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ARS Home Midwest Area Columbia, Missouri Plant Genetics Research Research





- Gillman, Jason
- Krishnan, Hari
- Bilyeu, Kristin
- 2023
- 2022
- 2021
- 2020
- 2019
- 2018
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Gene Discovery and Designing Soybeans for Food, Feed, and Industrial Applications **Location:
                            Plant Genetics Research** 2021 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**
                                

Objective 1:  Research continued on understanding the ability to combine oil and meal traits in soybean germplasm targeted to different maturity groups (MG) across most US soybean production environments. We completed the molecular selection and generation of soybean germplasm with the oil and meal trait combination targeted to MG I and V. Seeds of MG 0, I, II, III/IV, and V were produced in our MG III field environment. We produced and harvested seed, analyzed seed composition, and distributed seed to university and seed company partners with combinations of oil and meal value traits. We analyzed seed yield for soybean germplasm with oil and meal trait combinations targeted to MG III/IV, and determined there is need for genetic improvement compared to released soybean variety yields.  Soybean lines with the semi-determinate trait were low ranking in the trial. We conducted an experiment to investigate the interaction of oil and meal traits, and determined that there was no negative interference for the seed composition traits. Additionally, we generated a bi-parental mapping population to refine the genetic basis of higher seed protein content as the next step from our association experiment with our developed soybean accession panel.

We are well on the way to fully meeting all milestones for Objective 2. The 36-month Milestone (2.2) was completed one year ahead of schedule and the final crosses to generate our unique 8-way Multi-Parent Advanced Generation Inter-Cross (MAGIC) population will be completed during FY2021. When complete, the MAGIC population will have unprecedented genetic potential for beneficial alleles for 1) improved abiotic stress tolerance (drought and elevated temperature), 2) high seed yield and 3) improved seed composition (elevated seed protein/oil; increased seed oil stability due to elevated oleic acid and reduced linolenic acid; and more digestible seed carbohydrates). We are also on track to fully meet the 48-month (2.3) and 60-month (2.4) milestones. 

Objective 3:  Research continued on improving the overall sulfur amino acid content of soybeans. We have demonstrated that transgenic soybean plants overexpressing O-acetylserine sulfhydrylase (OASS) or Adenosine triphosphate sulfurylase (ATPS) accumulated significantly higher amounts of sulfur amino acid, methionine and cysteine. These two enzymes play crucial roles in sulfate assimilation and regulate the overall sulfur amino acid content of seed.  To study the effect of simultaneously overexpressing these regulatory enzymes, we have crossed OASS overexpressing transgenic soybean lines with ATPS overexpressing soybean lines. We monitored simultaneous overexpression in the hybrid lines at each successive generation (F2-F3) by measuring OASS and ATPS activity and immunoblot analysis. Our analysis demonstrated that the hybrid lines had several-fold increase in OASS and ATPS activity and content when compared to the non-transgenic controls.  Inductively coupled plasma-mass spectrometry analyses revealed that the hybrid lines (F3) developed in our study contained higher amounts of elemental sulfur in the seeds. SDS-PAGE and western blot analysis demonstrated that the accumulation of Bowman-Birk protease inhibitor and lunasin, two sulfur amino acid rich peptides, were elevated in the hybrid soybean lines. High-resolution 2D-gel electrophoresis and Delta2D gel analysis validated an overall increase in the Bowman-Birk protease inhibitor. Our results indicate that simultaneously overexpressing both OASS and ATPS could result in a significant improvement in the sulfur amino acid content of soybean seeds. **Accomplishments**
                                

1. Commercial launch of soybean high oleic acid seed oil trait. Significantly decreased utilization and demand for soybean oil, which was previously the largest source of cooking and frying oil, was the result of recent changes in food labeling requirements and ingredient use due to health concerns from trans fats, which were induced in hydrogenated soybean oil. Previous research by ARS researchers at Columbia, Missouri, and collaborators to develop a breeding method for high oleic soybean, which is the subject of multiple U.S. and foreign patents, was made available for research and development of new soybean varieties with oil that needs no hydrogenation and contains no trans fats. The altered oleic acid composition resulting from this method results in changes in the soybean oil properties for improved health properties, and functionality for use in food and industrial applications, where oxidative stability affects functionality such as frying oil and biodiesel fuel. This patented method to develop high oleic acid soybeans that shifts oxidatively unstable fatty acids into the mono-unsaturated oleic acid category of the oil led to the “Soyleic” trademark for the oil quality trait. Significant additional research investments from public and private funding sources have led to the development and commercial release of new soybean varieties by ARS partners. Upwards of 40,000 acres were planted commercially in 2021, not counting acres for seed increase. This research delivered a solution to a food health issue through collaborative innovation in research and partnership to assist the commercialization of research discoveries. 2. Discovery of a new Quantitative Trait Locus which enhances seed germination and emergence. Soybean is a high value seed crop but has a relatively low optimum reproduction temperature (22–24 °C or 72-75 °F) and the ancestors of high yielding cultivars lack substantive tolerance to elevated temperatures. In the Mid-South soybean growing region, earlier planting/harvest practices allow producers to avoid some drought stress but also result in unacceptable levels of seed damage and reduced economic seed value. Ongoing climate change may cause more widespread late-season drought and elevated temperatures resulting in increased incidence of seed damage. ARS researchers at Columbia, Missouri, and Stoneville, Mississippi, performed genetic mapping using a novel source of genetic tolerance to elevated temperatures and were successful in identifying a major-effect genomic region that enhances seed germination and emergence, and the improvement was most pronounced when seeds developed under elevated temperatures. This important discovery opens the possibility of marker assisted breeding and will facilitate efforts to genetically mitigate some of the deleterious effects of climate change on soybean seed quality, as well as improve soybean seed quality in the Mid-South region. **Review Publications**
                            *Piya, S., Lopes-Caitar, V., Kim, W., Pantalone, V., Krishnan, H.B., Hewezi, T. 2021. Title: Hypermethylation of miRNA genes during nodule development. Frontiers in Molecular Biosciences. 8. Article e616623. https://doi.org/10.3389/fmolb.2021.616623.*
                                *Niyikiza, D., Piya, S., Routray, P., Miao, L., Kim, W., Burch-Smith, T., Gill, T., Sams, C., Arelli, P.R., Pantalone, V., Krishnan, H.B., Hewezi, T. 2020. Interactions of gene expression, alternative splicing, and DNA methylation in determining nodule identity. Plant Journal. 103(5):1744-1766. https://doi.org/10.1111/tpj.14861.*
                                *Krishnan, H.B., Kim, W., Oehrle, N.W., Smith, J.R., Gillman, J.D. 2020. Effect of heat stress on seed protein composition and ultrastructure of protein storage vacuoles in the cotyledonary parenchyma cells of soybean genotypes that are either tolerant or sensitive to elevated temperatures. International Journal of Molecular Sciences. 21(13). Article 4775. https://doi.org/10.3390/ijms21134775.*
                                *Kim, W., Kim, S.N., Oehrle, N.W., Jez, J.M., Krishnan, H.B. 2020. Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the b-subunit of b-conglycinin in soybean seeds. Scientific Reports. 10. Article e14989. https://doi.org/10.1038/s41598-020-72134-z.*
                                *Gillman, J.D., Chebrolu, K., Smith, J.R. 2021. Quantitative trait locus mapping for resistance to heat-induced seed degradation and low seed phytic acid in soybean. Crop Science. 61(3):2023–2035. https://doi.org/10.1002/csc2.20419.*
                                *Beche, E., Gillman, J.D., Song, Q., Nelson, R.L., Beissinger, T., Decker, J., Shannon, G., Scaboo, A.M. 2021. Genomic prediction using training population design in interspecific soybean populations. Molecular Breeding. 41. Article e15. https://doi.org/10.1007/s11032-021-01203-6.*
                                *Hagely, K., Konda, A.R., Kim, J., Cahoon, E., Bilyeu, K.D. 2021. Molecular-assisted breeding for soybean with high oleic/low linolenic acid and elevated vitamin e in the seed oil. Molecular Breeding. 41. Article e3. https://doi.org/10.1007/s11032-020-01184-y.*
                                *Kambhampati, S., Aznar-Moreno, J.A., Bailey, S.R., Arp, J.J., Chu, K., Bilyeu, K.D., Durrett, T.P., Allen, D.K. 2021. Temporal changes in metabolism late in seed development affect biomass composition. Plant Physiology. 186(2):874-890. https://doi.org/10.1093/plphys/kiab116.*
                                *Islam, N., Krishnan, H.B., Natarajan, S.S. 2020. Proteomic profiling of fast neutron-induced soybean mutant unveiled pathways associated with increased seed protein content. Journal of Proteome Research. 19:3936-3944. https://doi.org/10.1021/acs.jproteome.0c00160.*
                                *Willette, A., Fallen, B.D., Bhandari, H., Sams, C., Chen, F., Sykes, V., Smallwood, C., Bilyeu, K.D., Li, Z., Pantalone, V. 2021. Agronomic performance of high oleic, low linolenic soybean in Tennessee. Journal of the American Oil Chemists' Society. https://doi.org/10.1002/aocs.12517.*
                                *Zeng, S., Skrabisova, M., Lyu, Z., Chan, Y., Dietz, N., Bilyeu, K.D., Joshi, T. 2021. Application of SNPViz v2.0 using next-generation sequencing data sets in the discovery of potential causative mutations in candidate genes associated with phenotypes. International Journal of Data Mining and Bioinformatics. 25(1-2):65-85. https://doi.org/10.1504/IJDMB.2021.116886.*


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