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
NEW
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1026666
Grant No.
2021-51300-34895
Project No.
SC-2021-02927
Proposal No.
2021-02927
Multistate No.
(N/A)
Program Code
113.A
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2025
Grant Year
2021
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
0%
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 09/01/22 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.