 8 hemp cultivars for RNA-sequencing (RNA-seq). These cultivars will be selected based on the outcomes of our GC-MS experiments for the most diverse terpene composition. We will sequence three biological replicates per cultivar, for a total of 48 samples (25 each from hop and hemp). To measure the expression level of genes involved in VOC biosynthesis, fresh flowers will be selected at the developmental stage preceding standard harvest times for the flowers and flash-frozen in liquid nitrogen upon collection.we will identify genes that show differential expression (DE) across cultivars (intra-species), as well as orthologous pairs of genes in hop and hemp that show significant differences in expression across species (inter-species). Our DE analysis will be guided by alignment to the reference genome assemblies for each species