Research Project: Gene Discovery and Designing Soybeans for Food, Feed, and Industrial Applications

Location: Plant Genetics Research

2023 Annual Report

Objectives
Objective 1: Identify new soybean alleles, or effective combinations of existing genes, that positively impact commercially relevant oil or meal traits; work with breeders to incorporate them into modern backgrounds; confirm their expression or effectiveness under field conditions; and determine value in food or feed applications. Objective 2: Identify and verify new genomic regions in soybean associated with improved stress tolerance, seed constituent (oil and protein), and quality traits, and use genomic strategies such as genetic mapping and genome analysis to make new genes rapidly available to breeders. Objective 3: Develop novel strategies to increase concentrations of S-containing amino acids and to reduce levels of trypsin inhibitor and allergens; work with breeders to develop soybean germplasm that combine these genes in high protein backgrounds to meet the animal nutrient requirements.

Approach
Obj 1- New soybean germplasm will be developed with combinations of the high oleic-low linolenic oil trait and low raffinose oligosaccharide meal trait that is targeted to different maturity groups (MG). Seeds produced in an appropriate environment will be evaluated for trait interactions, environmental stability, protein and oil content, and yield. We will establish a novel panel of approximately 400 soybean accessions from the National Plant Germplasm System (NPGS) and conduct genome-wide association studies (GWAS) with protein and oil data. Mutant soybean lines will be screened to identify seed composition variants. Obj 2- We will use a four pronged approach in order to dissect the genetic architecture underlying soybean seed value (principally seed oil and protein content) and abiotic stress adaptation: 2.1) a new GWAS using a diverse panel of 380 MG III genotypes to maximize genetic diversity within a very narrow maturity range; 2.2) Genomic Prediction to estimate seed composition breeding values for all 2,011 MG III accessions; 2.3) Fine mapping of a heat-tolerance trait from an exotic landrace; and 2.4) Development of a Multi-Parent Advanced Generation Inter-Cross (MAGIC) population. We will evaluate the potential of Genomic Prediction to predict seed composition and select parents with maximal genetic potential for developing a MAGIC population. We will Fine-map a previously identified major effect QTL associated with tolerance to heat-induced-seed-degradation. Obj 3- We will develop and characterize soybean germplasm with increased sulfur (S)-containing amino acids and decreased anti-nutritional factors. To enhance the S amino acid content, we plan to overexpress an enzyme in the sulfur assimilation pathway. Additionally, high-protein soybean experimental lines lacking Kunitz trypsin inhibitor (KTI) and ß-conglycinin, will be developed using a traditional breeding approach. In order to verify if overexpression of tow enzymes simultaneously will further increase the overall S-amino acid content, we will characterize ATPS and OASS activity in greenhouse grown material from genetic crosses between overexpressing transgenic soybeans lines. To better understand the chilling stress responses in soybean, a comparative proteomic analysis will be performed.

Progress Report
This is the final report for this project which terminated in January 2023. The replacement project, 5070-21000-044-000D, “Redesigning Soybeans for a Resilient Future of Food, Feeds, and Bio-Industry” does not have reporting yet. The research conducted under Objective 1 was successful in developing knowledge and soybean germplasm targeted to different US production areas with a combination of oil and meal traits. Soybean germplasm was developed that combined the high oleic acid/low linolenic acid oil trait (HOLL) with the low, ultra-low or no raffinose family of oligosaccharides (RFO) traits in a maturity group (MG) III background, and seed composition analysis showed no interference between the oil and meal traits. Additionally, the HOLL trait was combined with either the low or no RFO meal traits with additional selection for recombination between seed composition genes linked to maturity genes in soybean germplasm targeted to MG 0, I, II, and V production environments; the seed composition phenotypes were stable when the seed was produced in the targeted environments. Analysis of seed yield in preliminary trials in two environments for modified seed composition soybean germplasm demonstrated the achievement of up to 94% of the high yielding check cultivars in the test for a line with the HOLL and no RFO traits combined. When semi-determinate soybean germplasm lines were developed with the seed composition traits, no lines were identified with competitive seed yields in two MG III environments. The second goal to develop knowledge that will enhance efforts to increase soybean seed oil and protein content while maintaining high yields in US production areas had substantial progress made, but more research is needed. Little variation for seed protein content was observed in the association panel, so research was adjusted to identify the genetic architecture for seed protein content in biparental mapping populations developed from the two related high protein accessions from the association panel (with the reference cultivar Williams 82 as the common low protein parent). The two mapping populations have been genotyped and verified for integrity, but final seed protein phenotype information and genetic mapping will be completed after FY23. Overall, the objective 1 research results pointed to the ability to create new soybean germplasm with greater value from the seed constituents, stable expression of the seed composition traits alone and in combination with each other, and competitive seed yields. The research under Objective 2 was successful in identifying and characterizing new genetic means to boost soybean seed composition/value and soybean’s tolerance to abiotic stresses. A genetic mapping study for tolerance to heat induced seed degradation revealed a new quantitative trait loci (QTL) that confers tolerance to elevated temperatures, and that two previously cloned seed phosphorus bioavailability genes are inextricably linked to increased temperature sensitivity. A fine-mapping population was developed for the novel beneficial QTL, but our efforts were unsuccessful due to low trait heritability and two separate field disasters. Despite this setback, other genome wide association mapping (GWAM) studies were fruitful: we identified important genomic regions controlling soybean seed composition, and the data was leveraged to create a genomic prediction model to identify the most divergent lines from the USDA-ARS germplasm collection for seed composition and a three-location field study validated our genomic prediction models for seed protein and oil. We also identified, confirmed and fine-mapped a new QTL from wild soybean that increases seed protein with no significant cost in seed oil, in stark contrast to other known high seed protein QTL. We completed several multidisciplinary GWAM studies for drought tolerance traits. All this new genetic information was leveraged to choose parents to create a new Multi-parent Advanced Generation InterCross (MAGIC) genetic mapping population, and final hybridizations were completed in 2021. The newly developed MAGIC population was advanced to F5 and is now ready for field studies, with unprecedented genetic diversity combining eight parents which have either value-added seed compositional traits or abiotic stress tolerance. In the coming years, field studies for seed composition and abiotic stress tolerance will be performed, and we will also leverage these populations to accelerate development of improved soybean germplasm for the benefit of the soybean breeding community. Research has demonstrated that consumption of soybean meal can elicit biological responses like inflammation, reduce nutrient absorption capacity and significantly affect the growth performance of livestock. This negative effect is attributed to the presence of allergens in the soybean meal. We have earlier identified soybean 7S globulin ß-conglycinin as a significant food and feed allergen. The ß-conglycinin is composed of three subunits namely a’-, a-, and ß. Among these, the a-subunit has been identified as the most dominant allergen. Additionally, the abundance of anti-nutritional factors such as the Kunitz trypsin inhibitor proteins (KTi), also affects the animal performance since they cause pancreatic hypertrophy, liver damage, and hypoglycemia in livestock and poultry. We have been focused on developing soybean lines that are devoid of allergens and anti-nutritional factors. We screened hundreds of soybean lines from the USDA soybean germplasm collection and identified mutants that are devoid of the a-subunit of ß-conglycinin, the most dominant allergen. Additionally, by employing RNAi technology we have also generated transgenic soybean lines that lack all the three subunits of ß-conglycinin. We have utilized these mutants in our breeding program to develop soybean lines that are devoid of the allergenic a-subunit of ß-conglycinin and anti-nutritional Kunitz trypsin inhibitor proteins. For this purpose, we have crossed a Kti null soybean line with a soybean line that is devoid of the major allergen. F1 seeds from the crosses were advanced three generation by single seed descent. The introgression of the desirable traits (lack of the a-subunit of ß-conglycinin and Kti) were monitored throughout the breeding scheme by gel electrophoresis and immunoblot analysis. Based on this biochemical analysis we have identified 6 breeding lines that appear to carry the desired traits. Currently, select soybean lines with reduced anti-nutritional proteins and low allergens are being evaluated for their agronomic performance. Additionally, we are planning to evaluate the potential of these materials to enhance the growth performance of livestock by conducting animal feeding trails. It is anticipated that our unique soybean lines lacking the a-subunit of ß-conglycinin and Kti will have a significant positive impact on the livestock and poultry industries.

Accomplishments
1. Defining soybean seed composition targets for alternative proteins to meet nutritional needs while reducing climate impact. To meet the ongoing full nutritional needs of the global human population, desirable high protein foods must become more accessible. ARS scientists in Columbia, Missouri, partnered with other ARS scientists as well as food scientists and engineers in both academia and industry to develop new soybean germplasm and perform a baseline study that combined sensory analysis with chemical characterization of seven novel ARS seed composition germplasm lines separated into six seed composition categories based on their targeted oil, carbohydrate, oxidizing enzyme (lipoxygenase) status, and aromatic attributes. The results of analyses with soybean slurries established significant differences for sensory attributes and chemical composition. This new information will be important for tracking changes that impact flavor and functionality after further processing of the seeds into oil and protein products. This work identifies soybean germplasm with desirable seed compositions for variety development, and agronomic testing, ultimately to sustainably meet the needs for human food.

2. New wild soybean genetic trait that boosts seed protein with no cost to seed oil. Modern soybean cultivars have low overall genetic variation. Wild soybean may contain genetic differences that are viable options to increase genetic diversity and traits of value for both public and private soybean breeding programs. ARS scientists at Columbia, Missouri, hybridized a domesticated soybean line with a wild soybean line and identified a new genetic trait that increases seed protein content ~ 0.79% without a significant decrease in seed oil content. Genetic markers were developed so that marker assisted selection could be used to accelerate introgression of the wild soybean trait into modern soybean cultivars. This new finding is different from other available genetic markers for elevated protein because the new trait does not decrease seed oil when seed protein is increased. ARS scientists are working with a soybean seed company that will develop this trait to maximize seed protein content for the non-genetically modified soy food ingredient market and food applications such as soy flakes and soy flour.

3. Development of soybean lines with increased protein and essential sulfur containing amino acids. Soybean is the preferred protein source for both poultry and swine feed, but competition from other alternative feed ingredients is growing. To compete effectively, breeders need to develop soybean cultivars that contain higher protein with better nutritional composition, including the essential sulfur containing amino acids cysteine and methionine. ARS scientists in Columbia, Missouri, have developed soybean lines that not only contain higher amounts of protein but also improved sulfur amino acid content. They crossed a transgenic soybean line with elevated levels of sulfur amino acid content with a high protein Korean soybean line. The resulting plants exhibited elevated protein content along with improved cysteine and methionine profiles. The development of such high protein soybean lines with enhanced sulfur containing amino acids will help U.S. soybean compete as a protein source for animal feed. These newly developed lines can be exploited by soybean breeders to develop elite soybean lines with desirable agronomic characteristics and marketable components.

Review Publications
Skrabisova, M., Dietz, N., Zeng, S., Chan, Y., Wang, J., Liu, Y., Biova, J., Trupti, J., Bilyeu, K.D. 2022. A novel synthetic phenotype association study approach reveals the landscape of association for genomic variants and phenotypes. Journal of Advanced Research. 42:117-133. https://doi.org/10.1016/j.jare.2022.04.004.
Kim, S., Krishnan, H.B. 2023. Chapter seven - A fast and cost-effective procedure for reliable measurement of trypsin inhibitor activity in soy and soy products. Methods in Enzymology. 680:195-213. https://doi.org/10.1016/bs.mie.2022.08.016.
Chan, Y., Dietz, N., Zeng, S., Wang, J., Flint Garcia, S.A., Salazar-Vidal, N.M., Skrabisova, M., Bilyeu, K.D., Joshi, T. 2023. The allele catalog tool: a web-based interactive tool for allele discovery and analysis. BMC Genomics. 24: Article 107. https://doi.org/10.1186/s12864-023-09161-3.
Kim, W., Nott, J., Kim, S., Krishnan, H.B. 2022. Chapter twelve - soybean seed proteomics: Methods for the isolation, detection, and identification of low abundance proteins. Methods in Enzymology. 676:325-345. https://doi.org/10.1016/bs.mie.2022.07.001.
Biová, J., Dietz, N., Chan, Y., Joshi, T., Bilyeu, K.D., Škrabišová, M. 2023. AccuCalc: A python package for accuracy calculation in GWAS. Genes. 14(1). Article 123. https://doi.org/10.3390/genes14010123.
Liu, S., Luo, T., Song, Y., Ren, H., Qiu, Z., Ma, C., Tian, Y., Wu, Q., Wang, F., Krishnan, H.B., Yu, W., Yang, J., Xu, P., Zhang, S., Song, B. 2022. Hypocholesterolemic effects of soy protein isolates from soybeans differing in 7S and 11S globulin subunits vary in rats fed a high cholesterol diet. Journal of Functional Foods. 99. Article 105347. https://doi.org/10.1016/j.jff.2022.105347.
Chamarthi, S., Kaler, A., Abdel-Haleem, H.A., Fritschi, F., Gillman, J.D., Ray, J.D., Smith, J.R., Purcell, L. 2022. Identification of genomic regions associated with the plasticity of carbon 13 ratio in soybean. The Plant Genome. 16(1). Article e20284. https://doi.org/10.1002/tpg2.20284.
Yang, Y., La, T., Gillman, J.D., Lyu, Z., Joshi, T., Usovsky, M., Song, Q., Scaboo, A. 2022. Linkage analysis and residual heterozygotes derived near isogenic lines reveals a novel protein quantitative trait loci from a Glycine soja accession. Frontiers in Plant Science. 13. Article 938100. https://doi.org/10.3389/fpls.2022.938100.
Pereira, A.E., Huynh, M.P., Paddock, K.J., Ramirez, J.L., Caragata, E.P., Dimopoulos, G., Krishnan, H.B., Schneider, S.K., Shelby, K., Hibbard, B.E. 2022. Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis. Scientific Reports. 12. Article 17858. https://doi.org/10.1038/s41598-022-22229-6.
Piya, S., Pantalone, V., Zadegan, S.B., Shipp, S., Lakhssassi, N., Knizia, D., Krishnan, H.B., Meksem, K., Hewezi, T. 2023. Soybean gene co-expression network analysis identifies two co-regulated gene modules associated with nodule formation and development. Molecular Plant Pathology. 24(6):628-636. https://doi.org/10.1111/mpp.13327.
Mcdonald, S.C., Bilyeu, K.D., Koebernick, J., Buckley, B., Fallen, B.D., Mian, R.M., Li, Z. 2023. Selecting recombinants to stack high protein with high oleic acid and low linolenic acid in soybean (glycine max). Plant Breeding. 142(4):477-488. https://doi.org/10.1111/pbr.13102.