Source: CLEMSON UNIVERSITY submitted to
SUSTAINABLE, HIGH-QUALITY ORGANIC PULSE PROTEINS: ORGANIC BREEDING PIPELINE FOR ALTERNATIVE PULSE-BASED PROTEINS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1026666
Grant No.
2021-51300-34895
Cumulative Award Amt.
$1,227,675.00
Proposal No.
2021-02927
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2025
Grant Year
2021
Program Code
[113.A]- Organic Agriculture Research & Extension Initiative
Project Director
Thavarajah, D.
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Plant & Environmental Sciences
Non Technical Summary
Organic plant-based proteins are popular because they are a clean source of protein without added chemicals. Contrary to consumer perception, organically grown pulse crops have lower protein content. This low protein content makes organic pulse proteins expensive, and cultivars bred for non-organic production are often not suited to organic protein production. The organic ingredients possess better protein quality, digestibility, color, texture, and flavor than conventional pulse proteins. These quality traits need to be included in the breeding pipeline to develop future organic cultivars suitable for organic pulse-based protein production. Our long-term goal is to breed field pea and lentil cultivars suitable for organic production with higher protein quality, digestibility, color, texture, flavor, and better adaptation to protein isolation. This project is built on our previous OREI and Good Food Institute-funded projects to refine the breeding pipeline to develop protein-biofortified organic pulse cultivars for plant-based protein applications that will lead to economically rewarding sustainable organic farms. The objectives of this project are to (1) develop protein-enriched organic field pea and lentil varieties using on-farm field selection, (2) combine genomic information and protein trait mapping to enable the best genotype-to-phenotype mapping, and (3) develop on-farm educational and extension activities using the existing Clemson University (CU) "Going Organic portal," Carolina Farm Stewardship Association, Good Food Institute, and e-Organic platform. This project is aligned with the OREI goals to (1) develop and improve organic agriculture production, breeding, and processing methods; (2) determine desirable traits; and (3) develop new and improved seed varieties for organic agriculture.
Animal Health Component
25%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011414108150%
2021412108050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1414 - Lentil; 1412 - Peas (dry);

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Supporting Objectives: Develop a breeding pipeline for protein-enriched organic field pea and lentil varieties using on-farm field selection to (i) increase crop adaptation toward sustainable organic farming systems and (ii) improve protein quantity and quality (sulfur amino acid balance, protein digestibility, color, texture, aroma, and flavor) toward plant-based protein production.Combine genomic information and protein trait mapping to enable the best genotype-to-phenotype mapping and lead to future breeding of high protein quality cultivars with increased economic returns.Develop on-farm educational and extension activities using the Clemson University (CU) "Going Organic," Carolina Farm Stewardship Association (CFSA), Good Food Institute (GFI), and eOrganic resources.
Project Methods
Plant Material: For the on-farm selections, ~49 lentil elite cultivars in production and advanced breeding line accessions from the USDA-ARS and ICARDA breeding programs will be used. For the association mapping study, a ~361-member lentil Global Diversity Panel (LGDP) from ICARDA with a diverse population structure and familial relationship will be used for gene discovery. Field pea on-farm trials have been finished, and the same seeds will use for protein trait phenotyping.Experimental design: In years 1 and 2, the ~49 lentil cultivars and advanced breeding lines will be planted in a complete α-lattice field design with commercial checks with three replicates at two on-farm locations in SC and NC (n=588). Cultivars will be sown in 1.2 m × 6 m plots at a rate of 90-95 seeds/m2 and a seeding depth of 5-7 cm. Each plot will contain four rows spaced 30 cm apart. On-farm variety performance will be evaluated using days to flower, days to harvest maturity, pods/peduncle, pod height, plant height, canopy height, 1000-seed weight, and grain yield. All field evaluations will record diseases, insects, and weeds every week using a scale developed by the Clemson Organic Pulse Breeding program.Protein Quality Analysis: Proteins will be extracted using 10-20 g of organic lentil and field pea seeds from the on-farm trials. The isolation procedure for organic field pea proteins and related composition has been filed as a provisional patent (US patent 63/106,015) by the PI's team.Genotyping: Illumina sequencing for the advanced field pea/lentil cultivars will be conducted in year 1. Processing raw sequencing data will be carried out in TASSEL. Genetic variants [i.e., single nucleotide polymorphism (SNP) markers] identified from genome-wide association mapping (GWAS) will be analyzed in advanced lines and hybrids to determine the number of favorable alleles at each informative marker site (i.e., locus). On-farm results from years 1 and 2 will determine the prediction accuracy and utility of genomics in organic grain breeding.Data Analysis: Raw phenotypic datasets from each location will be compiled in years 1 and 2 after completing grain quality analyses. Broad-sense heritability estimates will be calculated from multiyear data generated from both field sites. Spearman correlation coefficients and rankings will be generated to understand interrelationships among production and quality traits. ANOVA will be performed to understand the individual effects of genotype, environment, and genotype-by-environment interaction. Based on previous work under conventional management, yield and quality traits are anticipated to be highly heritable. Under this hypothesis, genetic analysis will be important to leverage advanced genomic technologies (e.g., genomics-assisted breeding) to increase genetic gain and breeding efficiency and rapidly develop varieties suited for organic production and management systems.Objective 2: Develop and utilize public genomic resources to identify marker-trait associations and advance economically efficient, high-protein-quality cultivars.Plant Material: A ~361-member LGDP from ICARDA, including the base collection of lentil germplasm maintained at the USDA-ARS, will be used to generate genomic resources and identify marker-trait associations (MTAs).Field design: The field design for the LGDP will be similar to Objective 1. These accessions will be planted in a complete α-lattice field design with commercial checks with three replicates at two on-farm locations in SC and NC for two years (n=1083 per location). Each incomplete block is augmented with CDC Redberry (check cultivar for lentil genome) at random positions to reduce special variability. LGDP collection seeds will increase from single plants at CU USDA Organic certified fields in 2021 before planting at on-farm locations, including WP Rawl and Sons (Pelion, SC) and Lomax Farm (Concord, NC).Genotyping: As a recently developed lentil reference genome is now available (https://knowpulse.usask.ca/lentil-genome), lentil researchers are able to develop foundational genomic resources for use in current and future breeding initiatives. To that end, a genomic resource composed of 361 lentil accessions from the LGDP will be built by performing whole-genome sequencing using an Illumina NovaSeq 6000 system to obtain 10× coverage in years 1 and 2 of this project. Accessions will be sequenced using lyophilized leaf tissue grown in a controlled greenhouse. Sequencing data will be aligned to the V1.2 lentil reference genome and processed according to Genome Analysis Toolkit (GATK) best practices (McKenna et al. 2010; DePristo et al. 2011) to maintain data quality standards and metadata descriptions of the experiments and promote reproducibility of the results.MTAs: Raw phenotypic datasets from each location will be compiled in years 1 and 2 after completing protein quality analyses (amino acid quality, color, texture, flavor). Broad-sense heritability estimates will be calculated from multiyear data generated from all four field sites to determine the degree to which genetic factors contribute to the quantity, quality, and profile of lentil proteins. Spearman correlation coefficients and rankings will be generated to understand the associations between production and quality traits. As most agronomic traits of interest are regulated by many loci, where each locus confers an effect of varying magnitude and range on a given trait, experiments must be designed to capture a broad range of genetic effects.High-density markers will facilitate the fine-grained resolution of informative markers via GWAS. GWAS will be performed using tools such as GAPIT and GEMMA, which allow for the rapid execution of an array of models. Statistical analyses of the PSPPC and LGDP will use linear mixed models to account for relationships among individuals within the panels and control the false-discovery rate. Associated genetic variants (i.e., SNP markers) will be used to identify favorable alleles and putatively causative genes using knowledge of genes in linkage disequilibrium.Objective 3: Develop on-farm educational and extension activities using CU "Going Organic," CFSA, GFI, and eOrganic resources.Communication Methods: Communication methods include writing news releases, producing videos, and creating audio files. Outlets for disseminating this material include a webpage, social networks, radio stations, television stations, and print media. A webpage has been created on the Clemson Public Service and Agriculture website to house print articles and video files https://www.clemson.edu/cafls/organic-breeding/orei/index.html).Education Activities: The PI's ongoing organic education initiative has three components: (1) incorporate undergraduate students through CU's Creative Inquiry (CI; https://www.clemson.edu/centers-institutes/watt/creative-inquiry/)) and University Professional Internship and Co-op (UPIC) programs; (2) recruit local, regional, and national high-school students who are planning to attend the College of Agriculture, Forestry, and Life Sciences (CAFLS) at CU via the SC Commissioner's School, SC Governor's School, and Emerging Scholar Programs; and (3) educate national and international students on organic pulse breeding via the ongoing virtual Organic Plant Breeding Summer Institute.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Target audience: The target audience includes organic vegetable and pulse crop producers (Specialty Crops), food processors, pulse breeders, consumers, chefs, culinary personals, and future generations of plant breeders, including underrepresented SC high school (4-H), graduate students, Plant and Environmental Sciences (PES) undergraduate students, South Carolina (SC) Commissioner School, Creative Inquiry (CI), and University Professional Internship and Co-op (UPIC) programs. These key stakeholders are from local, national, and international audiences from Africa, Asia, Europe and South America. Briefly, (1) Classroom education includes graduate students, undergraduate students, visiting fellows, plant breeders, CI: undergraduate students, beginning, non-traditional farmers, and veterans, an internship program for undergraduate students (first-year students from PES), underrepresented national and international high school students; consumers, food processors, healthcare workers, and local community; 4-H students from SC (middle and high school students); local food and technology industry partners for commercialization of patents and varieties, and global research community (International Food Legume Research Community (IFLRC); North American Pulse Improvement Association (NAPIA), (2) Extension and outreach: growers, food processors, consumers, chefs, restaurant communities, local food communities, marketing personnel, researchers, breeders, and future generations of plant nutritional breeding leaders. Changes/Problems:One of the field locations at Clemson University was destroyed by bad weather, which delayed the planting window. Therefore, we are hoping to add one extra organic on-farm location for advanced breeding lines testing for variety release. What opportunities for training and professional development has the project provided?PD-Thavarajah applied for the nationally competitive program, the Jefferson Science Fellowship, and was selected for the 2024-2025 class of Jefferson Science Fellows, which is the 21st class of Fellows selected since 2003 as an initiative of the Office of the Science and Technology Adviser to the U.S. Secretary of State. This program is designed to build capacity for science, technology, and engineering expertise within the U.S. Department of State and the U.S. Agency for International Development (USAID). PD-Thavarajah will serve USAID from Aug 2024 to Aug 2025 (https://www.nationalacademies.org/our-work/jefferson-science-fellowships/fellows). PD-Thavarajah also obtained training and professional development courses on diversity and equity, conflict of interest, HR/hiring procedures, and the CITI program on human Subjects protection courses, including investigators conducting social and behavioral science research. PD-Thavarajah was the chair of the organizing committee of the 2023 North American Pulse Improvement Association (NAPIA) and Bean Improvement Corporation (BIC) meeting held at Greenville, SC. Senior doctoral student Sonia Salaria was awarded a Wade Stackhouse Graduate Fellowship (2023-2024) and a Graduate student travel award by 2023 BIC/NAPIA. Sonia attended the plant breeding tool workshop for accelerated plant breeding and career-building professional workshops at the NAPIA/BIC meetings. She served as the graduate student representative for the 2023 NAPIA/BIC organizing committee at Greenville, SC. Sonia also served as a teaching assistant to PD-Thavarajah courses to develop professional teaching experience. Further, she attended several coding and computation courses during the reporting period for genomic analysis. Amod Madurapperumage, a doctoral student, and the field manager, Tristan Lawrence, have obtained the United States Department of Transportation - Federal Aviation Administration (part 107) license and Training to fly UAVs from geospatial information services at Clemson University, SC. Amod won the Graduate student oral presentation competition (2nd place) at the 2023 NAPIA/BIC conference - in Greenville, SC. Amod attended several professional development training sessions for high-throughput phenotyping from Phenome Network, Texas-AM, and Agilent Technologies. Two other doctoral students and the visiting postdoctoral fellow attended the NAPIA/BIC and the NAPB conferences and presented their research posters. Mark Dempsey won first place in the graduate student poster competition at the 2023 BIC/NAPIA. High school student activities include attending STEM sessions. These workshops include Mendelian breeding games, phenomics, mendelian laws, food tasting, and cooking experiences for the farm-to-table approach. Dr. Thavarajah offered a fall three-credit creative inquiry (CI) course and the University Professional Internship and Co-op (UPIC) course. These classes have four modules: i) hands-on breeding experience, ii) phenomics techniques, iii) building healthy food systems, and iv) developing a pulse cookbook. At the end of the semester, these students presented their projects at the CI Undergraduate Research Symposium, NAPIA/BIC meetings and honors college research symposium. The Organic Pulse Breeding team, including graduate students, undergraduate students, postdoctoral fellows and staff, had several workshops with the industry partners related to this project, including pulse growers, SC Department of Agriculture, SC Research Authority (SCRA), Ardent Mill, Anson Mill, WP Rawls and Sons, Agilent technologies from the UK, Clemson University Research Foundation (CURF), and several tech companies from SC, France, and Canada. How have the results been disseminated to communities of interest?Protein quality and grain yield rankings by genotype have been distributed to stakeholders via the grower meetings. On-farm results were disseminated directly to stakeholders (food processing, consumers,and marketing) using multiple social media outlets - website, YouTube, and social media. As such, Mr. Cory Tanner fromClemson Extension has regularly coordinated data releases and publications in addition to updating social media networks. Dr. Erin Clayton has coordinated protein quality data release to the plant-based food processing and manufacturingcommunity. We developed a new global partnership with FoodShot Global for precision protein. The results have beenpresented at the NAPIA meeting to update the pulse research community and communicated to national and localstakeholders via board meetings, peer-reviewed publications, webinars, and workshops. What do you plan to do during the next reporting period to accomplish the goals?The project team will continue the work related to all three objectives.

Impacts
What was accomplished under these goals? Our project aims to breed protein-enriched field pea and lentil cultivars for southeast organic cropping systems. Three breeding cycles are ongoing for organic field peas -15-20 crosses were made for each breeding cycle. For the fourth breeding cycle, 25 crosses will be carried out in fall 2024. In the 2023 fall, F4:6 plots (162) and parental plots (22) were threshed, the F4:7 seed was cleaned, and then nutrient lab analyses were conducted for each plot using FT-MIR for nutritional traits. F4:7 seed of 162 composited 2023 field plots (representing twelve 2020 cross populations) were cleaned and analyzed for protein, resistant starch (RS), total starch (TS), and protein digestibility using 1-2 seeds. Seed coat color and seed size were also determined for each of the 162 F4:7 breeding lines. Four 2024 Breeder Preliminary Tests (2 replications each) using a 7x7 lattice field design with 40 (T-1 and T-2) or 41 breeding lines (T-3 and T-4) in each test were planted at W.P. Rawl Farm, SC. For the 2025 field season, early generations will be selected for protein, sulfur-containing amino acids (SAAs), and digestibility using a high-throughput phenomics platform. Organic cultivars will be released in 2025-2026 after two years of yield trials at the organic on-farm locations. Dry pea seed yields were 3833 kg/ha. Seed protein ranged from 17 to 30%, with 80-85% protein digestibility. Total prebiotic carbohydrate concentrations ranged from 14.7 to 26.6 g/100 g; a 100-g serving of organic dry pea provides 73-133% of the recommended daily allowance. In collaboration with Dr. Diego Rubiales (Institute for Sustainable Agriculture, Spain), 58 breeding populations (F3-F8) were evaluated for 2023 and 2024 field seasons, and selected lines will be tested in 2025 for dual release in the USA and Spain. After the MTA with Spain and Clemson University, 7-8 F8 lines were selected for the 2024 fall crosses. Lentil heat-tolerant (~143-member) and global mapping (129-member) populations from ICARDA were screened to develop climate-resilient cultivars with improved protein quality and digestibility (under the international treaty agreement with ICARDA and Clemson University). This panel had six sub-groups. The mean concentration of 17 different amino acids (AAs) was quantified, including the SAAs methionine (0.21%) and cysteine (0.22%). Fifty significant SNPs were associated with 17 traits of AAs and protein digestibility. The Lentil association mapping panel (LAMP), composed of 446 accessions, was evaluated for protein quality with agronomic adaptation. LAMP has been evaluated under greenhouse conditions for two years using a complete randomized experimental design. In 2024, LAMP was planted in the fields at two locations in South Carolina. The experiment was an α-lattice design with two replications per location and the check CDC Maxim. The data collection for agronomic traits (days to germination, days to flowering, days to maturity, plant height and height of the lowest pod) and protein quality traits (protein, sulfur-containing amino acid concentrations and protein digestibility) has been in progress. The lentil accessions with early maturity, tall plant height, good yield and protein quality have been found and used for the lentil crossing block. Fifteen parents were used for 27 crosses. The panel has also been genotyped, and the sequencing data will be processed and utilized to conduct GWAS in the fall of 2024. 96 LAMP accessions (not genotyped earlier) were genotyped with 10x coverage, and genotyping data analysis is in progress. The preliminary quality check for the genotyping data has been completed. Non-destructive phenotyping tools have been developed to measure eight nutritional traits from one scan for speed breeding. Fourier transform mid-infrared (FT-MIR) spectroscopy is a high-throughput, cost-effective method to quantify nutritional traits. FT-MIR spectroscopy coupled with an attenuated total internal reflectance sampling interface was used to develop multivariate models to measure protein, SAAs, TS, RS, fats, saturated fats, unsaturated fats, and moisture concentrations in a 30-s scan. Total N data from combustion analysis and SAA data from high-performance liquid chromatography (HPLC) analysis following acid hydrolysis were used for model calibration and validation. We used FT-MIR spectroscopy to screen the early generations (F2 and F5) to speed up the selection process. Chemometric models were developed with partial least square regression (PLSR) to determine total proteins and protein digestibility in field peas and lentils and, further, total fatty acids. These regression models had 70 % of samples for calibration and 30 % for validation. We have developed infrared (IR) cameras and unmanned aerial vehicles (UAVs) to select the best breeding populations in the field and greenhouse. High throughput phenotyping is further required for agronomic traits (i.e., plant height, lodging, days to maturity etc.). Remote sensors based on unmanned aerial vehicles (UAVs) are used to phenotype agronomic traits. For high-throughput phenotyping, spectral details can be unpacked and processed with photogrammetry tools, such as crop surface modeling (CSM) and normalized difference spectral indices. Accordingly, a photogrammetry study with remote sensing has been initiated on field pea breeding plots. A Phantom 4 UAV with a 20-megapixel camera was employed to collect images. The UAV was flown across Rawls farm (Advanced trials (F7): 24.3 m x 162.5 m) and Cherry Farm (Advanced trials (F7): 42.6 m x 54.8 m) fields at Pelion (33.7946 0N, 81.2713 0W) and Clemson (34.6527 0N, 83.8360 0W). Images were collected at 25 m and 30 m per location. These images were subjected to ortho mosaic to generate 2D maps to extract appropriate spectroscopic indices for photogrammetry modeling the agronomic traits above. For 2023-2024, 6 high school student workshops (Pulses 4-Health: Science of Peas, Lentils & Chickpeas) for 4-H were conducted, covering 200-250 students. The annual 2024 field day was conducted at the WP Rawls on-farm location in Pelion, SC, and 62 participants attended the field workshop. >120 extension activities, including training programs, short courses, workshops, presentations, meetings and farm contacts related to the project, were completed for the 2023 -2024 reporting time. The Thavarajah team organized an "Eat Pulses" workshop for consumers and the CU health team--this involved pre-and post-tests after lectures, round table discussions, and cooking experiences at the CU kitchen. Barriers to pulse consumption were discerned, with half the attendees indicating pulses were not part of their usual (habitual) food intake, almost one-third reporting the long cooking time was a barrier, and one-fifth noting they were unfamiliar with preparation and recipes. Less common barriers included not liking the taste and having gastrointestinal upset. Attendees were open to learning more, especially after attending this workshop and tasting authentic pulse dishes, and all were open to having pulse ingredients in their regular foods. The attendees were convinced the benefits of pulses, including health effects and reduced cost compared to animal protein, would be necessary for those with limited incomes. Regular courses were carried out for undergraduate students, including the Creative Inquiry and UPIC Internship programs. Students attend North American Pulse Improvement Association and National Association of Plant Breeders meetings, present their research, and publish a pulse cookbook in 2024. The program appeared on public radio, TV, and in several news publications on organic pulse breeding. This project trains five doctoral students (two new students), three research associates, two full-time technicians, two postdoctoral fellows, and > ten undergraduates. Substantial extension and outreach activities were done, and three new utility patents were submitted.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Amod Madurapperumage , Nathan Johnson, Pushparajah Thavarajah, Leung Tang, Dil Thavarajah!, 2023. Fourier-transform mid-infrared (FT-MIR) spectroscopy as a high-throughput phenotyping tool for measuring total fatty acids in chickpeas (Cicer arietinum L.). ACS Food Science & Technology, 3:1568-1576. https://doi.org/10.1021/acsfoodscitech.3c00239.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Nathan Johnson , J Lucas Boatwright, William Bridges, Pushparajah Thavarajah, Shiv Kumar, Dil Thavarajah!, 2023. Targeted improvement of plant-based protein: Genome-wide association mapping of lentil (Lens culinaris Medik.) diversity panel. Plants, People, Planet (PPP), 6(3):640-655 https://doi.org/10.1002/ppp3.10470.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Nathan Johnson , Pushparajah Thavarajah, Nathan Windsor, Leung Tang, Dil Thavarajah!, 2023. Fourier-transform infrared spectroscopy (FTIR): an inexpensive, rapid, and non-destructive tool for starch and resistant starch analysis. The Plant Phenome Journal, 2023; 6: e20086. https://doi.org/10.1002/ppj2.20086
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Sonia Salaria , J. Lucas Boatwright, Nathan Johnson, Priyanka Joshi, Pushparajah Thavarajah, George Vandemark, Dil Thavarajah!, 2023. Fatty acid composition and genome-wide associations of a chickpea (Cicer arietinum L.) diversity panel for biofortification efforts. Scientific Report, 13(1):14002. https://doi.org/10.1038/s41598-023-41274-3.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Sonia Salaria, Dil Thavarajah, 2024. Lentils (Lens culinaris Medik.): A potential new pulse crop for South Carolina. CAFLS Research Symposium, Garrison Expo Center, August 19-20, Clemson, SC.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Chamodi Senarathne, Pushparajah Thavarajah, Dil Thavarajah. 2024. Flavor chemistry towards biofortification and palatability of pulse-based food. CAFLS Research Symposium, Garrison Expo Center, August 19-20, Clemson, SC.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Amod Madurapperumage, Pushparajah Thavarajah, Leung Tang, Dil Thavarajah, 2024. Designing Fourier transform mid-infrared spectroscopy (FT-MIR) for high throughput phenotyping macro nutritional traits in pulses. CAFLS Research Symposium, Garrison Expo Center, August 19-20, Clemson, SC.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Amod Madurapperumage , Nathan Windsor , Nathan Johnson, Pushparajah Thavarajah, Leung Tang, Dil Thavarajah!, 2024. Fourier-transform mid-infrared (FT-MIR) spectroscopy for in vitro protein digestibility measurement of pulse crops, Crop Science, 02 July 2024. https://doi.org/10.1002/csc2.21300.
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Mark Dempski , Dil Thavarajah! 2024. Low Molecular Weight Carbohydrates and Stress Tolerance in Lentil (Lens culinaris Medikus): A Review. Frontiers in Plant Sciences, section Plant Breeding, 1408252.
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Amod Madurapperumage , M.Z. Nazer, J. Lucas Boatwright, William Bridges, George Vandermark, Dil Thavarajah!, 2024. High-throughput Phenotyping (HTP) Platforms for Pulse Crop Biofortification. Plants, People, Planet, PPP-R-2024-00942, Accepted.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Nathan Windsor , Lucas Boatwright, Richard Boyles, William Bridges, Diego Rubiales, Diego, Dil, Thavarajah!, 2024. Biofortifying Dry Pea (Pisum sativum L.) for Improved Performance and Nutrition. Legume Science, LEG3-2024-036, Accepted.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Mark Dempsey, Dil Thavarajah, 2024. Toward adapting lentil (Lens culinaris Medik) for Organic Production in the Southeastern U.S. CAFLS Research Symposium, Garrison Expo Center, August 19-20, Clemson, SC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Johnson, J., Kresser, C., Hazards, D., Windsor, N., Salaria S., Dil Thavarajah, 2024. From practicing pulse breeding to extension outreach: An extended approach for awareness of pulse crops. Clemson University 19th Annual Focus on Creative Inquiry Forum, Clemson, SC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Charles Kresser, Samadhi Thavarajah, Catherine Barry, Sonia Salaria, Dil Thavarajah 2023. How can a pulse-rich diet make an impact on human health? North American Pulse Improvement Association, Greenville, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Ash Terry, Jacob Johnson, Sonia Salaria, Tristan Lawrence, George Vandemark, Dil Thavarajah, 2023. Exploring Chickpeas (Cicer arietinum L.) for South Carolina Cropping Systems. North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Nathan Windsor, Dil Thavarajah 2023. Biofortifying dry peas for improved protein quality and adaptation to the southern United States. North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mark Dempsey, Dil Thavarajah, 2023. First Steps Toward Adapting Lentil for Organic Production in the Southeastern U.S. North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Amod Madurapperumage, Pushparajah Thavarajah, Leung Tang, George Vandemark, Dil Thavarajah 2023. Designing Fourier transform mid-infrared spectroscopy (FT-MIR) for high throughput phenotyping macro nutritional traits in pulses. North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Priyanka Joshi, Dil Thavarajah, George Vandemark, 2023. Genome-wide association mapping for seed protein concentration in chickpea (Cicer arietinum L.). North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Joshua Kay, Dil Thavarajah, 2023. Why Are We Interested in the Prebiotic Content in Dry Peas (Pisum sativum L.). North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Hidden hunger quickens the 'pulse' at Clemson's Tiger Gardens. SC Public Radio, July 12, 2024. https://www.southcarolinapublicradio.org/sc-news/2024-07-12/hidden-hunger-quickens-the-pulse-at-clemsons-tiger-gardens
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Market Bulletin, SC Department of Agriculture, June 6, Clemson students write a cookbook to help fight hidden hunger.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sonia Salaria, George Vandemark, Dil Thavarajah, 2023. Genome-wide association Studies (GWAS) to explore the fatty acid composition of a chickpea (Cicer arietinum L.) diversity panel for biofortification. North American Pulse Improvement Association, GV, SC, Nov 5-7, 2023.
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Morning AgClips, https://www.morningagclips.com/clemson-students-write-cookbook-to-help-fight-hidden-hunger/
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Clemson students write cookbook to help fight hidden hunger https://news.clemson.edu/clemson-students-write-cookbook-to-help-fight-hidden-hunger/
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Organic Farming Research Foundation (OFRF), Organic Researcher Spotlight: Organic pulse crops with Dr. Dil Thavarajah, https://www.youtube.com/watch?v=1glgpxEn9ic.
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Organic Farming Research Foundation (OFRF); A breeding pipeline is developing improved pulse crops for organic farmers in the southeast https://ofrf.org/news/organic-researcher-spotlight-dr-dil-thavarajah/
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Florence Morning News; going organic webinar series 2024: https://scnow.com/business/agriculture/clemson-sets-datestopics-for-2024-going-organic-webinar-series/article_847e7c14-2d3b-5088-b1ac-113689baf0a6.html
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Times and Democrat going organic webinar series 2024, https://thetandd.com/business/agriculture/clemson-sets-datestopics-for-2024-going-organic-webinar-series/article_96d67b22-b973-11ee-9b73-2fc6e5cce11a.html.
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Clemson announces dates/topics for 2024 Going Organic webinar series, https://news.clemson.edu/clemson-announces-dates-topics-for-2024-going-organic-webinar-series/
  • Type: Other Status: Published Year Published: 2024 Citation: 2024: Going organic webinar series, Jan 18, Market Bulletin. SC Department of Agriculture, SC.
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: A South Carolina chickpea variety? Nov. 16 Market Bulletin, SC. Department of Agriculture.
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: New crop development at Clemson University could lead to a boost in SC's economy, https://www.wyff4.com/article/south-carolina-clemson-chickpeas/45997308#
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Research could lead to SC chickpea variety, Index-Journal, https://www.indexjournal.com/townnews/fruit_farming/research-could-lead-to-sc-chickpea/article_ff3134e7-d5d1-582b-aded-f4d9394f38bc.html
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Research could lead to SC chickpea variety, Times and Democrat, https://thetandd.com/business/agriculture/research-could-lead-to-sc-chickpea/article_caf62532-7761-11ee-8c9c-2f0d506a1c8b.html
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Farms.com, https://www.farms.com/news/clemson-scientists-discover-plant-genes-to-help-grow-s-c-economy-202426.aspx
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Seed Today, https://www.seedtoday.com/article/307335/clemson-university-scientists-discover-plant-genes-to-help-grow-south-carolina-economy
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Research could lead to SC chickpea variety SC Now/Florence Morning News, https://scnow.com/business/agriculture/research-could-lead-to-sc-chickpea/article_de1b9765-d37e-5099-835e-e15b736424da.html
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: News Break, https://original.newsbreak.com/@us-updates-1671025/3210908520671-local-clemson-scientists-uncover-chickpea-genes-to-boost-south-carolina-s-economy
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Clemson scientists discover plant genes to help grow S.C. economy https://news.clemson.edu/clemson-scientists-discover-plant-genes-to-help-boost-s-c-economy/


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Target audience: The target audience includes organic producers, food processors, pulse breeders, consumers, and futuregenerations (underrepresented SC high school, graduate, and undergraduate students - Emerging scholars program, SCCommissioner School, Creative Inquiry, and UPIC programs). These key stakeholders are from local, national, andinternational audiences from Africa and Asia. Briefly, (1) Classroom education includes graduate students, visiting fellows,plant breeders, CI: undergraduate students, beginning, non-traditional farmers, and veterans, an internship program forundergraduate students (first-year students from PES), underrepresented national and international high school students;consumers, food processors, health workers, and community; 4-H students from South Carolina (middle and high schoolstudents); and global research community; (2) Extension and outreach: growers, food processors, consumers, marketingpersonnel, researchers, breeders, and future generations. Changes/Problems:Two North Carolina on-farm locations were dropped due to the weed and disease pressure. We will continue to focus on organic pulse breeding for SC. Other than the weather conditions, we are not expected to change any of the planned work for 2024. What opportunities for training and professional development has the project provided?Four doctoral students, one master's student, four undergraduates, two postdoctoral fellows, a part-time breeder, a food processing research scientist, a full-time field technician, and a lab technician were trained in this project. One doctoral student (Nathan Johnson) and a master's student (Tony Reta) graduated in Aug 2022. A master's student becomes an assistant breeder at a large vegetable company in California. Dr. Nathan Johnson is a postdoctoral fellow and starting medical school in Aug 2023. In the past year, Dr. Johnson has submitted two first-author articles, has published one co-authored article, and is a co-author on several articles under preparation and review. He has also helped plan and administer several field days and workshops offered by our breeding program, including serving as the master of ceremonies for one. He has attended several R and Python workshops offered by Clemson's Research Computing and Data Engagement team. He is currently teaching an informal class for lab members on bioinformatics using R and Linux command line. Dr. Johnson also developed the Breedbase. Both doctoral students and a new postdoctoral fellow attended the 2023 National Association of Plant Breeders Conference and presented their work. High school student activities include attending STEM sessions via the Commissioner's School (SC) and 4-H (SC). These workshops include Mendelian breeding games, phenomics, and cooking experiences for the farm-to-table approach. Dr. Thavarajah offered a fall three-credit creative inquiry (CI) course and the University Professional Internship and Co-op (UPIC) course for seven undergraduate students. These classes have three modules: i) hands-on breeding experience, ii) phenomics techniques, and iii) building healthy food systems. At the end of the semester, these students will present their projects at the CI Undergraduate Research Symposium. How have the results been disseminated to communities of interest?Protein quality and grain yield rankings by genotype have been distributed to stakeholders via the grower meetings. Aftercomplete data analysis, On-farm results will be disseminated directly to stakeholders (food processing, consumers, andmarketing) using multiple social media outlets - website, YouTube, and social media. As such, Mr. Cory Tanner from ClemsonExtension has regularly coordinated data releases and publications in addition to updating social media networks. Dr. ErinClayton has coordinated protein quality data release to the plant-based food processing and manufacturing community. Wedeveloped a new global partnership with FoodShot Global for precision protein. The results have been presented at theNAPIA meeting to update the pulse research community and communicated to national and local stakeholders via boardmeetings, peer-reviewed publications, webinars, and workshops. What do you plan to do during the next reporting period to accomplish the goals?We will continue the work related to all three objectives in the proposal.

Impacts
What was accomplished under these goals? Objective 1: Dry pea: breeding cycle 1: F1 seeds were increased in 2021 using single seed descent (SSD), F4 nurseries were planted, and F6 progenies were tested in 2023 and selected for yield testing in 2024. F5 seeds were selected for protein quality and to advance (F5-F8) to release organic cultivars in 2025. Dry pea seed yields were up to 3833 kg/ha. Seed protein ranged from 17 to 30%, with 80 to 85% protein digestibility. Total prebiotic carbohydrate concentrations ranged from 14.7 to 26.6 g/100 g; a 100-g serving of organic dry pea provides 73.5 to 133% of the recommended daily allowance. Heritability estimates for individual prebiotic carbohydrates ranged from 0.27 to 0.82. Organic dry peas are rich in minerals (Fe: 1.9-26.2 mg/100 g; Zn: 1.1-7.5 mg/100 g) with low to moderate concentrations of phytic acid (18.8-516 mg/100 g). Selected F4:6 progeny plots (162) were harvested in late May 2023 based on visual appearance for date of maturity, height, lodging resistance, disease resistance, and overall uniform appearance. Single plants were selected from 16 progeny plots segregated for maturity and/or plant height. F4:7 seed will be composited from selected plots. Harvested lines will be further evaluated for seed appearance and compositional traits during Summer and Fall 2023. Selected F4:7 Clemson University breeding lines will be evaluated for seed yield, disease resistance, and general agronomic traits in 2024 field tests in two USDA organic-certified on-farm fields. In cooperation with Dr. Diego Rubiales (Institute for Sustainable Agriculture, CSIC, Avenida Menéndez Pidal s/n, Campus Alameda del Obispo, Spain), fifty-eight breeding populations (F3 - F8) were evaluated in the 2023 Pelion SC on-farm field nursery. Marked differences in disease resistance, particularly powdery mildew, and suitable agronomic traits were observed among the populations. Selected populations will be further evaluated in the 2024 field nursery.Breeding cycle two was established in 2022- eighteen crosses were made in the GH. Parents utilized six commercial cultivars, six high-protein plant introductions (PIs), and two PIs with improved phosphorus uptake. F1 seeds (384) were planted in a GH on February 13, 2023, with resulting F2 seeds harvested from 371 F1 plants from mid-April to mid-May 2023. F2 seed from nine selected crosses is now being advanced F2 - F3 via SSD, with the resulting F4 seed to be planted in the Pelion on-farm field nursery in January 2024. F2 seed from the other nine 2022 crosses of adapted high-protein PI parents will be planted in the field in 2024, with agronomically suitable F2 plants selected for advancement. Breeding cycle 3: F1 seeds are harvested from fifteen crosses made during April-May 2023 in a Clemson GH. There were three commercially available, adapted cultivars, five "old" cultivars from the USDA field pea germplasm collection, and one PI. The five cultivars and a single PI from USDA Germplasm Collection were selected based on field evaluations for agronomic and seed composition traits at Pelion, SC (2019) and Clemson, SC (2022). Lentil: For organic lentil breeding, nine lentil cultivars were tested in production in 2022 (3 locations - NC site was dropped due to disease and weed pressure) and 2023 (2 locations), and the lentil global diversity panel with 299 accessions was tested in two locations in 2022 and 2023. Organic lentil breeding is limited due to severe root rot incidences without fungicide treatments. In year 2, nine elite cultivars currently in production were planted at two certified organic locations in Clemson and Pelion, SC. The Pelion trial had the best agronomic performance, adaptability, and yield. Pelion had minimal disease pressure, while Clemson had significant disease pressure, with almost all plots showing a significant loss in the stand. Fifteen lentil parents were selected from ICARDA lentil materials to develop organic cultivars adapted for organic production with short duration, disease resistance, and yield. Twenty-eight possible lentil crosses will be made in Nov 2023. Root rot diseases were the primary production constraint, including Fusarium spp., Verticillium wilt, and an unidentified oomycete. Among the 298 accessions evaluated, 12 accessions combined the favorable characteristics of higher canopy cover (>10%), disease incidence ≤85% (assessed 74 days after planting), and were mature by harvest: PI 178940, PI 283604, PI 289079, PI 297286, PI 298357, PI 298645, PI 320936, PI 320952, PI 426797, PI 431666, PI 432271, PI 518731 Objective 2: Lentil: A ~143-member lentil heat-tolerant GWAS population and 129-member global mapping population from ICARDA were screened to develop climate-resilient organic lentil cultivars with improved protein quality and digestibility to determine population structure. This panel had six sub-groups, and the mean concentration of 17 different amino acids (AAs) was quantified, including the sulfur-containing amino acids (SAAs): methionine (0.21%) and cysteine (0.22%). Fifty significant SNPs were associated with 17 traits (number of SNPs): alanine (3), arginine (2), aspartate (2), aspartate: TotalAA ratio (7), cystine (7), glutamate: TotalAA ratio (7), glycine: TotalAA ratio (7), histidine: TotalAA ratio (7), isoleucine (1), leucine (3), lysine (3), methionine (7), methionine: TotalAA ratio (7), phenylalanine (2), threonine (1), valine (2), and protein digestibility. The Lentil association mapping panel (LAMP), composed of 446 accessions, was evaluated for protein quality with better agronomy. The LAMP was screened in the greenhouse in a completely randomized design (CRD) with three replications per accession during Spring-Summer 2022 and 2023. The elite lentil germplasm from ICARDA was also obtained, including 15 parents for the crossing program. This germplasm was planted for seed increase in the greenhouse at the end of the fall of 2022. 96 LAMP accessions (not genotyped earlier) were genotyped with 10x coverage, and genotyping data analysis is in progress. The preliminary quality check is in progress for the genotyping data. Pea: Genome-wide association studies have been performed for total protein, sulfur-containing amino acids (SAA), and protein digestibility (PDg). The study included 299 genotypes, of which 267 had been previously genotyped. These were grown in three location years: Clemson 2021, Clemson 2022, and WP Rawls 2020. Total protein ranged from 15.7-36.5%, with a mean of 23.6%. SAA ranged from 0.11-0.56% with a mean of 0.23%. PDg ranged from 81.9-87.4%, with a mean of 84.6%. The BLINK model from GAPIT was used. The analysis was performed on the combined environments as well as individual environments. In summary, 27 unique SNPs were identified across the three traits. Protein had 18 SNPs, SAA had 8 SNPs, and PDg had 1 SNP. Food processing: Three inventions have been developed: 1) an organic protein isolation process, 2) prebiotic carbohydrate enrichment, and 3) liquid protein and carbohydrates for beverages and semi-solid food applications. A US Patent application for invention 1 (#73/106,015), titled "Methods of isolating plant protein and related composition," was published in April 2022. It describes the isolation of organic proteins to create organic protein compositions balanced with all essential AAs that are highly digestible, free of agricultural residues, and with no added sodium or chlorides. We will use this patented technology to screen advanced breeding lines of pulse crops that provide the highest protein yield, digestibility, and functional food properties to apply in liquid (e.g., milk), semi-solid (e.g., yogurt), and solid (e.g., burgers) food applications. Objective 3: Following were conducted: 9 webinars, four student workshops, three undergraduate courses, a field day, an advisory board meeting, 10-12 extension talks, and a consumer workshop. Approximately 500 stakeholders were reached for organic pulse breeding and nutrition.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Dil Thavarajah, Tristan Lawrence, Lucas Boatwright, Nathan Windsor, Nathan Johnson, Joshua Kay, Emerson Shipe, Shiv Kumar, Pushparajah Thavarajah, 2023. Organic dry pea (Pisum sativum L.): a sustainable alternative pulse-based protein for human health. PLoS ONE 18(4): e0284380. https://doi.org/10.1371/journal.pone.0284380.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Sonia Salaria, J. Lucas Boatwright, Nathan Johnson, Priyanka Joshi, Pushparajah Thavarajah, George Vandemark, Dil Thavarajah!, 2023. Fatty acid composition and genome-wide associations of a chickpea (Cicer arietinum L.) diversity panel for biofortification efforts. Scientific Report. Submission ID c3b96c8f-2d78-45fe-ad8b-da64ca0f29a7
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Nathan Johnson, Pushparajah Thavarajah, Nathan Windsor, Leung Tang, Dil Thavarajah!, 2023. Fourier-transform infrared spectroscopy (FTIR): an inexpensive, rapid, and non-destructive tool for starch and resistant starch analysis. The Plant Phenome Journal, TPPJ-2023-05-0013-OA
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Nathan Johnson, J Lucas Boatwright, William Bridges, Pushparajah Thavarajah, Shiv Kumar, Dil Thavarajah, 2023. Targeted improvement of plant-based protein: Genome-wide association mapping of lentil (Lens culinaris Medik.) diversity panel. PPP, PPP-MS-2023-00644
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sonia Salaria, Dil Thavarajah, 2023. Genome-Wide Association Studies (GWAS) to explore the fatty acid composition of a chickpea (Cicer arietinum L.) diversity panel for biofortification. National Association of Plant Breeders, GV, SC. July 19-20, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Van Grouw, N., *Kittel, A., *Terry, A., *Miller, A., *Pussella, K., *Johnson, J., *Windsor, N., Salaria S., Dil Thavarajah, 2023. Fighting Food Insecurity with Innovative Gardening Methods. Poster presentation at Clemson University 18th Annual Focus on Creative Inquiry Forum, Clemson, SC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mark Dempsey, Dil Thavarajah, 2023. Adapting and optimizing lentil for organic production in the Southern US. National Association of Plant Breeders, GV, SC. July 19-20, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Priyanka Joshi, Dil Thavarajah, George Vandermark, 2023. Genome-wide association mapping for seed protein concentration in chickpea (Cicer arietinum L.). National Association of Plant Breeders, GV, SC. July 19-20, 2023.
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Lentil (Lens culinaris Medik.) Prebiotic Carbohydrates and Protein Quality: Uncovering Genomic Associations and Developing Rapid FTIR Phenotyping Methods Author: Nathan Johnson- http://tigerprints.clemson.edu/all_dissertations/3116 Publication
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Pre-breeding of Kale (Brassica oleracea var. acephala)  Organic adaptation and shelf life Author: Craig Reda- https://tigerprints.clemson.edu/do/search/?q=Kale%20quality&start=0&context=4572410&facet= Publication
  • Type: Other Status: Published Year Published: 2023 Citation: 2023: Thavarajah receives Clemsons 2023 Godley-Snell Award. https://news.clemson.edu/thavarajah-receives-clemsons-2023-godley-snell-award/
  • Type: Other Status: Published Year Published: 2022 Citation: 2022: Clemson webinar series promotes Going Organic when breeding pulse crops. https://news.clemson.edu/clemson-webinar-promotes-going-organic-when-breeding-pulse-crops/
  • Type: Other Status: Published Year Published: 2022 Citation: 2022: American Society of Agronomy, Society Science, CSA News, High-Throughput Phenotyping Tool for Protein Biofortification in Pulses. https://doi.org/10.1002/csan.20845
  • Type: Websites Status: Published Year Published: 2022 Citation: https://www.clemson.edu/cafls/organic-breeding/index.html
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Thavarajah Dil, 2023. Opening talk, Organic Pulse Crop Breeding and Nutrition, "Eating Pulses" Workshop, Clemson University, June 10, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Thavarajah Dil, 2023, Pulse 101 lecture, Pulse Crop Field day, May 2, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Invited Speaker, Breeding for seeds traits, ASA-CSSA-SSSA, Baltimore, MD, Nov 6-9, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Invited Speaker, Organic Dry Pea, and Lentil Adaptation to South Carolina for Plant-based Protein Production: NIFA-OREI project update. E-Organic webinar. Oct 20, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Speaker, OREI: Sustainable organic pulses, Organic pulse breeding team meeting, July 28, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Thavarajah Dil, 2023. Breeding Biofortified Pulses for human health: A high-throughput phenotyping platform for quantifying nutritional traits. International Conference on Pulses: Smart Crops for Agricultural Sustainability and Nutritional Security, Invited Talk, New Delhi, India, February 11, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Thavarajah Dil 2023. Breeding protein enriched organic dry peas, Protein symposium 2023, University of Rwanda, Rwanda. May 2, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Pulse Breeding for Human Health, Invited Seminar, College of Agriculture, Animal Science and Veterinary Medicine (CAVM), Busogo Campus, University of Rwanda, Rwanda. August 20, 2022.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Target audience:The target audience includes organic producers, food processors, pulse breeders, consumers, and future generations (underrepresented SC high school, graduate, and undergraduate students - Emerging scholars program, SC Commissioner School, Creative Inquiry, and UPIC programs). These key stakeholders are from local, national, and international audiences from Africa and Asia. Briefly, (1) Classroom education includes graduate students, visiting fellows, plant breeders, CI: undergraduate students, beginning, non-traditional farmers, and veterans, internship program for undergraduate students (first-year students from PES), underrepresented national and international high school students; consumers, food processors, health workers, and community; 4-H students from South Carolina (middle and high school students); and global research community; (2) Extension and outreach: growers, food processors, consumers, marketing personnel, researchers, breeders, and future generations. Changes/Problems:Two on-farm locations in North Carolina (NC) were unsuccessful due to the weed and disease pressure. Also, the current lentil cultivars in production are not adapted to climatic conditions in NC. We acquired new germplasm from ICARDA, Morocco, to test in NC, but we are expected to plant dry pea in NC instead of lentils in the 2023 winter. A new organic producer (Del Valle Fresh Inc, Roebuck, SC) will be added as the third on-farm location in SC to test organic lentil and dry peas in the 2023 winter. Other than the weather conditions or COVID-19, we are not expected to change any of the plan work for 2023. What opportunities for training and professional development has the project provided?Three doctoral students, 2-3 undergraduates, part-time technicians, part-time breeders, part-time scientists, and full-time field technicians were trained. >100 high school students attended the breeding workshops. The PD participated in the Breeding for Quantitative Traits in Plants 3-credit course offered by Dr. Rex Bernardo in Spring 2022. Nathan Johnson: From Aug 2021 to Aug 2022, Nathan Johnson analyzed a lentil diversity panel for protein digestibility using an enzymatic assay and sulfur concentration using hydrogen peroxide digestion and ICP spectroscopy. He developed partial least squares models for rapid quantification of protein, sulfur-containing amino acids, resistant starch, and total starch using Fourier Transform Infrared Spectroscopy (FTIR) for pulse crops. He performed genome-wide association studies for protein quality traits and discovered significant genetic markers for 17 of these traits. He also performed admixture analysis and analysis of variance for the lentil diversity panel. Nathan completed one class, "Pulse Genomics for Nutrition," in Spring 2022, led a high school 4-H group event, gave two oral presentations (Clemson Going Organic Webinar series and the North American Pulse International Association Biennial Meeting), and gave two poster presentations (Clemson Growers Meeting and McCall Farms Gift Announcement). He published one co-first-authored paper describing his protein FTIR work and drafted a first-author paper describing his protein GWAS work. He prepared and successfully defended his dissertation on July 14. Sonia Salaria and Mark Dempsey (first year doctoral students) - They have taken regular graduate-level classes, completed first year field and greenhouse studies, developed the lentil association mapping population for protein quality, and currently developing a MAGIC population for lentil protein quality. A UPIC undergraduate intern trained on plant breeding and FTIR technology for protein quality measurement. How have the results been disseminated to communities of interest?Protein quality and grain yield rankings by genotype have been distributed to stakeholders via the grower meetings. After complete data analysis, On-farm results will be disseminated directly to stakeholders (food processing, consumers, and marketing) using multiple social media outlets - website, YouTube, and social media. As such, Mr. Cory Tanner from Clemson Extension has regularly coordinated data releases and publications in addition to updating social media networks. Dr. Erin Clayton has coordinated protein quality data release to the plant-based food processing and manufacturing community. We developed a new global partnership with FoodShot Global for precision protein. The results have been presented at the NAPIA meeting to update the pulse research community and communicated to national and local stakeholders via board meetings, peer-reviewed publications, webinars, and workshops. What do you plan to do during the next reporting period to accomplish the goals?We will continue the work related to all three objectives in the proposal.

Impacts
What was accomplished under these goals? Objective 1: Advanced Lentil Breeding Trial: In year 1 of the organic lentil evaluations, nine elite cultivars in production were planted at three certified organic locations in Pelion, South Carolina (SC), Concord, and Mills River, North Carolina (NC). The Pelion, SC trial was planted on 1 February 2022, and the two NC locations were planted on 15 February 2022. The cultivars were planted in a randomized complete block design with four replicates, one check variety per rep, and encompassed by border plots. The trials were planted using an Almaco cone plot planter, sown into 4.65 m2 (50 ft2) plots with seven rows spaced 7.5 inches apart at a seeding rate of 130 seeds/m2. Agronomic data were collected using the Pheno App' Field Book' to record quantitative and visual assessment data, including germination, vigor, days to flower, days to maturity, canopy height, plant height, the height of the first pod, pods per peduncle, lodging, disease/insect damage, frost tolerance, and canopy closure. Visual evaluations and qualitative data were given by assigning ratings relative to other test entries over three reps on a scale of 0/1 to 5 according to predefined criteria. The Pelion trial was harvested on 1 June 2022 with an Almaco SPC 20 configured with a platform head. The Concord trial was harvested on 16 June 2022, and the Mills River trial was not harvested due to poor organic management. The seed was stored in a controlled drying barn to attain the optimal moisture content before being weighed, cleaned, sampled, and ground for nutritional analysis. Data analysis for the yield, agronomic traits, and nutritional quality is still in progress. The Pelion trial had the best agronomic performance, adaptability, and yield. The highest yielding cultivars in Pelion were 'Avondale,' 'CDC Imvincible,' and 'CDC Proclaim.' The highest yielding cultivars in Concord were 'CDC Peridot, 'CDC Imvincible,' and 'CDC Impala.' The three lowest yielding cultivars were the same at both locations, with 'CDC Dazil' as the lowest, followed by 'Pardina' and 'CDC Impress.' The study will be replicated again in 2022-2023 at three locations. LSP: The Lens Single Plant (LSP) collection was increased and evaluated for the first time at the certified organic location in Pelion, SC. The collection comprised 300 accessions from the Lens Single Plant Collection, obtained from the USDA-ARS germplasm in Pullman, Washington. The germplasm collection was planted on 1 February 2022 in an alpha-lattice design into ten-foot plots, consisting of four rows spaced 15 inches apart using an Almaco' head-row' plot planter. Accessions were evaluated on agronomic adaptability and performance using the Pheno App' Field Book' to record quantitative and visual assessment data. The collection was hand-harvested at maturity in increments starting on 20 May 2022 and ending on 3 June 2022. The harvested plants were then moved to a drying barn and threshed with an Almaco Belt Thresher. The seed will be used for protein quality and genomic analysis, and the remaining seed will be planted in 2023. The agronomic and nutritional data analysis will be used to identify accessions with exceptional agronomic adaptability and protein content for parental selection and implementation into the breeding pipeline. The study will be replicated again in 2022-2023 for two locations for the genomic prediction study. PSP: A subset of the Pea Single Plant (PSP) collection was selected and planted on 31 January 2022 at the organic transition farm location in Clemson, SC, to serve as observational plots. Ninety-eight accessions were selected for their high protein content obtained from nutritional analysis from previous trials. The germplasm collection was planted in a randomized complete block design into ten-foot plots, consisting of four rows spaced 15 inches apart using an Almaco' head-row' plot planter. Observational notes and agronomic ratings were recorded and used to identify accessions with desirable traits and adaptability as breeding parents. The trial was hand harvested on 7 June 2022 and moved to the drying barn before being threshed. Seed samples were collected and ground for seed composition nutritional analysis. For breeding cycle 2, Spring of 2022, eighteen crosses were made on protein quality and adaptability. Parents utilized were six commercial cultivars, six high-protein plant introductions (PI's), and two PI's having improved phosphorus uptake under organic growing conditions. The resulting F1 seed has been harvested and planted in the GH and then advanced F2 - F3 via SSD in the GH, with the resulting F4 seed planted in the Pelion field nursery. Objective 2: Phenotyping Pipeline (partnership with the USAID): Fourier-transform mid-infrared (FT-MIR) spectroscopy is a high-throughput, cost-effective method to quantify nutritional traits, total protein, and sulfur-containing amino acids SAA concentrations in plant matter. We used FT-MIR spectroscopy coupled with an attenuated total internal reflectance (ATR) sampling interface to develop multivariate models to measure total protein and SAA concentrations. Total nitrogen (N) data from combustion analysis and SAA data from high-performance liquid chromatography analysis following acid hydrolysis were used for model calibration and validation. Protein Quality and digestibility: A ~143-member lentil heat-tolerant GWAS population and 129-member global mapping population from ICARDA were used for screening to develop climate-resilient lentil cultivars with improved protein quality and to determine population structure for lentils. This panel had six sub-groups. The mean concentration of 17 different amino acids (AA) was quantified, including the sulfur-containing amino acids (SAA): methionine (0.21%) and cysteine (0.22%). Fifty significant SNPs were associated with 17 traits (Johnson & D. Thavarajah in preparation for PNAS, 2023). Lentil Protein Mapping Panel: A lentil association mapping panel (LAMP) composed of 446 accessions was assembled to evaluate high protein quality in cultivated germplasm with better agronomy.The LAMP was screened in the greenhouse in a completely randomized design with three replications per accession during Spring-Summer 2022. LAMP will be genotyped in the fall of 2022 to perform Genome-wide association studies and identify significant SNPs linked to agronomic and protein quality traits. The combined phenotyping and genotyping approach will facilitate the identification of genomic regions impacting the agronomy and quality, providing the best lines for agronomy and quality to develop the lentil breeding pipeline using genomic prediction. Food processing pipeline: We developed three inventions: 1) an organic protein isolation process, 2) prebiotic carbohydrate enrichment, and 3) liquid protein and carbohydrates for beverages and semi-solid food applications. Invention 1 has been submitted to the US Patent and Trademarks Office (#73/106,015), and the other two are undergoing commercial value evaluation at the Clemson University Research Foundation (CURF:#2022-033). The US Patent application is titled "Methods of isolating plant protein and related composition" and was published in April 2022. Objective 3: See the accomplishments under the products and other products. We developed a social media outlet, the "Going Organic" webinar series, several grower events, and 4-H and SC commissioner school workshops (500 contacts).

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Sonia Salaria , Jon Lucas Boatwright, Pushparajah Thavarajah, Shiv Kumar, Dil Thavarajah!, 2022. Protein biofortification in lentils (Lens culinaris Medikus.) toward human health. Frontiers in Plant Science, Plant Breeding 13:869713. 05 April 2022 | https://doi.org/10.3389/fpls.2022.869713.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Amod Udayanga , Nathan Johnson , Pushparajah Thavarajah, Leung Tang, Dil Thavarajah!, 2022. Fourier-transform infrared spectroscopy (FTIR) as a high-throughput phenotyping tool for quantifying protein quality in pulse crops. The Plant Phenome Journal, 5, e20047. https://doi.org/10.1002/ppj2.20047.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hasnae Choukri, Noureddine El Haddad, Khawla Aloui, Kamal Hejjaoui, Adil El-Baouchi, Abdelaziz Smouni, Dil Thavarajah, Fouad Maalouf, Shiv Kumar, 2022. High-temperature stress during the reproductive stage effects on grain yield and nutritional quality of lentil (Lens culinaris Medikus). Front. Nutr., 15 April 2022 | https://doi.org/10.3389/fnut.2022.857469.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Amod Udayanga , Leung Tang, Pushparajah Thavarajah, William Bridges, Emerson Shipe, George Vandemark, Dil Thavarajah!, 2021. Chickpea (Cicer arietinum L.) as a source of essential fatty acids  a biofortification approach. Frontiers in Plant Science, Plant Breeding. 12:734980. doi: 10.3389/fpls.2021.734980.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: J Lucas Boatwright, Sarah Powers, Dil Thavarajah, 2021. Developing Genome-wide Resources for Mineral Biofortification of Pea (Pisum sativum L.). North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nathan Johnson, Dil Thavarajah, 2021. Toward a high-throughput molecular breeding pipeline for lentil protein quality. North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Amod Udayanga, Dil Thavarajah, 2021. Fourier Transform Mid Infrared (FT-MIR) Spectroscopy as a tool to measure nutritional traits in pulses. North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Joshua Kay, Dil Thavarajah, 2021. Are Organic Peas just as Nutritious as Conventional Peas? North American Pulse Improvement Association, Nov 2-4, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Sonia Salaria, Dil Thavarajah, 2021. Lentil Protein Biofortification using Conventional Breeding and Association Mapping Approaches. North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mark Dempsey, Dil Thavarajah, 2021. Adapting Organic Lentil to the Southeastern US Climate and Soil. North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Tristan Lawrence, Dil Thavarajah, 2021. Going Organic  Breeding organic dry pea cultivars. North American Pulse Improvement Association, Nov 2-4, 2021.
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Nathan Johnson, LENTIL (LENS CULINARIS MEDIK.) PREBIOTIC CARBOHYDRATES AND PROTEIN QUALITY: UNCOVERING GENOMIC ASSOCIATIONS AND DEVELOPING RAPID FTIR PHENOTYPING METHODS, August 2022, Clemson University
  • Type: Websites Status: Awaiting Publication Year Published: 2022 Citation: Website: clemson.edu/cafls/organic-breeding/orei/
  • Type: Websites Status: Published Year Published: 2022 Citation: YouTube Channel: Clemson Pulse Breeding https://www.youtube.com/channel/UCvfFSQSuo12_tNy7qZFATwg
  • Type: Other Status: Published Year Published: 2022 Citation: Twitter Handle: @CpulsesBreeding https://twitter.com/Cpulsesbreeding
  • Type: Other Status: Published Year Published: 2021 Citation: Organic Pulse Breeding, Market Bulletin, SC Department of Agriculture, Vol 25, 10.
  • Type: Other Status: Published Year Published: 2021 Citation: Clemson doctoral student determines lentil genetic markers vital for global nutrition security. Clemson University. https://news.clemson.edu/clemson-doctoral-student-determines-lentil-genetic-markers-vital-for-global-nutrition-security/
  • Type: Other Status: Published Year Published: 2021 Citation: Clemson research aims to help S.C. farmers meet the demand for more nutritious legumes https://news.clemson.edu/clemson-research-aims-to-help-s-c-farmers-meet-demand-for-more-nutritious-legumes/
  • Type: Other Status: Published Year Published: 2021 Citation: Atlanta Business Journal https://atlantabusinessjournal.com/clemson-research-aims-to-help-sc-farmers-meet-demand-for-more-nutritious-legumes-agriculture/
  • Type: Other Status: Published Year Published: 2021 Citation: The Times and Democrat - The 'poor man's meat': Research aims to produce more nutritious legumes https://thetandd.com/business/agriculture/the-poor-mans-meat-research-aims-to-produce-more-nutritious-legumes/article_fabfd5de-311b-529f-8979-787422b412cd.html
  • Type: Other Status: Published Year Published: 2021 Citation: Florence Morning News https://scnow.com/agriculture/clemson-research-aims-to-help-sc-farmers-meet-demand-for-more-nutritious-legumes/article_821baf76-1ce6-11ec-ac47-3bdacfb13994.html
  • Type: Other Status: Published Year Published: 2021 Citation: Southeast Farm Press Daily email newsletter. https://enewspro.penton.com/preview/farmpress/SEFP-01/20210923_SEFP-01_75/display?utm_rid=CPG02000002564174&utm_campaign=61999&utm_medium=email&elq2=eebf46faba394f53b3d072ab587ddcdb
  • Type: Other Status: Published Year Published: 2021 Citation: Carolina Farm Steward Association  https://us12.campaignarchive.com/?u=d23f69ef676d1fb458eadcd63&id=42d1cd7c4f&e=c5e879a6d2
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Alternative proteins, Plant-Based Foods, and the City: From Seed to Take-Out. Invited Panel/presentation, New York Botanical Garden "food dialogues" program. NY, Oct 1, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Organic pulse biofortification program, Seminar, PES, Clemson University, SC, Oct 15, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Pulse Crops and Human Health, CU School of Health Research, Invited Seminar, Nov 4, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Pulse breeding towards N fixation (Part-2), Invited lecture PES 8900: Plant Nitrogen Metabolism, Nov 23, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Invited talk, Organic dry pea breeding for SC, Carolina Golden Rice Foundation, Nov 30, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2022. Invited Panel Speaker, Plant-based protein - UNC CleanTech Summit, UNC, Chapel Hill, March 30, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Invited Speaker, Organic Pea Breeding, Upstate Vegetable Grower Meeting, Greenville, SC, April 11, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Speaker, OREI: Sustainable organic pulses, Organic pulse breeding team meeting, July 28, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Organic Pulse Breeding for Human Health, Invited Seminar, College of Agriculture, Animal Science and Veterinary Medicine (CAVM), Busogo Campus, University of Rwanda, Musanze, Rwanda. August 2, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Thavarajah Dil, 2021. Invited talk, Nutritional importance of lentil in fighting back malnutrition - experiences from a model work. Legume Systems Innovation Lab Event on Lentil Sector in Nepal, USAID  Nepal, Dec 16, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thavarajah Dil, 2022. Invited talk, Biofortification of pulses-recent Developments. Western Ag- Professional Development conference 2022, Saskatoon, Canada. March 2, 2022.