Progress 01/15/24 to 01/14/25
Outputs
Target Audience:During this reporting period, target audiences included: 1) The scientific research community in plant science, food science, and nutrition, reached through dissemination of project findings at conferences. 2) Graduate students directly participating in the project, who received advanced research training and professional development opportunities. 3) The private sector, engaged via a student internship and industry-relevant conference presentations. These groups were targeted to advance student training, share scientific progress, and facilitate potential application and workforce development. Changes/Problems:A collaborator who agreed to conduct the secondary metabolite analysis suddenly left the institute and cannot conduct the liquid chromatography-mass spectrometry (LC-MS) analysis. PD Obata inherited the instruments and is currently setting them up in his laboratory space. However, it took much longer to establish the analytical workflow, which is still ongoing. The secondary metabolite samples have been prepared and properly stored. The analysis will be conducted once the method is established. What opportunities for training and professional development has the project provided?During the reporting period, this project continued to provide significant opportunities for training and professional development, primarily benefiting the graduate students actively engaged in the research. TRAINING ACTIVITIES: Ongoing hands-on training and mentorship were provided to Ph.D. student Rajnee Hassan (Center for Biological Chemistry, UNL) and Master's student Prabhashis Bose (Food Science & Technology Masters' Program, UNL). They continued to apply and enhance their proficiency in the project's interdisciplinary research skills, including metabolite profiling, data analysis relevant to quantitative genetics, and gastrointestinal digestion simulation methods. In addition, Rajnee had further opportunity to enhance her expertise by attending the prestigious Cold Spring Harbor Laboratory's Metabolomics course in June 2024, and Prabhashis gained valuable industry experience through an internship in the private sector during the Fall 2024 semester. PROFESSIONAL DEVELOPMENT: The project supported extensive professional development activities for the trainees during this period, facilitating the dissemination of their research and recognizing their achievements. Students actively presented project findings at internal seminars and external scientific conferences. Rajnee Hassan received multiple accolades for her presentations, including a poster award at the Biochemistry & Complex Biosystems Graduate Student Recruitment Event (UNL, Feb 2025), 2nd place in the oral presentation competition at the Plant Science Retreat (Nebraska City, Oct 2024), 3rd place in the poster presentation competition at the Nebraska Plant Science Symposium (UNL, Mar 2024), and was recognized with the Plant Science Innovation (PSI) Heuermann Graduate Recognition Award (UNL, May 2024). Prabhashis Bose successfully defended his Master's thesis in December 2024 and received significant recognition for his work, including the highly competitive 2024-2025 Folsom Distinguished Master's Thesis Award (UNL), the John and Louise Skala Fellowship (UNL) for the 2024-2025 academic year, and the Widaman Distinguished Graduate Assistant Award from the Institute of Agriculture and Natural Resources (UNL). Prabhashis also presented in the Nutraceutical and Functional Foods (NFF) Division at the IFT FIRST Annual Meeting & Expo in July 2024. Additionally, Dr. Musa Ulutas (Department of Agronomy and Horticulture, UNL), who completed his Ph.D. just prior to this reporting period based on project research, presented work derived from this project at the Maize Genetics Conference in March 2024. These combined training and development activities highlight the project's contribution during this specific reporting period to developing the skills, professional visibility, and career progression of the graduate students and recent graduates involved. How have the results been disseminated to communities of interest?The results were disseminated to the local and externalscientific community via poster and oral presentations, includingBiochemistry & Complex Biosystems Graduate Student Recruitment Event (Poster; UNL, Feb 2025), Plant Science Retreat (Oral; Nebraska City, NE, Oct 2024), Nebraska Plant Science Symposium (Poster; UNL, Mar 2024), American Society of Plant Physiologist Midwest Section Annual Meeting (Oral and Poster; Lincoln, NE, Mar 2025), Nutraceutical and Functional Foods (NFF) Division at the IFT FIRST Annual Meeting & Expo (Poster; Chicago, IL, July 2024) and Maize Genetics Conference (Poster; Raleigh, NC, March 2024). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: During this reporting period, progress was made towards enhancing the maize health benefits by studying its wild relatives (teosinte) and landraces. Bioassays showed higher antioxidant and anti-inflammatory potential in teosinte kernels digested by human gastrointestinal (GI) tracts compared to modern and landrace maize, supporting the hypothesis that health-beneficial traits were lost during domestication. Detailed chemical analysis of digested kernels identified candidate compounds potentially responsible for these bioactivities. We linked key kernel metabolites (quinic and caffeic acids) to specific candidate genes (a glycosyltransferase and a beta-glucosidase) located within genetic regions known to have lost diversity during domestication. This work provides specific chemical and genetic targets for breeders aiming to naturally restore these lost health benefits in modern maize varieties via genome-enabled selection. Objective 1: Characterize the nutritional properties in raw kernels of maize and its relatives at diverse stages of maize domestication and improvement. MAJOR ACTIVITIES: Correlation analyses between raw kernel metabolite data and kernel digesta bioactivity (Obj 2) identified potential compounds associated with the health-beneficial traits in kernels. The relevance of quinic acid in antioxidant activity of the kernel digest was tested by enriching B73 maize kernel material with synthetic quinic acid. Metabolite profiling of these quinic acid-enriched digests is underway. DATA COLLECTED: Results from correlation analyses linking raw kernel metabolite contents to digested kernel bioactivities. Antioxidant activity data for quinic acid-enriched vs. non-enriched B73 kernel digests. DISCUSSION: Raw kernel contents of quinic acid, caffeic acid, chlorogenic acid, and 5-hydroxyindole-3-acetic acid correlated positively with antioxidant activity in digests and quinic acid and 3,4-dihydroxybenzoic acid correlated with anti-inflammatory activity. These compounds may be antioxidants/anti-inflammatories themselves or precursors converted to bioactive molecules during GI digestion. However, enriching B73 kernels with quinic acid did not increase digest antioxidant activity, suggesting raw kernel quinic acid level alone is not the direct driver of teosinte's superior antioxidant activity. Anti-inflammatory testing of the enriched samples is ongoing. The workflow correlating raw kernel components with digest bioactivity and using enrichment studies remains valuable for assessing the relevance of specific kernel molecules, despite of the initial negative result. KEY OUTCOMES AND ACCOMPLISHMENTS: Established and utilized a workflow correlating raw kernel metabolite levels with post-GI digestion bioactivity to identify potential precursors of health-beneficial compounds. Evidence suggests raw quinic acid level alone doesn't drive high antioxidant activity in teosinte digests, prompting investigation into its potential role as a precursor to other molecules or synergistic effects. Objective 2: Determine chemical compositions and antioxidative and anti-inflammatory activities of the digested kernels in the human GI tract. MAJOR ACTIVITIES: Anti-inflammatory activity assays using HT-29 human intestinal cells and comprehensive peptide profiling were completed for the GI digested kernels from the 76 full panel genotypes. Correlation analyses were conducted between chemical contents in the digests and the bioactivities of those digests, as well as between compound contents in raw kernels (Obj 1) and those in the digests. Validation of bioactivities of candidate molecules using synthetic compounds is underway. DATA COLLECTED: Anti-inflammatory activity data and the profiles of 94 dipeptides, 7 tripeptides, and 17 larger peptides for 76 kernel digests. Results from correlation analyses identified 39 antioxidant and 8 anti-inflammatory candidate compounds in the digests. DISCUSSION: The teosinte group overall exhibited higher anti-inflammatory potential compared to landraces and modern maize. This indicates teosinte kernels possess higher potential for both bioactivities post-digestion, supporting the hypothesis of trait loss during domestication. Correlation analyses pinpointed 39 metabolites in digests positively associated with antioxidant activity and 8 with anti-inflammatory activity, providing a list of candidate bioactive molecules for future validation. Except for quinic acid, most raw kernel compounds correlating with bioactivities (caffeic acid, chlorogenic acid, etc.) were not detected in digests, suggesting they can be transformed into bioactive molecules during digestion. Positive correlations between potential raw kernel precursors and digest metabolites suggested possible transformations. Peptide analysis identified peptides including MM and IH are specifically enriched in teosinte digests and correlate with antioxidant activity. Larger peptides, SSIIPQ, SSPLPQ, were specifically detected in teosinte digests. These results suggest they may contribute to bioactivities. KEY OUTCOMES AND ACCOMPLISHMENTS: Completed the bioactivity assays for the panel, confirming higher health-beneficial potential in teosinte GI digests. Generated peptide and metabolite profiles and identified potential bioactive peptides and compounds in digesta. Strengthened the understanding of chemical transformations during GI digestion by linking potential raw kernel precursors to compounds found in digests. Further developed the workflow for identifying bioactive compounds and their precursors in digested crop materials. Objective 3: Identify genes associated with health-beneficial traits using population genomic approaches. MAJOR ACTIVITIES: Existing Quantitative Trait Loci (QTL) data for raw kernel metabolite content, generated in a parallel USDA-HATCH project using a modern maize diversity panel (n=283), was analyzed in the context of the selective sweep regions previously identified in this project. DATA COLLECTED: Identification of specific QTLs for raw kernel quinic acid and caffeic acid contents that physically overlap with genomic regions identified as selective sweeps associated with maize domestication. DISCUSSION: Health-beneficial bioactivities appear to have been lost during domestication (Obj 2), strongly suggests that the underlying alleles reside within domestication selective sweeps. A QTL associated with kernel quinic acid contents was found within a domestication sweep region and contains a glycosyltransferase gene. Similarly, a QTL associated with kernel caffeic acid contents was located in a different domestication sweep containing a beta-glucosidase gene. Both glycosyltransferases and beta-glucosidases are enzyme classes known to be involved in the biosynthesis and modification of specialized metabolites, including phenolic compounds related to quinic acid and caffeic acid. Their location within domestication sweeps suggests their variation might have been directly affected by the domestication process, potentially altering metabolic flux and contributing to the observed differences in phenolic acid precursors between teosinte and modern maize. Functional testing of these candidate genes is planned for future work. KEY OUTCOMES AND ACCOMPLISHMENTS: Identified specific candidate genes (a glycosyltransferase and a beta-glucosidase) located within domestication selective sweep regions that are associated with variation in key phenolic acid precursor levels (quinic and caffeic acid). This provides strong candidate genetic targets potentially responsible for trait loss and suitable for future efforts to re-introduce beneficial alleles from teosinte into modern maize.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Bose P, Hasan R, Obata T, Majumder K. Understanding how domestication changed maize's bioactive properties. IFT FIRST Annual Meeting & Expo 2024, Nutraceutical and Functional Foods (NFF) Division, Chicago, IL, July 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Hasan R, Bose P, Gen X, Yang J, Majumder K, Obata T. Shikimate Dehydrogenase Modulates Homoserine Biosynthesis in Maize Kernels: Insights from Metabolite-based GWAS and Metabolic Network Modeling. American Society of Plant Physiologist Midwest Section Annual Meeting, Lincoln, NE, March 2025.
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Progress 01/15/21 to 01/14/25
Outputs
Target Audience: The Scientific Research Community: This primary audience includes plant geneticists, plant breeders, evolutionary biologists, plant biochemists, metabolomics researchers, food scientists, and nutrition scientists. They were served through the dissemination of project findings at scientific conferences and symposia and through the planned release of genomic and metabolomic datasets. This audience utilize the project's fundamental knowledge, such as the impact of domestication on nutritional traits, the identification of novel bioactive compounds in digested kernels, candidate health-beneficial genes from wild relatives, and improved analytical methodologies, to advance scientific understanding, inform new research directions, and develop tools for crop improvement. Maize Breeders: Breeders are a crucial target audience for the translational aspects of this research. The project provided them with information on valuable lost traits in maize ancestors, specific chemical compounds linked to health benefits, and, importantly, candidate genes and genomic regions from teosinte and landraces associated with these traits. This knowledge is vital for developing strategies to re-introduce these beneficial characteristics into modern maize lines through genome-enabled selection, ultimately leading to more nutritious and health-promoting cultivars. Graduate Students and Postdoctoral Researchers: The students (four Ph.D. and one Master's) and two postdoctoral researcher directly involved in this project were a core target audience. They benefited from intensive, hands-on training in an interdisciplinary research environment, acquiring advanced skills in areas like comparative genomics, metabolomics, in vitro digestion models, cell-based bioassays, and complex data analysis. These experiences, along with opportunities to present research and achieve academic milestones, including degree completions, awards, academic and industrial positions, were designed to develop them as the next generation of scientists equipped to tackle complex agricultural and nutritional challenges. Food Industry and Nutraceutical Sector: While an indirect audience for much of the project period, this sector was reached through presentations at relevant conferences and the potential future publication of findings. The identification of maize lines or specific compounds with enhanced health-beneficial properties post-digestion is of interest for developing novel food products, ingredients, or supplements with improved nutritional profiles and consumer appeal. These audiences were engaged to ensure the project's findings contribute broadly to scientific knowledge, practical crop improvement, workforce development, and potentially new health-focused food applications. These contributions ultimately broadly benefit U.S. citizens' well-being and the U.S. agricultural economy. Changes/Problems:The project had a one-year no-cost extension due to the unexpected delay in bioactivity testing caused by the defective commercial enzyme lot and trouble in establishing the intestinal cell culture. What opportunities for training and professional development has the project provided?This project provided substantial opportunities for training and professional development, significantly enhancing the skills and career trajectories of the graduate students and postdoctoral researcher involved. Training Activities: Three Ph.D. students (Rajnee Hassan, Center for Biological Chemistry, UNL; Semra Palali Delen and Musa Ulutas, Department of Agronomy and Horticulture, UNL) and one Master's student (Prabhashis Bose, Food Science & Technology Masters' Program, UNL) received direct, hands-on training through their work on this interdisciplinary project. Mentoring focused on developing proficiency in key research areas integral to the project goals, including advanced metabolite profiling techniques (e.g., GC-MS), quantitative genetics analysis, and the application of the semi-high throughput in vitro gastrointestinal digestion method developed within the project. This training equipped them with valuable skills spanning biochemistry, food science, and plant genetics. Professional Development Activities: The project supported extensive professional development for the trainees: Dissemination and Networking: All graduate students actively presented their project research at various internal seminars and external scientific conferences, enhancing their communication skills and professional networks. Specific highlights include: Rajnee Hassan presented award-winning posters at the Biennial Cellular & Molecular Biology of the Soybean Conference (2nd prize, Aug 2023), the UNL Biochemistry & Complex Biosystems Graduate Student Recruitment Event (Feb 2025), and the Nebraska Plant Science Symposium (3rd place, Mar 2024), as well as a 2nd place oral presentation at the Plant Science Retreat (Oct 2024). Prabhashis Bose presented an award-winning flash talk (1st prize) and poster at the 3rd International Symposium of Bioactive Peptides (Sep 2023), presented a poster at the American Oil Chemists' Society annual Meeting (May 2023) with support from a UNL Larrick Graduate Student Travel Fund and at IFT First Annual Meeting & Expo (July 2024). Musa Ulutas received 2nd place for his short presentation at a Nebraska Plant Science Symposium (Mar 2021). Advanced Skills Development: Rajnee Hassan was selected for and attended the prestigious Cold Spring Harbor Laboratory's Metabolomics course (June 2024), providing specialized training in a key area of her research. Recognition and Awards: The high quality of the trainees' work and their academic achievements were recognized through several prestigious awards: Prabhashis Bose received the Widaman Distinguished Graduate Assistant Award (IANR, UNL), the highly competitive 2024-2025 Folsom Distinguished Master's Thesis Award (UNL), and the John and Louise Skala Fellowship (UNL) for 2024-2025. Rajnee Hassan received the Plant Science Innovation (PSI) Heuermann Graduate Recognition Award (May 2024 UNL). Academic Milestones: The project supported students through critical academic milestones. Rajnee Hassan successfully completed her comprehensive exam to advance to Ph.D. candidacy. Semra Palali Delen successfully defended her Ph.D. dissertation in December 2023. Musa Ulutas also successfully defended his Ph.D. dissertation in December 2023. Prabhashis Bose successfully defended his Master's thesis in December 2024 and continues his research as a Ph.D. student. Career Advancement: A postdoctoral researcher, Dr. Michael Meier (Department of Agronomy and Horticulture, UNL), participated in the project early on (Jan-Apr 2022), gaining experience in quantitative genetics analysis before transitioning to an industry position at Rancho Biosciences. These combined training and development activities demonstrate the project's significant contribution to fostering the next generation of scientists in plant science, nutrition, and bioinformatics. How have the results been disseminated to communities of interest?The results and insights derived from this study were primarily disseminated through active participation in scientific conferences, symposia, and workshops at international, national, regional, and local levels. Project personnel, particularly graduate students, shared key findings on maize domestication's impact on kernel chemistry, the identification of bioactive compounds in digested kernels, and associated genetic loci through numerous oral presentations, poster presentations, and flash talks. Notable examples include presentations at: International meetings such as the IFT FIRST Annual Meeting & Expo (July 2024, focusing on food science and nutrition aspects like bioactive properties) and the 3rd International Symposium of Bioactive Peptides (September 2023, highlighting peptide-related discoveries). Specialized national conferences like the Maize Genetics Conference (March 2024, sharing genetic findings with maize researchers), the Biennial Cellular & Molecular Biology of the Soybean Conference (August 2023, presenting comparative biological insights where Rajnee Hassan won 2nd prize for her poster), and the American Oil Chemists' Society Annual Meeting (May 2023). Prestigious training workshops such as the Cold Spring Harbor Laboratory's Metabolomics course (June 2024), where Rajnee Hassan not only received training but also shared project-related perspectives with peers and leading researchers. Regional and local university symposia, including the Nebraska Plant Science Symposium (where students won presentation awards) and the Plant Science Retreat, which facilitated sharing of progress and fostering collaborations within the local research community. Internal departmental seminars and graduate student recruitment events (e.g., UNL Biochemistry & Complex Biosystems Graduate Student Recruitment Event, February 2025, where Rajnee Hassan received a poster award), provided platforms for discussion and feedback. These presentations ensured that emerging data on chemical diversity, in vitro bioactivities, candidate genes, and novel analytical workflows reached researchers in plant genetics, biochemistry, food science, nutrition, and crop improvement. In addition to presentations, whole-genome sequencing data for teosinte and landrace accessions generated by this project are planned for submission to public repositories, making them accessible to the broader research community for further studies. Manuscripts detailing the project's major findings are also in preparation for submission to peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT This project explored maize wild relatives (teosinte) and landraces for the health-beneficial traits potentially lost during their domestication to reintroduce them into modern maize to enhance its health values. We found that health-beneficial antioxidant and anti-inflammatory activities in the human gut are statistically significantly higher in teosinte compared to modern maize. Key findings pinpointed specific chemical compounds generated during digestion in the human gut responsible for these effects and identified candidate genes controlling precursor compound levels in kernels. This research provides crucial knowledge and specific genetic targets for breeding new maize varieties with enhanced health benefits. The project also generated valuable genomic resources and research pipelines for the scientific community. Ultimately, this work paves the way for the development of crop varieties that contribute to better human nutrition and health. Objective 1 MAJOR ACTIVITIES: Kernels from 26 modern lines, 25 landraces, 25 teosintes representing maize evolutionary history were analyzed. Polar metabolites were profiled using GC-MS. Correlations between raw kernel metabolites and digested kernel bioactivities (Obj 2) were analyzed. DATA COLLECTED: Quantitative profiles of 103 polar metabolites (sugars, amino acids, organic acids, secondary metabolites) for 76 representative lines. DISCUSSION: Significant differences in metabolite profiles were observed between domestication groups. Teosinte kernels consistently showed higher levels of phenolic compounds (quinic acid, caffeic acid, chlorogenic acid), while modern maize and landraces generally had higher sugar levels, consistent with maize selection for increased grain yield, primarily composed of starch. Specific raw kernel phenolics correlated positively with post-GI digestion antioxidant activity (quinic, caffeic, chlorogenic, 5-hydroxyindole-3-acetic acids) and anti-inflammatory activity (quinic acid, 3,4-dihydroxybenzoic acid), suggesting they might be the precursors to bioactive molecules formed during GI digestion. However, direct quinic acid enrichment in B73 kernels failed to increase the antioxidant activity of digests, indicating that raw kernel quinic acid level alone is not the primary factor underlying bioactivities. KEY OUTCOMES AND ACCOMPLISHMENTS: Quantified significant metabolite shifts in kernels during maize domestication and recent improvement processes, including loss of phenolics and gain of carbohydrates, providing baseline data highlighting the distinct chemical diversity and nutritional potential present in teosinte and landrace maize kernels. Identified specific phenolic compounds in raw kernels as potential precursors for health-beneficial compounds generated during digestion. Revealed that raw kernel quinic acid content alone does not confer the high antioxidant activity seen in digested teosinte kernels. The developed workflow correlating raw kernel composition with post-digestion bioactivity to identify potential precursor molecules is applicable to other crops for enhancing health benefits. Objective 2 MAJOR ACTIVITIES: A semi-high-throughput in vitro GI digestion protocol simulating human digestion was developed and optimized for small sample amounts and processing capacity. Kernels from the diverse panel were processed using this method. Metabolite (GC-MS) and peptide profiles of the digests were determined. Antioxidant and anti-inflammatory activitiesof the digests were assessed in HT-29 intestinal cells. Correlations between digest composition and bioactivity were analyzed. DATA COLLECTED: Total peptide content and profiles of 71 metabolites and 119 peptides in GI digests for 76 genotypes; Quantitative antioxidant and anti-inflammatory bioactivity data for kernel digests from all genotypes; Identified 39 potential antioxidant and 8 potential anti-inflammatory compounds in digests. DISCUSSION: Peptide levels in digests were surprisingly comparable between teosinte and modern maize, despite much higher raw protein in teosinte, suggesting lower protein digestibility in teosinte kernels. Kernel digests from teosinte showed significantly higher antioxidant and anti-inflammatory activities compared to landrace and modern maize digests in the HT-29 intestinal cell model. These results strongly support the hypothesis that health-beneficial activities were lost during maize domestication. Antioxidant and anti-inflammatory activities of the digests were positively correlated with the levels of 39 and 8 metabolites and 45 peptides were predicted to be bioactive by a deep-learning model, suggesting them potential bioactive molecules. Correlations were found between specific candidate precursor molecules in raw kernels (Obj 1) and potential bioactive molecules identified in the digests. KEY OUTCOMES AND ACCOMPLISHMENTS: Demonstrated significantly higher bioactivity in digested teosinte kernels compared to landraces and modern maize, providing strong evidence for the loss of these health traits during domestication. Characterized chemical changes occurring during digestion and identified dozens of specific compounds within the digested material potentially responsible for the observed bioactivities, establishing targets for future validation. Established links between precursor compounds in raw kernels and potential bioactive compounds in digests. Developed and implemented an improved, higher-throughput simulated GI digestion method suitable for genetic studies with limited material, enabling large-scale analysis. Objective 3 MAJOR ACTIVITIES: Whole-genome sequences (WGS) of 8 landrace and 23 teosinte lines were sequenced to supplement existing sequence data to cover the full panel (n=74 total). Approximately 70 million SNPs were identified across the panel using GATK4. Following selection scans, 292 and 439 selective sweeps were identified during the domestication and improvement processes, respectively. Additionally, using a modern maize panel to link genetics, GWAS was conducted to associate kernel metabolites (Obj 1) with genetic markers. DATA COLLECTED: WGS data for the project accessions; SNP dataset (~70M SNPs) across the panel; mapped selective sweep regions; GWAS results linking SNPs to metabolite levels. DISCUSSION: Identified selective sweeps included regions containing known key domestication/improvement genes (e.g., tb1, tga1, flowering time genes), suggesting the power of the approach. The observed loss of bioactivities in Obj 2 align with the hypothesis that relevant alleles reside within these domestication sweeps. GWAS identified QTLs for key metabolites: a QTL for quinic acid content was mapped to a glycosyltransferase gene located within a domestication sweep, and a QTL for caffeic acid content was mapped to a beta-glucosidase gene, also in a domestication sweep. Both genes are plausibly involved in specialized metabolite biosynthesis relevant to these acids. KEY OUTCOMES AND ACCOMPLISHMENTS: Provided genomic evidence supporting the loss of health-beneficial alleles during domestication. Identified specific candidate genes (a glycosyltransferase and a beta-glucosidase) located within domestication selective sweeps that are associated with the accumulation of phenolic acids (quinic and caffeic acid), potentially linked to health benefits, providing concrete genetic targets for future research and breeding efforts aimed at re-introducing lost beneficial traits into modern maize. Generated valuable WGS resources for diverse teosinte and landrace accessions available to the research community of maize genetics and evolution.
Publications
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The significantly higher antioxidant activity in the gastrointestinal digests of teosinte kernels indicates that teosintes have alleles that improve the health benefits of maize kernels. This information can be helpful for maize breeders in generating genetic resources for the production of maize kernels with superior health benefits. The whole genome sequences of the landrace and teosinte and the identified selective sweeps provide critical information to maize geneticists to understand the domestication and improvement events and to characterize the gene functions. Changes/Problems:We have asked for a year no-cost extension of this project. It allows us to complete most of the proposed experiments by the end of the extended project period. What opportunities for training and professional development has the project provided?Two Ph.D. students (Rajnee Hassan, Center for Biological Chemistry, UNL, Musa Ulutas, Department of Agronomy and Horticulture, UNL) and one master's student (Prabhashis Bose, Food Science & Technology Masters' Program, UNL) have been trained through this project during the reporting period. They showed significant improvement in their research skills in biochemical, food science, and plant genetics in this interdisciplinary project. Rajnee completed her comprehensive exam to be a Ph.D. candidate. She presented her results at the Biennial Cellular & Molecular Biology of the Soybean Conference in August 2023, and her presentation was selected as the second prize in the poster competition. She has also been selected to attend the Cold Spring Harbor Laboratory's Metabolomics course in June 2024. She has also presented at four internal conferences and seminars. Prabhashis plans to defend his master's thesis in the summer 2024 semester. He presented a poster and flash talk at the 3rd International Symposium of Bioactive Peptides (ISBP) in September 2023 and won the first prize in the flash talk competition. He also presented a poster in another external research meeting and oral and poster presentations internally. According to his significant academic accomplishments, Prabhashis received the Widaman Distinguished Graduate Assistant Award from the Institute of Agriculture and Natural Resources at the University of Nebraska-Lincoln. Musa presented his research during the NE plant science symposium and won the 2nd place award for the short presentation. In December 2023, he successfully defended his dissertation and obtained his Ph.D. degree. How have the results been disseminated to communities of interest?The results of gastrointestinal digestion and metabolite profiling were presented in the 3rd International Symposium of Bioactive Peptides (ISBP), AOCS Annual Meeting & Expo 2023, 2023 UNL Plant Science Symposium, and other internal seminars. What do you plan to do during the next reporting period to accomplish the goals?We will soon complete the anti-inflammatory activity assays for the digested kernel materials. We will also determine the specialized compounds and oligo peptides in the digested kernels using the LC-MS/MS analyses. The correlation analyses between the contents of individual chemicals and antioxidant and anti-inflammatory activities will identify the potential bioactive molecules. Their bioactivities will be evaluated using the authentic compounds by the same bioactive assays used for the kernel digests (Objective 2). We will also analyze the elemental compositions and specialized compound profiles in raw kernels (Objective 1). The alleles associated with the chemical contents and bioactivities in digested kernels will be identified. We will mainly focus on the 292 genetic sweep regions found between teosinte and landrace since the results in this reporting period indicate that the antioxidant activity has been lost during the domestication. We will calculate genetic load at the gene level within and across populations to identify candidate genetic loci underlying the metabolite accumulation and health-beneficial bioactivities (Objective 3).
Impacts
What was accomplished under these goals?
PROBLEMS TO BE ADDRESSED The bioactivities of the food materials depend on the availability of the bioactive compounds in the digested foods. We need to determine the bioactivities and chemical contents in the digested kernels to elucidate the compounds associated with the health-beneficial bioactivity of kernels. The comparative analysis among modern maize, open-pollinated landraces, and maize wild ancestor (teosinte) populations allows the identification of health-beneficial chemicals lost during maize domestication and improvement processes and the genomic regions related to their accumulation. MAJOR ACTIVITIES In the previous reporting periods, we have digested kernels of 26 modern maize, 25 landraces, and 25 teosinte varieties in vitro under the condition simulating human gastrointestinal (GI) digestion. In this reporting period, we analyzed the metabolite compositions of the digested kernel materials by a GC-MS metabolomics approach. The antioxidant activities of the same materials have been evaluated with HT-29 cultured human intestinal cell line treated with pro-oxidant 2,2′-azobis (2-methylpropionamide)-dihydrochloride (AAPH) to induce oxidative stress. To analyze the genomic regions associated with the metabolite levels and bioactivities, the genome of the eight landrace and 23 teosinte lines have been sequenced. We obtained the genomic sequence of other lines (in total, n=26 for modern maize, n=25 for landraces, and n=23 for teosinte) from our previous experiments and NCBI database. DATA COLLECTED Objective 2: GC-MS analysis detected 66 metabolites in kernel digests, including amino acids, sugars, and organic acids. We tend to detect more amino acids and di- and tripeptides than in raw kernels, most likely due to those derived from protein degradation. These compounds were more accumulated in digests from teosinte kernels than those from maize, most likely due to higher protein contents in raw teosinte kernels. Interestingly, our results in the previous reporting period showed similar peptide levels between digested teosinte and maize kernels. These results indicate that the proteins in teosinte kernels can be more susceptible to peptidases to produce smaller peptides and free amino acids. Objective 2: We have completed the antioxidant activity assays of the digested kernels from all genotypes. The HT-29 human intestinal cell line was incubated with 100 mg of kernel GI digesta for 3 h, and oxidative stress was induced by AAPH treatment. Reactive oxygen species (ROS) generation was monitored by the DCFDA fluorogenic dye to determine oxidative stress levels. The digests of three modern maize, four landrace, and 17 teosinte kernels showed statistically significant antioxidant effects. The antioxidant activities of the teosinte kernels were significantly higher than those of modern and landrace maize lines. The highest antioxidant activities in the teosinte kernel digests were equivalent to that of a known antioxidant, quercetin (100 mM), and reduced the ROS levels by 30%. On the other hand, the kernel digest of the Mo18W modern maize inbred increased the ROS levels by 10%, although the increase was not statistically significant. Thus, the kernel digests from the tested population showed significant variation in antioxidant activity. Objective 3: We have sequenced eight landrace and 23 teosinte lines to obtain whole genome sequences of all tested genotypes. Around 70 million single nucleotide polymorphisms were identified by SNP calling using GATK4 software among all modern maize, landrace, and teosinte genotypes. The sequence comparisons between teosinte/landrace and landrace/modern maize genotypes identified 292 and 439 genomic regions where the genetic diversity had been lost during the domestication and improvement processes. DISCUSSION The activities in this reporting period analyzed the chemical profiles and antioxidant activities in the kernel materials digested by in vitro gastrointestinal digestion. We have also sequenced and analyzed the genomic sequences of teosinte, landrace, and modern maize genotypes. Metabolite profiling showed distinct chemical contents in the kernel digests from teosinte lines, corresponding with their higher antioxidant activities than the other two groups. These results indicate that the critical genetic components regulating the accumulation of antioxidative compounds could be lost during the maize domestication process. These genetic components can be included in the 292 selective sweeps that lost diversity during the domestication process. Further analysis of the correlation between chemical contents and antioxidant activities, antioxidant activities and genomic sequence, and chemical contents and genomic sequence will indicate the antioxidative compounds and the genes associated with the accumulation of the antioxidative compounds. It is interesting that the kernel digests of the landrace lines did not show significant differences in antioxidant activities compared to those of the modern maize. The antioxidant activities are often attributed to the accumulation of antioxidative pigments such as anthocyanin. Some landrace lines have colored kernels due to anthocyanin accumulations. However, the antioxidant activities were not directly related to kernel colors. These results indicate that the anthocyanins with known antioxidant activities may not be the major antioxidants in maize kernels that are effective in the human intestine, and other unknown chemicals are likely responsible for their bioactivities. KEY OUTCOMES AND ACCOMPLISHMENTS The metabolite profiling and antioxidant activity data will lead to the identification of potential antioxidant chemicals in maize kernels. The whole genome sequences of the landrace and teosinte genotypes are valuable resources for analyzing the genetic events that occurred during the maize domestication and improvement and for identifying genetic resources to improve the modern maize varieties.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Palali Delen, S., Lee, J. & Yang, J. Improving the metal composition of plants for reduced Cd and increased Zn content: molecular mechanisms and genetic regulations. CEREAL RESEARCH COMMUNICATIONS (2023). https://doi.org/10.1007/s42976-023-00453-8
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Nolasco E., Krassovskaya I., Hong K., Hansen K., Alvarez S., Obata T., Majumder K. Sprouting alters metabolite and peptide contents in the gastrointestinal digest of soybean and enhances in-vitro anti-inflammatory activity. Journal of Functional Foods (2023) Volume 109:105780, https://doi.org/10.1016/j.jff.2023.105780.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Ulutas M., Delen Y., Hao J., Ge Y., Schachtman D., Yang J., High Throughput Phenotyping of Field Excavated Roots of Maize Lines Under Different N Conditions. NE Plant Science Symposium, April, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bose, P, Hasan, R., Obata, T., Majumder, K. Impact of domestication on the quality and quantity of maize proteins and metabolites. 3rd International Symposium of Bioactive Peptides (ISBP), September 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bose, P.; Hasan, R.; Obata, T.; Majumder, K. Effect of Domestication on the Digestibility of Maize Protein. AOCS Annual Meeting & Expo 2023, Proteins and Co-products Division, May 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hasan, R., Bose, P., Majumder, K., Obata, T. Enhanced Bioactivity of Day-4 Soybean Sprouts: A Potential Functional Food for Inflammation-Associated Diseases. Biennial Cellular & Molecular Biology of the Soybean Conference, August 2023
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The major target audiences in this reporting period were food and nutritional scientists, plant biochemists, and plant geneticists. The mechanisms of health benefits are significant topics in food and nutritional sciences and biochemistry. The variations in protein digestibility and metabolite and peptide contents in digested kernels are presented to scientists atscientific conferences. Changes/Problems:We had an unexpected problem with the digestive enzyme needed for simulated gastrointestinal digestion. It took some time to figure out the problem, which caused the delay of the entire project since the majority of the chemical determinations used digested materials. We probably need an extension of the project period to recover the delay. What opportunities for training and professional development has the project provided?Two Ph.D. students (Rajnee Hassan, Center for Biological Chemistry, UNL; Semra Palali Delen, Department of Agronomy and Horticulture, UNL) and one master's student (Prabhashis Bose, Food Science & Technology Masters' Program, UNL) have been involved in this project. They have been trained to conduct metabolite profiling, quantitative genetics analysis, and semi-high throughput GI digestion. Semra acquired her Ph.D. degree in December 2023. All of them presented their results in internal seminars and scientific conferences. Prabhashis was awarded the Larrick Graduate Student Travel Fund from the UNL to attend and give a poster presentation at the American Oil Chemists' Society Annual Meeting (May 2023). How have the results been disseminated to communities of interest?The results of kernel digestibility were presented in the American Oil Chemists' Society Annual meeting (May 2023). What do you plan to do during the next reporting period to accomplish the goals?We will finalize the GC-MS metabolite profiling of the digested kernel material (Objective 2). Profiles of secondary metabolites, including phenolic compounds in raw and digested kernels, will be analyzed using an LC-MS protocol, which is currently under development (Objectives 1 and 2). Additionally, the elemental composition of the raw kernels will be analyzed by ICP-MS (Objective 1). The antioxidative and anti-inflammatory activities in digested kernels will be determined using Caco2 and HT-29 intestinal cell lines using the protocol developed in the previous reporting period (Objective 2). We will finalize the genome sequence assembly early in the next reporting period. The genome sequence data will be used to identify the alleles affecting each biochemical trait of kernels determined in Objectives 1 and 2 by the genomic evolutionary rate profiling approach (Objective 3).
Impacts
What was accomplished under these goals?
GOAL The long-term research goal of the research team is to enhance nutritional and health-beneficial traits of maize kernels through genome-enabled selection. We intend to use a selection approach rather than a transgenic approach considering consumer perception of food derived fromtransgenic plants. T IMPACT In this reporting period, the kernels of modern maize, open-pollinated maize landraces, and the wild maize ancestor, teosinte, were processed, and a metabolomics approach were undertaken to analyze their chemical contents. The data provide the information to elucidate how the kernel nutritional values have been altered through maize domestication and improvement. We will identify the important regions in the maize genome which affect the nutrition content in maize kernels by elucidating the chemical abundances and the genomic regions which showed related changes in different domestication groups in the following reporting period. A part of kernel materials was further processed by the semi-high throughput digestion method simulating the human gastrointestinal digestion developed in the previous reporting period. We determined the protein digestibility of these kernel materials, which showed significant differences among teosinte, landrace, and modern maize varieties, with improved digestibility through the domestication processes. The peptide contents in the digested kernels were higher in teosinte. These results indicate that the kernels of ancestral teosinte are more challenging to digest, and incomplete gastrointestinal digestion may produce more bioactive peptides. The ongoing bioassays will test this possibility. These results are expected to identify the genomic regions related to the chemical properties of maize kernels, which will be helpful information for developing maize varieties to produce kernels with improved nutritional and health-beneficial values. Major activities/experiments The kernel materials of 26 modern maize, 25 landraces, and 25 teosinte varieties were frozen and pulverized for further analysis. Whole genome sequencing of unsequenced eight landraces and 25 teosinte have been conducted and the genome sequence assembly is currently ongoing. Soluble metabolites were extracted from aliquots of 25 mg kernel materials using methanol: methyl tert-butyl ether for GC- and LC-MS analyses. The 103 metabolites, including sugars, amino acids, organic acids, and small molecular weight secondary metabolites, were determined by a semi-targeted metabolite profiling using GC-MS. ANOVA showed the contents of most of the metabolites significantly differed between domestication groups. Among them, phenolic compounds including quinic acid, caffeic acid, and chlorogenic acid were highly accumulated in teosinte than in landrace and modern maize. Contrary, the contents of amino acids, sugars, sugar acids, and sugar alcohols were generally lower in teosinte than the other two groups. Thus, the metabolite analysis showed significant differences in metabolite contents in kernels of maize at different domestication stages, which may be associated with their health-beneficial bioactivities. The kernel material was processed by the semi-high throughput digestion method simulating the human gastrointestinal digestion developed in the previous reporting period. The peptide contents in the digested kernels were significantly higher in teosinte and modern maize than in the landraces. The peptide contents varied among genotypes within the domestication groups, with modern lines showing the largest variety. The metabolite profiling of the digested kernel materials is currently ongoing. Data collected The profiles of 103 metabolites in the kernels of 76 lines of modern maize, landraces, and teosinte were obtained. The peptide contents in the digested kernels in these genotypes were determined. The whole-genome sequencing data has been produced. Discussion The metabolite profiling of raw kernels and peptide contents in digested kernels in this reporting period highlighted the great diversity of chemical properties of kernels among and within the domestication groups of maize. The higher contents of multiple phenolic compounds in teosinte is probably due to the higher phenylpropanoid biosynthesis activity in teosinte. The phenylpropanoid profiling in the following reporting period will clarify this trend. However, the phenolics can be accumulated in the kernel shell, which is lost in the landraces and modern maize. We will conduct metabolite profiling of dissected shells of teosinte kernels to clarify if the differences in phenolics accumulation are due to the morphological diversity. It is widely known that kernel carbohydrate contents increased and protein contents decreased through maize domestication and improvement. This trend is also apparent in the current study as the kernels of modern maize and landraces showed generally higher contents of sugars, sugar acids, and sugar alcohols than teosinte. Interestingly, the contents of free amino acids in the raw kernels are lower in teosinte despite teosinte accumulating much higher amounts of protein in kernels. We have no clear explanation for this trend, but the compositions of the storage proteins can cause the inconsistency between amino acids and protein contents. The peptide contents in the gastrointestinal digested kernels are surprisingly diverse within the domestication groups. We had expected more significant differences between the teosinte and other groups considering the presence of physically hard shells on teosinte kernels. However, the peptide levels in digested teosinte kernels were close to the modern and landrace maize despite nearly three times higher protein contents in raw kernels. This is likely due to the low digestibility of teosinte kernels, and we are determining the remaining protein contents in the digested kernels to evaluate their digestibility. Protein digestibility is a complex trait affected by many chemical and physical factors. The genetic comparison in the following reporting period may indicate the potential mechanism affecting the digestibility of the kernels. Key outcomes and accomplishments We have determined the metabolite profiles and gastrointestinal digestibility of the kernels of maize and its relatives. These data indicated the chemical diversity in the kernels of these genotypes. Once the whole genome sequences of the remaining landrace and teosinte lines are completed, the genomic loci related to the nutritional facts of maize kernels will be identified.
Publications
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The majortarget audiences in this reporting period were food and nutritional scientists, plant biochemists, and plant geneticists. The mechanisms of health benefits are significant topics in food and nutritional sciences and biochemistry. The genetic materials and genome sequence data of teosinte and landrace maize lines produced in this reporting period are valuable resources for plant geneticists and maize breeders. Changes/Problems:Since we could not identify a postdoc responsible for kernel material production before the summer nursery season, Co-PD Yang spent one month for plant care in the summer. Two graduate research assistants are hired to work on this project instead of a technician and a postdoc. These will have no significant impact on the project schedule and expenditure. What opportunities for training and professional development has the project provided?Two Ph.D. students (Rajnee Hassan, Center for Biological Chemistry, UNL; Semra Palali Delen, Department of Agronomy and Horticulture, UNL) and one master's student (Prabhashis Bose, Food Science & Technology Masters' Program, UNL) have joined to work on this project. They have been trained to conduct metabolite profiling, quantitative genetics analysis, and semi-high throughputGI digestion. One postdoc (Michael Meier, Department of Agronomy and Horticulture, UNL) participated in this project to conduct a quantitative genetic analysis from the beginning of 2022. He got a position at Rancho Biosciences, San Diego, CA, in April 2022. How have the results been disseminated to communities of interest?The manuscript describing the results of genome-wide mediation analysis has been accepted for publication in Genetics (https://doi.org/10.1093/genetics/iyac057). The book chapter describing the analytical procedure of metabolite profiling by high-resolution GC-MS used in this project has been accepted to be published in the Methods in Molecular Biology book series. What do you plan to do during the next reporting period to accomplish the goals?We will finalize the metabolite analysis of raw kernels (Objective 1) and whole-genome sequencing (Objective 3) soon. The results will be used for the quantitative genetic analysis to identify the genes associated with the accumulation of nutrient contents in kernels (Objective 3). The kernel materials will be treated by simulated GI-digestion using the high-throughput procedure developed in this reporting period. The digested kernels will be the subject of metabolite analysis and bioactive assays during the next reporting period (Objective 2).
Impacts
What was accomplished under these goals?
IMPACT In this reporting period, we harvested the kernels from lines of modern maize, open-pollinated maize landrace, and the wild maize ancestor, teosinte. Whole-genome sequences of these lines will be analyzed to reveal the relationships between genomic sequences and nutritional traits of these kernels. The kernels are used to determine nutritional traits, including the contents of dietary compounds and the health-beneficial bioactivities in the human intestine. These analyses in the following reporting periods will determine the beneficial genes regulating nutritional values of maize kernels in maize ancestors, which were lost during maize domestication. We have also developed multiple experimental and analytical procedures to enhance the project activities. First, a semi-high throughput method to simulate gastrointestinal digestion was developed, which will allow faster processing of a large number of samples. Second, assay systems to assess the biological (anti-inflammatory and antioxidative) activities of digested food materials were developed to simulate the responses of the human intestinal cells. Third, a novel quantitative genetic approach was developed to elucidate the relationships between genes, compound accumulation, and plant traits. This approach identified the genes related to the contents of multiple compounds in the leaves of maize juveniles and the agronomic traits using the previous and public data. These procedures will be beneficial for the scientific community to investigate the effects of foods in the human intestine and identify genes determining food crops' nutritional values. Major activities/experiments We have selected the plant lines to be analyzed, including 26 modern maize, eight landraces (17 have been selected previously), and 25 teosintes, to maximize the geographic distribution and genetic diversity. Seeds of all these lines were propagated through the summer 2021 nursery. Genomic DNA was extracted from the kernels, and whole-genome sequencing analysis is currently ongoing. The metabolite profiles of the kernels of modern maize lines have been analyzed. We have developed a gastrointestinal digestion procedure enabling higher throughput and saving sample amounts. The method we used to employ requires grams of samples, which is not ideal, especially for teosinte that produces a limited quantity of kernels. The old procedure is also labor-intensive and can process only one sample/day. The newly developed method allows us to process 12 samples at once with as low as 100 mg of material. The kernel samples are currently being processed for bioactivity and metabolite assays with this procedure. Initial bioactivity assessment will be conducted with the intestinal cell lines (Caco2 cells) since it allows high throughput bioactivity tests. However, Caco2 cells sometimes fail to reflect the physiological responses of normal intestinal cells. To confirm the bioactivity of the kernels and candidate chemicals, we have established a human induced pluripotent stem cell (iPSC)-derived intestinal cell culture, which better resembles the normal intestinal cell. We have also established the procedure using an additional intestinal cell line, HT-29, in parallel and combination with Caco2 cells to reduce the risks of artifacts. We have also developed a novel quantitative genetic approach to map genetic loci associated with phenotypes, including metabolite accumulations, by high dimensional mediation analysis. This approach was applied to identify the genes regulating metabolite accumulation in the leaves of juvenile maize. The metabolite profiling data was previously generated from the same population of modern maize inbreds analyzed in this project. The mediation analysis identified many genes mediating the metabolite accumulation and agronomical phenotypes. Among them, the Bx13 gene mediated 15 agronomical and 25 metabolic traits. This gene encodes 2-oxoglutarate-dependent dioxygenase enzyme modifying benzoxazinoids, defense compounds abundant in maize seedlings. Bx13 gene expression had been associated with the accumulation of not only benzoxazinoids, and our results indicated it also influences the accumulation of central carbon metabolites such as glutamine, trans-aconitate, and malic acid. Data collected The whole-genome sequencing data will be generated soon. The GC-MS chromatography data of the modern maize kernels have been collected. Discussion The landrace maize and teosinte genetic materials are valuable tools for analyzing genetic modifications during the maize domestication process. These materials also serve as the genetic resources of the beneficial crop traits overlooked in the selection history. We will take advantage of these materials to elucidate genetic loci associated with the nutritional properties of maize kernels lost during maize domestication in the following reporting period. The Caco2 is a cell line derived from human colorectal adenocarcinoma cells. While it is a handy tool to test the physiology of intestinal cells, Caco2 sometimes shows abnormal behavior derived from its cancerous origin. The iPSC-derived intestinal cell line has a non-cancerous origin and will more accurately represent the diversity and physiological characteristics of human gastrointestinal epithelial cells. Since the iPSC cells require much more effort to maintain and test, we will use Caco2 cells for the initial test and iPSC cells for confirmation. It should be noted that the anti-inflammatory activity of soybean sprouts detected in Caco2 cells was confirmed in vivo using a mouse model in our previous study (unpublished), indicating the reliability of the Caco2-based assays. The parallel use of Caco2 with another intestinal cell line (HT-29) will reduce the risk of detecting Caco2-specific responses. The newly developed genome-wide mediation approach can detect the mediator genes for a trait of interest. This approach will be a powerful tool for revealing the metabolite mediating bioactivities for the phenotype and further identifying the genes associated with nutritional values and health beneficial bioactivities. Key outcomes and accomplishments We have selected modern maize, landrace, and teosinte lines and generated kernel materials for the analyses. Metabolite profiling and whole-genome sequencing of these materials will provide valuable data for the research community to analyze the genetic basis of kernel nutrient accumulations and their acquisition and loss through the domestication history. The higher throughput procedure for the simulated GI digestion and reliable assay systems for the intestinal bioactivities developed in this reporting period will enhance the downstream analyses. The newly hired graduate research assistants have been trained and started to process the samples.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Zhikai Yang, Gen Xu, Toshihiro Obata, Qi Zhang, and Jinliang Yang. Genome-wide mediation analysis: an empirical study to connect phenotype with genotype via intermediate transcriptomic data in maize. Genetics. (accepted) DOI:10.1093/genetics/iyac057
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2022
Citation:
Nishikant Wase, Nathan Abshire, Toshihiro Obata. High Throughput Profiling of Metabolic Phenotypes Using High-Resolution GC-MS. Methods in Molecular Biology (In press)
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