Source: UNIVERSITY OF MISSOURI submitted to NRP
A SURVEY OF METABOLITES ASSOCIATED WITH FLAVOR IN NEW-MAKE WHISKEY USING LANDRACES OF MAIZE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032676
Grant No.
2024-67011-42960
Cumulative Award Amt.
$179,488.00
Proposal No.
2023-11514
Multistate No.
(N/A)
Project Start Date
Aug 15, 2024
Project End Date
Aug 14, 2027
Grant Year
2024
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
(N/A)
Non Technical Summary
Corn has traditionally not been grown for specific products or uses in the US. Instead, the generic yellow dent corn has been cultivated over the last 100 years to serve every purpose as an ingredient or product. With the new tools we have in plant research, we can now try to look back at locally adapted corn that was present before the yellow dent displaced them in the early 20th century and select them for specific products that can compete with the current seed we have now.My research primarily focuses on the whiskey market and how we can make better corn for that market. To do that, I will be growing two populations of corn currently used at the small scale for whiskey making that was previously used by distillers before the onset of yellow dent corn in the US and prohibition. I will then cross the parents together to make a population and analyze that population after two rounds of self-pollunation to look at flavor diversity in this population and how it relates to the genetics of the plants. Overall, this project will help inform us how flavor traits are inherited in corn and will help begin the process of identifying and growing corn for specific uses in the American whiskey market.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115101040100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Maize has been a staple ingredient for a diverse range of products that we have come to depend on in the modern economy. While the number of products derived from maize have been integral to the development of the agricultural economy, we have failed to acknowledge the value of diversity and a spectrum of maize lines to address modern challenges in agriculture including production needs, quality, and health. Landraces of maize grown before the genetic bottleneck of modern breeding offer exciting opportunities to look for traits that can be valuable to markets that use maize as a raw material. My research focuses on one such market: the craft distilling industry that primarily utilizes maize as the main ingredient. Using maize landraces, I hypothesize that there is more metabolomic diversity in the landraces compared to yellow-dent corn that are associated with flavor, and that there are genetic controls to these metabolites that we can select for future breeding efforts. These hypotheses will be addressed through two objectives:1. Using untargeted metabolomics approaches, I will characterize the metabolite composition of two landraces with known, distinct taste profiles in whiskey.2. Using Quantitative Trait Loci (QTL) mapping methods, I will generate a population derived from the landrace parents and map the genomic regions associated with these metabolites.Through this work, we will conduct foundational research on the identification of the genetic components of flavor metabolites in maize, all while producing lines better suited to the needs of the craft whiskey industry.
Project Methods
I will produce new-make whiskey from two landraces and explore their metabolites. The landraces will be diverse colored landraces already used for craft whiskey production: Bloody Butcher and Hopi Blue. When fermented and distilled commercially, these landraces present two dramatically different taste profiles, and I can qualitatively assess that there are flavor (and presumably metabolite) differences between the two. 100 g of seed is milled into a flour and added to a spinner flask along with water and α-amylase. After mixing, the solution (mash) cooks to gelatinize the starch. Once cooked, distiller's yeast is added to the mash with gluco-amylase. This addition allows for simultaneous conversion of dextrins to simple sugars to feed the yeast while not over-saturating the mash initially with simple sugars which can inhibit yeast. The mash ferments for 3 days and then is rapidly cooled. Once ready for distillation, the beer is added to a 1 L short path distillation column and heated to vaporize ethanol (and metabolites) and collected for analysis. This process is analogous to how distillers fractionate the volatiles and produce whiskey.To cover the full range of metabolites potentially present in the maize parent populations, I will use a two-pronged approach using both GC and LC. Using Solid Phase Micro-Extraction (SPME), which captures volatile metabolites.I will extract volatile molecules and inject the sample directly into the GC (Agilent 7890 GC QToF Mass Spectrometer (MS)) for analysis [16-17]. For LC, I will inject 1 mL of sample into the LC (Bruker Impact II QToF MS) for quantification. I will use known quantities of internal standards provided by the MU Metabolomics core to quantify each metabolite.I will use 3 technical replicates for both the GC/MS and LC/MS to ensure consistency within the batch and to eliminate outlier data. I will then use analysis of variance (ANOVA) to look for significant differences between landraces. Once I have each sample analyzed, I will compare the data to an established NIST metabolite library and look for metabolites correlated with flavors using a whiskey flavor lexicon. I will genotype each F2 individual by identifying single nucleotide polymorphisms (SNPs) throughout the genome. Genomic DNA will be extracted from leaf samples using a CTAB DNA Miniprep. I will then genotype the population using the Illumina Maize SNP50k genotyping array, which provides sufficient coverage of the maize genome to study SNP diversity while at the same time, being significantly cheaper and faster for our experiments compared to low-depth whole genome sequencing approaches. In tandem with the genotyping array, I will phenotype to identify and quantify metabolites extracted from pooled seed derived from each of the F3 families. I will produce new-make whiskey from F3 family seed (see above) and target the metabolite(s) identified previously (see above) associated with flavor. As a cheaper alternative to metabolomic assays, I may be able to use an enzymatic or spectrophotometric assay to quantify the target molecule(s).For mapping metabolite traits, I will use composite interval mapping (CIM) to map interval regions between SNP markers and look for significant correlations, while also accounting for unlinked marker effects (cofactors) to screen for false positives and increase the accuracy of QTL identification. To perform this analysis, I will first plot our phenotypic data as-is, then I will conduct a permutation analysis where I randomize the phenotypic data and repeat the same analysis 1000 times. This randomization will form a null distribution that I can then plot and apply a significant p-value cutoff for accepting false positives (0.05) and that cutoff will determine the LOD threshold that I will use to determine if a QTL is statistically significant. Once I have identified significant regions, I will use the B73 version 5 maize genome assembly to look for genes within the regions that may be promising candidates that explain the differences in metabolite quantity I see in our QTL population. Afterwards, one could utilize fine-mapping approaches to narrow down QTL regions to find genes associated with our phenotype, but this is likely due to the 3-year timeline of my proposal. For evaluation, I will work with Dr. Flint-Garcia and Dr. Braun to evaluate my objectives and goals using the IDP and weekly progress meetings. This process will provide a wide array of feedback from both my primary and collaborating mentors with different perspectives and will facilitate my research success. I will also present my findings at local and national conferences through poster presentations, the MU Plant Talks series, and at least one scientific talk to communicate the significance of my research to the scientific community. I will also give one talk to the broader public to explain the significance of my research. These events will solidify my graduate experience and education, and will prepare me for a career as a research scientist in plant breeding and quantitative genetics.

Progress 08/15/24 to 08/14/25

Outputs
Target Audience: This project's audience during this reporting period are other scientists that listened to presentations regarding this project and the advising committee that provided feedback to the project as it progressed in an annual meeting for the PD. Presentations for this project to other scientists include at the aforementioned committee meeting for the PD, as well as multiple presentations of a poster of the research in 3 scientific meetings/symposia, and a field presentation of the work in a field demo for the lab the PD is a member of. The PD also disseminated the signifincance of the work to members of the community. The presentations for this project to the communityinclude a booth of the different projects of the lab for the general public as part of a college-wide outreach event at the university farm and an outreach project for Future Farmers of America (FFA) to disucss research as an avenue of working in agriculture. The first outreach event allowed for the sharing of this project to people of all ages and allowed for a dynamic presentation to explain the project to everyone. The second outreach event had a range of high-school students interested in agriculture as a career and were interested in learning about the opportunities that research in agriculture can offer. Changes/Problems:Due to unforseen inbreeding depressionwith the QTL inbred lines, we were unable to generate suffiecient seed to couduct two replicates for the QTL analysis in the same year. Instead, we had to pool all replicate seedof an F2 derived F3 familyand include it as one replicate. We then will have to grow out the population for a second year this summer to generate a second replicate the same way as the firstto achieve suffecient statisical power for the QTL mapping. This means that the project progressionwill be delayed, but still is projected to be complete by the end of the grant cycle in 2027. Additionally, there have been significant problems with the machine used to quantify sugar for validation of the ethanol extraction method over the course of the year, meaning that the publication of this method has been delayed. After multiple attempts at repairing the machine, the PD concluded that no more work could be done to fix the machine and has already implemented alternative methods to validate the ethanol extraction method using spectrophotometric approaches.The publication is anticipated to be completed and submitted for publication this year. What opportunities for training and professional development has the project provided?This project has allowed for oppurtunities to network and connect with other scientists in the program. Already, there has been online meetings for the cohort to learn about the program and the future requirements of filling out forms and conducting annual reviews in compliance with the USDA policies and reporting reqirements. The cohort has also formed a community to introduce and get to know each other and our projects. Along with the cohort, I have also connected with other scientists at a networking event to increase visibility of the project as well as advertise my professional skills for post-doctoral training oppurtunities. Through the research itself, I have been able to share the research project in both a scientific and generalized manner through poster and scientific presentations and outreach, respectively. I have shared the project at university symposia, including the annual IPG symposium at the University of Missouri, and the annual Maize Genetics Conference. In outreach, I have shared my work through Science on Wheels, an organization aimed at sharing research with the broader community in Missouri. I have also shared my work through presenation of lab work to adults and children through an FFA field outreach event hosted by university extension offices, and through participation in the CAFNR showcase as well as a maize genetics field day, highlighting research from the college of agriculture for the public and other stakeholdersto enjoy.These activites, whether as scientific presentations or as outreach, allow for newtorking oppurtunites for me to grow my professional network while also imporiving my communications skills to better demostrate the significance and progress of my work. Finally, the formalized meetings I have with my advisory committee have allowed me to present my progress and forecast the next year of resarch to meet the goals of this project in a timely manner. How have the results been disseminated to communities of interest?As previously described, I have been able to share my project in both a scientific way through posters and talks, but also through outreach bypresenting the project to a general audience. Scientifically, I have presented a poster on the progress of this work, specifically on the development of a high throughput method to obtain whiskey through corn, I have given this poster presentation at local symposia including the annual University of Missouri Interdisciplinary Plant Group (IPG) symposium and also a student led symposium sponsered by Corteva Agriscience here at the university. I plan to give an updated version of this poster to the upcoming symposia this year as well as at the Maize Genetics Conference in St. Louis, Missouri and other future conferences. Through outreach, I have given a variety of talks to descibe my research to a variety of audiences. As part of the unversity extension program, I have talked to the public in two major instances about the project. The first was an event for the Future Farmers of America (FFA) where we presented oppurtunities for research in agricuture to high-school students. At this event, I highlighted the oppurtunities for research and what research my lab conducts to get students interested in a future of reseach in agricuture. The other event was a college of agriculture outreach event where a similar program was presented about what research was being conducted but in a way to show the benefits of the research for the community. We emphasized the impact the research has on the agricultural market and how the research impacts people's daily lives. Through the CAFNR showcase and our maize genetics field day, we are able to highlight the significance of the research conducted at the University of Missouri in maize, as well as present about the projects and how they benefit stakeholders in the community. What do you plan to do during the next reporting period to accomplish the goals?In the near future, the data for the scaled fermentation method will be generated and published. The publication of this method will allow for the project to accelerate into the main stage where the QTL population samples will begin to be converted into ethanol and assayed for metabolite markers. These markers will be determined through the forthcoming untargeted metabolomics assay from the parent landraces that will occur in the spring. This process will lead up to various symposia and conferences were I will present the progress of this work to the scientific community. Finally, the summer will be the period where the second replicate of the QTL population will be grown and then harvested to conduct the second round of fermentation and flavor metabolite assays.

Impacts
What was accomplished under these goals? After the 2024 summer field season, one replicate of the QTL population was completed. The second replication of this population has been planted and will be collected this summer. Additionally, the method for extracting ethanol from maize made significant progress during this period, with the publication almost complete. After the completion of the method, the replicatesof the QTL population will begin to be produced into ethanol in preparation for metabolite analysis for QTL mapping. In conjunction with the phenotyping of the QTL population being prepared, the leaf tissue of the F2 generation has been processed and shipped for genotyping the QTL population. This will allow for the combination of genotypic and phenotypic measurement for the mapping of flavor-associated traits. The parent landraces have been successfully increased and are ready for metabolite analysis. A collaboration has been established with the metabolomics core at the University of Missouri and preparations are being made to perform untargeted metabolomics on the parents once they have been processed into distillate.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Traylor, Z. B., Fitzsimmons, S. L., Draves, M. A., Salazar-Vidal, M. N., Tracy, W. F., & Flint-Garcia, S. (2025). Planting, pollinating, harvesting, and monitoring growth in maize (Zea mays) for research. Cold Spring Harbor Protocols.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Salazar-Vidal, M. N., Draves, M. A., Fitzsimmons, S. L., Traylor, Z. B., Tracy, W. F., & Flint-Garcia, S. (2025). How to monitor growth and identify developmental stages of maize (Zea mays). Cold Spring Harbor Protocols.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Draves, M. A., Traylor, Z. B., Salazar-Vidal, M. N., Fitzsimmons, S. L., & Flint-Garcia, S. (2025). Field preparation and planting corn (Zea mays). Cold Spring Harbor Protocols.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Fitzsimmons, S. L., Salazar-Vidal, M. N., Traylor, Z. B., Draves, M. A., & Flint-Garcia, S. (2025). How to pollinate corn (Zea mays). Cold Spring Harbor Protocols.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Salazar-Vidal, M. N., Draves, M. A., Fitzsimmons, S. L., Traylor, Z. B., & Flint-Garcia, S. (2025). How to harvest and store corn (Zea mays). Cold Spring Harbor Protocols.