Source: CLEMSON UNIVERSITY submitted to
SEED GRANT ON GENOMICS OF PREBIOTIC CARBOHYDRATES AFFECTING LENTIL NUTRITIONAL QUALITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015284
Grant No.
2018-67014-27621
Project No.
SC-2017-07720
Proposal No.
2017-07720
Multistate No.
(N/A)
Program Code
A1141
Project Start Date
Feb 15, 2018
Project End Date
Feb 14, 2020
Grant Year
2018
Project Director
Thavarajah, D.
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Plant and Environmental Sci
Non Technical Summary
Non-technical summary: Obesity has become a global epidemic. A prebiotic-rich, low-caloric diet is important in combating obesity. Consumption of prebiotics stimulate the growth and activity of hind gut bacteria, provides energy source for colonocytes, strengthen the gut mucosal barrier, and suppress pathogens. As such, products enriched with prebiotics are becoming popular in the American diets. Our results indicate that US-grown lentils provide 15g of prebiotics per 100 g serving. Significant genotype and genotype × location effects contributed to the variability in the concentrations of prebiotics, a feature that is useful in breeding lentils for climate change. Therefore, US-grown pulses offer new market opportunities by identification of genetic markers associated with high levels of prebiotic carbohydrates that could significantly accelerate nutritional breeding for different environments and consumer preference. The objectives of this seed grant are (1) determine the type and concentration of prebiotic carbohydrates in global lentil association mapping population, and 2) develop a multi-State collaborative AFRI standard research grant (2020 submission).
Animal Health Component
0%
Research Effort Categories
Basic
33%
Applied
33%
Developmental
34%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011414108050%
2011414108150%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1414 - Lentil;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Project goals: Characterize the type and concentration of prebiotic carbohydrates (sugar alcohols, fructooligosaccharides, raffinose-family oligosaccharides, and starches) in global lentil association mapping populationDevelop a multi-State collaborative AFRI standard research grant (July, 2020) to identify candidate genes of prebiotic carbohydrates responsible for increased lentil nutritional quality using genome-wide association studies (GWAS).
Project Methods
Methods: A total of ~459 diverse lentil accessions, comprised of a global association mapping population (129 accessions), USDA core collection (180 accessions), and association mapping population for heat and drought tolerance collection (150 accessions) from ICARDA will be used. The experiment will be a complete randomized block design with 2 replicates (n=918) for the experiment and one replicate for seed increase. Plants will be grown under controlled environment conditions. The pots will be watered to pot field capacity every day and 250 mL of nutrient solution (20% total N, 20% total P, 20% soluble K, 0.02% B, 0.05% chelated Cu, 0.1% chelated Fe, 0.05% Mo, 0.05% Zn, and 1% EDTA; Plant Products Co. Ltd., Brampton, ON, Canada) will be added to all pots every two weeks, as per standard procedures for lentil at the Clemson University Pulse Quality and Nutrition program. The experiment will take place at the Clemson University greenhouse complex (PGW 36, Controlled-Environment Ltd., Winnipeg, MB, Canada), with day/night temperatures of 21/18 °C, photosynthetic active radiation levels of 300-400 µmol/m2/s, and a 16 h photoperiod beginning at 0600 local time. Lentil will be grown to seed physiological maturity (~90-110 days). At physiological maturity, lentil plants will be hand harvested, and then total biomass and grain yield will be measured. Biomass will be recorded as dry weight after air-drying at 40 °C. Plants will be hand-threshed and the total seed weight per pot will be recorded. Seeds will be stored at -40 °C until analysis.Prebiotic carbohydrate analysis: Ground lentil seed samples will be weighed (~500 mg) into 15 mL polystyrene conical tubes. Extraction will be carried out using a previously described method (Muir et al., 2009). Samples will be combined with 10 mL of ddH2O, will agitated to assure proper homogenization, will incubated in a water bath at 80° C for 1 hour. This will be followed by centrifuging at 3000 x g for 15 minutes at 22° C using an Eppendorf 5810 R centrifuge. After being centrifuged, 1 mL of the supernatant will be diluted with 10 mL of ddH2O and will passed through a 13 mm x 0.45 µm nylon syringe filter. Analysis of SA, monosaccharide, disaccharide, and oligosaccharides will be performed on a high performance anion exchange chromatography system (ICS-5000 Dionex, Sunnyvale, CA, USA) using a method previously described by Feinberg (2009). Carbohydrates will be separated by a CarboPac PA-100 4 x 250 mm column in series with a CarboPac PA-100 4 x 50 mm guard column (Dionex, Sunnyvale, CA, USA). Detection will be carried out using a pulsed amperometric detector (PAD, Dionex) in congruence with a working gold electrode (Thermo Fisher Scientific, Inc.) with a silver-silver chloride electrode at 2.0 μA. RS and total starch analysis will be performed by a method approved by AOAC International, previously described (McCleary & Monaghan, 2002; Megazyme, 2012).

Progress 02/15/18 to 02/14/20

Outputs
Target Audience:Target audience: National and international pulse growers National and international pulse researches/breeders/nutrition experts National and international pulse processers and nutritional quality experts Nutritional food and health research community The above target audience reached via: North American Pulse Improvement Association (NAPIA), Fargo, ND, Nov 6-8, 2019. Target audience: 50-75 researches, breeders, pulse growers, and nutrition experts Institute of Food Technologist - 19, New Orleans, LA, June 2 - 5, 2019: >500 food processors and food researchers GFI's Good Food Conference, San Francisco, Sep 4-6, 2019 : >700 food processors, researches, and private companie Research workshop at Washington State University - Emerging Opportunities for Pulse Production: Genetics, Genomics, Phenomics, and Integrated Pest Management" Pullman, WA, June 24-25, 2019. Nutrition 2019, Baltimore, Maryland, June 8-9, 2019 - reached out to national and international nutritional experts via delivering a keynote International Food Legume Research Conference, Morocco, April 2018 - >100 international researchers, breeders via delivering a keynote speech. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI: Dr. Thavarajah completed the Plant Breeding two course modules at the Tucson Plant Breeding Institute, Jan 7-12, 2018. Dr. Thavarajah completed two modules on plant breeding tools, statistical tools, GWAS, and R analysis. Dr. Thavarajah is the current president of the North American Pulse Improvement Association, the American representative for the International Steering Committee (ISC) of the 8th International Food Legume Research Conference (IFLC-VIII) in 2022, Nairobi, Kenya. Graduate Student: Through the support of this NIFA grant, Nathan Johnson has been able to further his professional development. He is soon to complete 24 class credit hours toward a masters degree including courses such as Advanced Genetics, Statistical Genetics, and Design and Analysis of Experiments. He has co-led our lab's Emerging Scholars Program for two years, in which he taught underrepresented high school juniors and seniors. This program included guided lab experiments, tours of Clemson's greenhouse complex and organic farm, and lecture and discussion about pulse crops, biofortification, and healthy eating habits. In the course of his research, he has completed lab and greenhouse safety training and the required Responsible Conduct of Research training. He has attended introductory R and Python workshops. He has received individualized training in high-performance liquid chromatography, bioinformatics, and statistics. In addition to attending numerous departmental seminars, he has participated in two conferences: the Emerging Opportunities for Pulse Production Conference (June 24-25, 2019, Pullman, WA) at which he presented a research poster and the North American Pulse Improvement Association (NAPIA) Biannual Meeting (Nov. 6-8, 2019, Fargo, ND) at which he gave an oral presentation of his research. Nathan has also co-authored two peer-reviewed papers and is working on his second first-author manuscript. In preparation for medical school, Nathan has taken prerequisite undergraduate courses in Biology, Biochemistry, and Organic Chemistry as well as over 20 hours of shadowing. Two undergraduate students (Agronomy and Biochemistry Majors): Two undergraduate students were trained in this project. They learned the following: (1) greenhouse maintenance, (2) fertilization protocol, (3) pest and disease control, (4) engage in high throughput chemical phenotyping of carbohydrates using anion exchange chromatography, (5) data entry (5) SEM imaging and (6) manuscript writing. Micheal Smathers (Dr. Thavarajah' Advisee) wrote the first author publication as an undergraduate student intern (SEM imaging on lentil seed development during moisture stress). Micheal is starting his graduate studies at the University of California Davis in August 2020. High school students: Two workshops were conducted for high school scholars from SC. How have the results been disseminated to communities of interest? Project results were presented at the 7th International Food Legume Research Conference (IFLC-VII), Morocco, the North American Pulse Improvement Association (NAPIA) meeting in November 2019, Nutrient 2019, IFT 2019, and NIFA Project Director meetings Dr. Thavarajah has been communicating with the American Pulse Association, and the USA Dry Pea and Lentil Council. Upon the completion of the graduate student thesis and the submission of the full proposal to NIFA in 2021, all raw data will be available to the general public via Clemson University, the Germplasm Resources Information Network (GRIN), and ICARDA websites. Dr. Kumar has made several lentils crosses to develop cultivars for SC. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project Goal 1: Heat Tolerance Mapping Population: The heat tolerance population included 150 accessions. Each accession had two greenhouse replicates. Sorbitol (Sor) concentrations ranged from 8.2 to 498.9 mg/100g with a mean ± standard deviation of 206.6 ± 60.53 mg/100g. The heritability estimate is 74.4% of the total variation in Sor concentrations. The accessions with the five highest Sor concentrations were ILL4778, ILL10865, ILL8614, ILL7084, and ILL1005. The accessions with the five lowest Sor concentrations were ILL10862, ILL10825, ILL10810, ILL2230, and ILL1474. Mannitol (Man) concentrations ranged from not detectable to 261.9 mg/100g with a mean ± standard deviation of 46.4 ± 42.2 mg/100g. The heritability estimate is 72.7% of the total variation in Man concentrations. The accessions with the five highest Man concentrations were ILL5418, ILL6925, ILL595, ILL918, and ILL956. The accessions with the five lowest Man concentrations were ILL10862, ILL33117, ILL33115, ILL7012, and ILL1097. Glucose (Glu) concentrations ranged from 31.3-206.4 mg/100g with a mean ± standard deviation of 95.9 ± 28.7 mg/100g. The heritability estimate is 63.9% of the total variation in Glu concentrations. The accessions with the five highest concentrations of Glu were ILL890, ILL191, ILL158, ILL590, and ILL34113. The accessions with the five lowest concentrations of Glu were ILL6994, ILL6925, ILL468, ILL4164, and ILL550. Fructose (Fru) concentrations ranged from 8.4-234.3 mg/100g with a mean ± standard deviation of 62.6 ± 37.9 mg/100g. The heritability estimate is 62.0% of the total variation in Fru concentrations. The accessions with the five highest concentrations of Fru were ILL5261, ILL890, ILL158, ILL33104, and ILL1005. The accessions with the five lowest concentrations of Fru were ILL468, ILL7084, ILL4881, ILL918, and ILL6925. Sucrose (Suc) concentrations ranged from 292.3-798.0 mg/100g with a mean ± standard deviation of 469.2 ± 82.7 mg/100g. The heritability estimate is 57.5% of the total variation in Suc concentrations. The accessions with the five highest concentrations of Suc were ILL494, ILL4606, ILL1861, ILL590, and ILL6644. The accessions with the five lowest concentrations of Suc were ILL257, ILL10053, ILL4164, ILL6994, and ILL7012. Stachyose + raffinose (Sta/Raf) concentrations ranged from 373.0-1018.0 mg/100g with mean ± standard deviation of 553.5 ± of 88.2 mg/100g. The heritability estimate is 50.3% of the total variation in Sta/Raf concentrations. The accessions with the five highest concentrations of Sta/Raf were ILL597, ILL590, ILL6644, ILL890, and ILL556. The accessions with the five lowest concentrations of Sta/Raf were ILL502, ILL4152, ILL10661, ILL1474, and ILL10315. Verbascose + kestose (Ver/Kes) concentrations ranged from 122.1-498.2 mg/100g with mean ± standard deviation of 305.1 ± 79.3 mg/100g. The heritability estimate is 74.9% of the total variation in Ver/Kes concentrations. The accessions with the five highest concentrations of Ver/Kes were ILL595, ILL468, ILL312, ILL598, and ILL623. The accessions with the five lowest concentrations of Ver/Kes were ILL624, ILL494, ILL5261, ILL719, and ILL6415. Preliminary RS concentrations ranged from 13.14-17.91 g/100g with a mean ± standard deviation of 15.70 ± 1.03 g/100g. A strong correlation was found for Glu:Fru (r = 0.76). Weak correlations were found for Man:Fru (r = -0.33), Man:Ver/Kes (r = 0.34), Fru:Ver/Kes (r = -0.36), Sta/Raf:RS (r = -0.30), and Ver/Kes:RS (r = -0.31). Sor and Suc had no correlations above ±0.3. Global Mapping Population: The global mapping population from ICARDA included 129 accessions. Each accession had two greenhouse replicates. Sor concentrations ranged from 4.7-467.9 mg/100g with a mean ± standard deviation of 200.7 ± 49.6 mg/100g. The variability due to accessions explained 46.7% of the total variation in Sor concentrations. The accessions with the five highest concentrations of Sor were ILL5384, ILL4841, ILL129, ILL5424, and ILL4782. The accessions with the five lowest concentrations of Sor were ILL890, ILL2406, ILL4926, ILL7745, and ILL5519. Man concentrations ranged from 1.7-524.9 ± 40.8 mg/100g and a standard deviation of 55.1 mg/100g. The variability due to accessions explained 90.2% of the total variation in Man concentrations. The accessions with the five highest concentrations of Man were ILL5418, ILL550, ILL4782, ILL7084, and ILL4841. The accessions with the five lowest concentrations of Man were ILL2406, ILL224, ILL2150, ILL598, and ILL3517. Glu concentrations ranged from 21.6-699.9 mg/100g with a mean ± standard deviation of 92.1 ± 54.0 mg/100g. The variability due to accessions explained 38.9% of the total variation in Glu concentrations. The accessions with the five highest concentrations of Glu were ILL7084, ILL4881, ILL1013, ILL4409, and ILL7090. The accessions with the five lowest concentrations of Glu were ILL224, ILL556, ILL490, ILL129, and ILL257. Fru concentrations ranged from 5.6-797.0 mg/100g with a mean ± standard deviation of 78.7 ± 73.1 mg/100g. The variability due to accessions explained 43.4% of the total variation in Fru concentrations. The accessions with the five highest concentrations of Fru were ILL7084, ILL5968, ILL5384, ILL7650, and ILL598. The accessions with the five lowest concentrations of Fru were ILL1890, ILL2181, ILL224, ILL5519, and ILL2297. Suc concentrations ranged from 311.9-1330.8 mg/100g with a mean ± standard deviation of 519.3 ± 155.9 mg/100g. The variability due to accessions explained 65.9% of the total variation in Suc concentrations. The accessions with the five highest concentrations of Suc were ILL5384, ILL129, ILL5424, ILL1861, and ILL4345. The accessions with the five lowest concentrations of Suc were ILL1890, ILL2181, ILL5416, ILL1949, and ILL257. Sta/Raf concentrations ranged from 369.4-2313.4 mg/100g with a mean ± standard deviation of 595.7 ± 230.1 mg/100g. The variability due to accessions explained 82.2% of the total variation in Sta/Raf concentrations. The accessions with the five highest concentrations of Sta/Raf were ILL5384, ILL129, ILL5424, ILL4345, and ILL7774. The accessions with the five lowest concentrations of Sta/Raf were ILL490, ILL4152, ILL550, ILL556, and ILL4791. Ver/Kes concentrations ranged from 82.2-1328.9 mg/100g with a mean ± standard deviation of 327.4 ± 120.5 mg/100g. The variability due to accessions explained 84.2% of the total variation in Ver/Kes concentrations. The accessions with the five highest concentrations of Ver/Kes were ILL129, ILL5424, ILL4345, ILL5645, and ILL5653. The accessions with the five lowest concentrations of Ver/Kes were ILL4791, ILL858, ILL6185, ILL86, and ILL597. Nystose (Nys) concentrations ranged from not-detectable - 6.34 mg/100g with mean ± standard deviation of 0.49 ± 0.90 mg/100g. The variability due to accessions explained 72.2% of the total variation in Nys concentrations. The accessions with the five highest concentrations of Nys were ILL4345, ILL129, ILL7774, ILL5384, and ILL1861. The accessions with the five lowest concentrations of Nys were ILL4830, ILL4471, ILL494, ILL4857, and ILL4738. Strong correlations were found for Sor:Sta/Raf (r = 0.73) and Suc:Sta/Raf (r = 0.85). Moderate correlations were found for Sor:Suc (r = 0.67), Sor:Ver/Kes (r = 0.55), Glu:Fru (r = 0.60), Suc:Ver/Kes (r = 0.61), Suc:Nys (r = 0.58), Sta/Raf:Ver/Kes (r = 0.64), Sta/Raf:Nys (r = 0.61), and Ver/Kes:Nys (r = 0.62). Weak correlations were found for Sor:Nys (r = 0.45), Fru:Suc (r = 0.34), and Fru:Sta/Raf (r = 0.34). Project Goal 2: NIFA full proposal will be submitted on the 2020-2021 funding cycle. ICARDA lentil breeder, Dr. Shiv Kumar made 28 lentil crosses, and F2 seeds were sent to Clemson University.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Nathan Johnson, Casey Johnson, Pushparajah Thavarajah, Shiv Kumar, Dil Thavarajah 2020. Lentil prebiotic carbohydrates play a vital role in human and plant health. Plants, People, Planet (PPP3_10103) Accepted.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Siva, N., Thavarajah, P., Thavarajah Dil, 2020. Prebiotic carbohydrate concentrations of common bean and chickpea change during cooking, cooling, and reheating. Journal of Food Science Accepted JFDS-2019-0570. https://doi.org/10.1111/1750-3841.15066
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Siva, N, Thavarajah P., Kumar, S., Thavarajah, Dil, 2019. Variability in Prebiotic Carbohydrates in Different Market Classes of Chickpea, Common Bean, and Lentil Collected From the American Local Market Frontiers in Nutrition, 6:38.
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Thavarajah Dil, Mcswain, M. CR. Johnson, SK. Agrawal, P. Thavarajah, 2019. Pulses, Global Health, and Sustainability: Future Trends. In: Health Benefits of Pulses. Ed: Wendy Dhal, Springer Nature.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johnson N, Thavarajah P, McGee R, Kumar S, Kresovich S, Thavarajah Dil 2019. Prebiotic Carbohydrate Enriched Lentil Cultivars to Combat Obesity, Malnutrition, and Climate Change. North American Pulse Improvement Association (NAPIA), Fargo, ND, Nov 6-8, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Siva, N, Thavarajah, Dil 2019. Precooked shelf-stable lentil as a source of prebiotic carbohydrates to combat obesity" (ID number 23059), IFT19, New Orleans, LA, June 2 - 5, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Thavarajah Dil. 2018. Keynote Presentation, Pulse Crop Biofortification Global Strategies, International Food Legume Research Conference, May 5-8, 2018, Morocco.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Siva, N, P. Thavarajah, Thavarajah, Dil. 2018. The impact of processing and cooking on prebiotic carbohydrates in lentil. Journal of Food Composition and Analysis, 70:72-77.
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Michael Smathers, Nathan Johnson, Pushparajah Thavarajah, Shiv Kumar, Dil Thavarajah! 2020, Moisture Stress Influences Starch Granule Formation in Lentil (Lens culinaris Medikus). Plants, Plants-684660.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Siva, N. and Thavarajah, Dil. 2018. Lentil (Lens culinaris Medikus) diet changes body fat and gut microbiome. Graduate Research and Discovery Symposium. Clemson University. USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Thavarajah, Dil 2019. Invited Panel, Emerging Opportunities for Pulse Production: Genetics, Genomics, Phenomics, and Integrated Pest Management Pullman, WA, June 24-25, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Thavarajah, Dil 2019. Invited Panel, American Society for Nutrition (ASN), ASN breakfast program Plant-based proteins in the Dietary Guidelines for Americans 2020-2025: Positioning pulses for success, Nutrition 2019, Baltimore, Maryland, June 8-9, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johnson, N, Thavarajah, P, Kumar, S, Kresovich, S, Thavarajah Dil, 2019. Lentil Nutrigenomics, NIFA-AFRI PD Meeting, National Association of Plant Breeders meeting, August 25-29, 2019, Callaway Gardens, GA, USA.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Siva, Niroshan 2019. Doctoral thesis, Clemson University, SC. Prebiotic Carbohydrate Profiles of Lentil, Chickpea, and Common Bean Bean. https://tigerprints.clemson.edu/all_dissertations/2442/


Progress 02/15/18 to 02/14/19

Outputs
Target Audience:Target audiences: Pulse growers - national (Washingtan State, ND, SD, and MT), and international (India, and Africa) Breeders - national (USDA-ARS - Washington State) and international (ICARDA, Morocco) Consumers and food processors - national and international Young scientists - a PhD graduate student (Nathan Johnson), a undergraduate student major in Agronomy Emerging scholars - Under representative high school scholars from SC (Jouniors and rishing seniors from high schools). Commissioner's School for Agriculture - Future plant breeders who are in high school interested on nutritional plant breeding Efforts: Pulse growers were updated with project progress via news release, e- news, meetings, presentations, and regular phone calls with the USA Dry Pea Lentil Council. e- news articles for growers, breeders, and consumers: 2018: Clemson to launch weekly farm market and expand research at campus farm, http://newsstand.clemson.edu/mediarelations/clemson-to-launch-weekly-farm-market-and-expand-research-at-campus-farm/. 2018: Farm to table: Meet 5 Tigers who are working to eliminate hunger, malnutrition and obesity, https://newsstand.clemson.edu/farm-to-table-meet-5-tigers-who-are-working-to-eliminate-hunger-malnutrition-and-obesity/?utm_source=homepage-feature 2018, S.C. farmers might one day grow 'poor man's meat' thanks to Clemson research, http://newsstand.clemson.edu/mediarelations/s-c-farmers-might-one-day-grow-poor-mans-meat-thanks-to-clemson-research/ 2018, Lentils: The Healthiest Food You Don't Eat-But Should, https://bottomlineinc.com/health/diet-nutrition/bottom-line-guide-to-lentils. 2018: Finding a solution for hidden hunger, The Council on Undergraduate Research https://www.cur.org/finding_a_solution_for_hidden_hunger/. 2018: Clemson University News Stand, http://newsstand.clemson.edu/finding-a-solution-for-hidden-hunger/ Presentations made to growers, consumers, and breeders: Thavarajah Dil, 2018. Keynote Presentation, Pulse Crop Biofortification Global Strategies, International Food Legume Research Conference, May 5-8, 2018, Morocco. Thavarajah Dil, 2018. Research update meeting at ICARDA, Morocco with Dr. Rebecca McGee (USDA-ARA lentil breeder) and Dr. Shiv Kumar (ICARDA lentil breeder), May 9, 2018. Thavarajah Dil 2018. Invited oral e-presentation, Nutrigenomics of prebiotics rich pulses to combat obesity: a two-year seed grant, The USA Dry Pea and Lentil Council, Pulse Growers Research Review, Feb 21, 2018. Thavarajah, Dil 2018. Invited Seminar, Pulse crops towards global food security and human health. Department of Plant Sciences, University of California at Davis, August 23, 2018. Thavarajah, Dil 2018. Invited Seminar, Pulse crops linking to global food security and human health. International Association of students in Agricultural and related Sciences (IAAS) National Summit 2018, SC, April 7, 2018. Teaching and Curriculum presentations (graduate and undergraduate teaching): Thavarajah, Dil 2018. Developed a Study Aborad Program - "Golabal Food Security in Sri Lanka" -May 5-25, 2019. Thavarajah, Dil 2018. Invited Lecture, Legume nitrogen Metabolism and Drought, PES 8900 - Plant Nitrogen Metabolism, Nov 15, 2018. Thavarajah, Dil 2018. Invited Panel, Lentil nutritional Breeding - Technical science writing for STEM fields, Clemson University, Sep 4, 2018. Thavarajah, Dil 2018. Invited Lecture, Food system: Crops, Diet and Human health. Plant Breeding Institute, Department of Plant Sciences, University of California at Davis, August 24, 2018. Thavarajah, Dil 2018. Invited Lecture. Future Plant Breeders to Feed the World. South Carolina Commissioner's School for Agriculture- Plant Science, July 23, 2018. Thavarajah, Dil 2018. Invited Lecture, Food systems linking to the world health challenges - biotechnology challenges in global food production, AgEd 4160 - Ethics and Issues in agriculture and the Food and Fiber system, Feb 8, 2018. Consumer meetings/intervews: Clemson University News Stand, Sep, 2018. Bottomlineinc.com - interview, July 12, 2018. Lentil Food Processer from Chicargo - enriching pulse carbohydrates, June 27, 2018. Good Food Institute, Pulses as whole foods for prossessing, research meeting, July 15, 2018 Clemson Life Workshop (Special-Need Students) for healthy eating, April 20, 2018. American Pulse Association, Board meeting, Knoxville, April 18, 2018. Farm Day, March 31st, 2018, Bart Garrison Agricultural Museum, SC. Teaching workshops: Thavarajah D. 2018. Pendleton Elementary School, Oct 8, 2018. Thavarajah, Dil 2018. Future plant scientists to feed the world. SC Commissioner's School for Agriculture, July 23, 2018. Thavarajah D. 2018. Emerging Scholars for Agriculture and Food Security, Juniors workshop, June 18-22, 2018. Thavarajah D. 2018. Emerging Scholars for Agriculture and Food Security, Rising Seniors workshop, July 9-13, 2018. Changes/Problems:All required growth chamber facilities and analytical chemistry instrumentation are available in the PI's program at Clemson University. However, plants are biological material, therefore may anticipate germination issues, unknown diseases, and plant nutritional disorders etc. In addition, instrumentation malfunction and human errors are also possible. So far, no major changes happned during the first year of the project. What opportunities for training and professional development has the project provided?The PI: Dr. Thavarajah attend plant breeding workshop at the Tucson Plant Breeding Institute, Jan 7-12, 2018. Dr. Thavarajah completed two modules on plant breeding tools, statistical tools, GWAS, and R analysis. Dr Thavarajah has been elected as the American representative for the International Steering Committee (ISC) of the 8th International Food Legume Research Conference (IFLC-VIII) in 2022, Nairobi, Kenya. Doctoral Student: Dr. Thavarajah hired a doctoral student in May 2018 - Nathan Johnson from MN, USA. Nathan enrolled in four graduate courses toward his PhD: (1) Advanced Genetics, (2) Statistical Genetics, (3) Statistical Methods I, and (4) Nutrigenomics - Journal Club (PI is the lead instructor). Nathan has also engaged in additional professional development activities - GWAS and statistical software training. He completed safety training for both laboratory and greenhouse as well as Phase 1 of the required Responsible Conduct of Research training. He co-led Dr. Thavarajah' Emerging Scholars Program, in which he taught underrepresented high school juniors and seniors about pulse crops, prebiotic carbohydrates, biofortification, healthy pulse eating practices, and guided through a laboratory experiment on lentil carbohydrate analysis. Nathan also participated in an introductory R workshop, a high-performance liquid chromatography workshop, two weeks full time training, and has attended several genetic/genomic/ agronomic seminars. Two undergraduate students (Agronomy and Biochemistry Majors): Two undergraduate students were trained in this project. They learned following: (1) greenhouse maintenance, (2) fertilization protocol, (3) pest and disease control, (4) engage in high throughput chemical phenotyping of carbohydrates using anion exchange chromatography, and (5) data entry and storage. Highschool students: Two workshops were conducted for high school scholars from SC. How have the results been disseminated to communities of interest?Preliminary project results were presented at the 7th International Food Legume Research Conference (IFLC-VII), Morocco, May 2018. Upon the completion of data analysis, results will be presented at the North American Pulse Improvement Association (NAPIA) meeting in November 2019. Dr. Thavarajah and Nathan Johnson will present project update at the NAPIA meeting that will benefit the US pulse researches, growers, and consumers. Dr. Thavarajah has been already communicating to the national stakeholders via the American Pulse Association, and the USA Dry Pea and Lentil Council. Upon the completion of the doctoral thesis, all project data will be available to the general public via Clemson University, the Germplasm Resources Information Network (GRIN), and ICARDA websites. What do you plan to do during the next reporting period to accomplish the goals? Complete carbohydrate analysis for remaining samples Complete greenhouse experiment for seed increase Complete statistical analysis, data interpretatio, and prepare another manuscript Prepare AFRI full proposal Attend two plant breeding meetings - (1) the National Association of Plant Breeders (NAPB) and Plant Breeding Coordinating Committee (PBCC) annual meeting, August 25-29, 2019 in Pine Mountain Georgia, and (2) North American Pulse Improvement Association (NAPIA) in Nov, 2019.

Impacts
What was accomplished under these goals? Project Goal 1: Characterize the type and concentration of prebiotic carbohydrates (sugar alcohols, fructooligosaccharides, raffinose-family oligosaccharides, and starches) in global lentil association mapping population. Greenhouse Experiment: First run of the greenhouse experiment included a total of ~279 diverse lentil accessions, comprised of a global association mapping population (128 accessions), and association mapping population for heat and drought tolerance collection (150 accessions) from ICARDA was completed. These lentil accession were grown in the greenhouse from Jan-May 2018. The green house experiment was a complete randomized block design with 2 replicates (n=558). Harvested lentil plants were threshed, and seed yield was recorded. Then, seeds were hand cleaned, and ground using pestle and motor. Ground seed samples were stored in -20C freezer until further analysis. The second run of the greenhouse experiment with two populations is currently growing in the green house for both carbohydrate analysis, and one replicate for seed increase. Second growing cycle of these accessions will be completed early February 2019. Then, the USDA core collection (180 accessions) with two replicates will be planted in mid-February 2019. Carbohydrate Measurements: Concentrations of sugar alcohols, fructooligosaccharides, raffinose-family oligosaccharides, and starches are still in the progress. We completed the prebiotic carbohydrate analysis for heat tolerant population, and the rest of the analysis will be completed in March 2019. Since starting in May 2018, Nathan Johnson (doctoral student) has been working on this research project. He analyzed 150 accessions from the heat tolerance population for 10 different prebiotic carbohydrates. The carbohydrate concentration ranges are as follows in mg/100g: Sorbitol (38.4-3631.1), Mannitol (ND-300.5), Glucose (193.3-3140.2), Fructose (15.0-864.2), Sucrose (5083.5-16405.7), Raffinose+Stachyose (2799.4-10079.7), Verbascose+Kestose (1625.4-9717.5), Nystose (7.4-630.1). He is currently growing the second run of these lentil populations (both the global mapping and heat tolerance populations) in the greenhouse, composed of 150 and 128 accessions, respectively. We found 6 -10x genetic variation for these carbohydrates indicating a significant heritability estimate especially for sugar alcohols and raffinose family oligosaccharides. Data analysis is still in progress and will be completed in May 2019. Project Goal 2: Develop a multi-State collaborative AFRI standard research grant (July 2020) to identify candidate genes of prebiotic carbohydrates responsible for increased lentil nutritional quality using genome-wide association studies (GWAS). Progress: Project team (Dr. Thavarajah; Dr. Kresovich; Dr. Shiv Kumar; Mr. Nathan Johnson) has been meeting every month since May 2018. We discussed project progress, future needs, and already developed the full proposal objectives. Dr. Thavarajah met with theUS lentil breeder (Dr. Rebecca McGee), and other North American pulse researches at the International Food Legume Research Conference which was held in Morocco - May 2018. We are expecting to finish 90% of the project data analysis in summer 2019. Depending on the seed project results, we might submit the full proposal in July 2019. Following activities were done towards developing full proposal. Preliminary Germplasm Development: Based on the preliminary carbohydrate data (heat tolerant population), our project collaborator ICARDA lentil breeder, Dr. Shiv Kumar made 28 lentil crosses (Table 1), and F2 seeds will be send to Dr. Thavarajah program to grow in Clemson University Simpson Farm field trials for AFRI full proposal. Dr. Kumar is also sending 10 ICARDA lentil genotypes (most suitable for tropical weather) to establish 2019 winter nursey in Clemson, SC. Our collaboration with ICARDA will be able to develop future high nutritional lentil varieties for SC, USA. Our project data will be available to the general public and other researches once the doctoral thesis is competed. Table 1: Crossing block made by ICARDA, Morocco. PN Carbohydrates Female Male ICBL-2019-101 Sorbitol ILL268 ILL1474 ICBL-2019-102 Sorbitol ILL623 ILL2230 ICBL-2019-103 Sorbitol ILL705 ILL2406 ICBL-2019-104 Sorbitol ILL719 ILL10281 ICBL-2019-105 Sorbitol ILL1097 ILL10661 ICBL-2019-106 Sorbitol ILL4778 ILL10810 ICBL-2019-107 Sorbitol ILL623 ILL10825 ICBL-2019-108 Sorbitol ILL4778 ILL10862 ICBL-2019-109 Mannitol ILL304 ILL1097 ICBL-2019-110 Mannitol ILL5418 ILL1460 ICBL-2019-111 Mannitol ILL5424 ILL7012 ICBL-2019-112 Mannitol ILL304 ILL10825 ICBL-2019-113 Mannitol ILL5418 ILL10862 ICBL-2019-114 Mannitol ILL5424 IG33115 ICBL-2019-115 Mannitol ILL5424 IG33117 ICBL-2019-116 Raff + Starch ILL597 ILL502 ICBL-2019-117 Raff + Starch ILL598 ILL956 ICBL-2019-118 Raff + Starch ILL619 ILL1474 ICBL-2019-119 Raff + Starch ILL6644 ILL10315 ICBL-2019-120 Raff + Starch ILL6644 ILL10661 ICBL-2019-121 Raff + Starch ILL6644 ILL10690 ICBL-2019-122 Verba+Kest ILL598 ILL494 ICBL-2019-123 Verba+Kest ILL623 ILL5151 ICBL-2019-124 Verba+Kest ILL598 ILL5595 ICBL-2019-125 Verba+Kest ILL623 ILL5968 ICBL-2019-126 Nystose ILL2684 ILL1005 ICBL-2019-127 Nystose ILL4881 ILL5509 ICBL-2019-128 Nystose ILL5553 ILL10864

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: Thavarajah Dil, Mcswain, M. CR. Johnson, SK. Agrawal, P. Thavarajah, 2018. Pulses, Global Health, and Sustainability: Future Trends. In: Health Benefits of Pulses. Ed: Wendy Dhal, Springer Nature
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Siva, N, Thavarajah P., Kumar, S., Thavarajah, Dil, 2019. Prebiotic carbohydrates in pulse crops towards improve human health. Frontiers in Plant Sciences, 441785.