Research Project: Designing Soybeans with Enhanced Seed Quality and Plant Health Traits

Location: Crop Production and Pest Control Research

2024 Annual Report

Objectives
Objective 1: Conduct research to identify genetic loci involved in soybean seed protein and oil accumulation, characterize resource partitioning of the new soybean genotypes, and work with breeders to develop new germplasm incorporating genes for improved protein and oil traits. Goal 1.A: Map genetic loci involved in soybean seed protein and oil accumulation. Goal 1.B: Work with breeders to evaluate and incorporate new meal and oil traits. Objective 2: Conduct research to elucidate pathogen biology, fungicide resistance, and virulence of Frogeye Leaf Spot disease, and identify novel host resistance to this threat to soybean production in the North Central region. Hypothesis 2.A: Frogeye leaf spot in the northcentral states is caused by distinct clonal lineages consisting of both mating types and wide-spread-QoI resistance. Hypothesis 2.B: C. sojina populations have high virulence diversity. Hypothesis 2.C: Low-level sexual recombination occurs in C. sojina. Objective 3: Conduct field and greenhouse research evaluations and statistical analyses to collect breeding-related data, and report results on testing of public sector soybean breeding lines evaluated in the Uniform Soybean Tests – Northern Region. Goal 3: Evaluate advanced public breeding lines for agronomic performance, disease resistance and quality traits throughout the northcentral U.S. and Canada.

Approach
Objective 1: Genes involved in soybean seed protein and oil accumulation will be isolated through phenotypic screening of seed composition traits. Mapping populations will be developed to isolate and characterize genes conferring improved composition phenotypes. Environmental and developmental characteristics will be examined in protein:oil mutants to further understand the control of resource partitioning in the seed. New and existing genes will be combined and evaluated for their utility to provide soybean breeders with new composition traits for current and future needs. Objective 2: Cercospora sojina isolates will be collected from soybean fields in Indiana and other states. The isolates will be examined for resistance to QoI fungicides by amplifying and sequencing their mitochondrial cytochrome b gene. Their mating types will be determined by amplifying Mat1-1 and Mat1-2 genes using gene-specific primers. The pathogen population will be genotyped using a panel of single nucleotide polymorphism markers. Isolates representing different genotypes will be used to inoculate a set of 14 soybean differentials to determine their virulence. Isolates with different mating type genes will be used to co-inoculate soybean. Pathogen will be re-isolated from plant tissue and genotyped to determine whether sexual recombination occurred. Objective 3: Public soybean breeders submit their soybean breeding lines for evaluation of agronomic performance, disease resistance and quality traits. Entries are separated by maturity group and assigned to either the ‘Preliminary Tests’ or the ‘Uniform Tests’. Seeds of each entry, along with those of the standard reference varieties, are packaged and distributed to collaborators throughout the U.S. and Canada for evaluation. In addition, entries will be evaluated at multiple locations in Indiana. Harvested seeds will also be tested for quality traits. Collaborators submit performance data from their locations to ARS after harvest. This data is compiled and analyzed by this research group following established protocols. The results will be published in an annual report book and online.

Progress Report
For Objective 1, in fiscal year 2024 rough map positions were determined for four mutants with high levels of seed oil or protein and one mutant with high linolenic acid. One high oil mutation maps to Chromosome 2, and the high linolenic acid trait maps to Chromosome 14. We are fine-mapping these two lines with single nucleotide polymorphism (SNP) and microsatellite markers to further delimit the genomic regions containing the mutations. This data will be used in concert with whole genome-resequencing and gene expression profiling approaches to identify the causative genes. We planted five new mapping populations in the field in 2024 to evaluate for segregation, collect tissue, and prepare for rough mapping in 2025. We will evaluate seed yield in 2-row replicated plots on of 33 high protein and high oil mutants for a second season, which will assist in prioritizing which high protein mutants to pursue on the basis of yield potential. For Objective 1.B, the project aims to introgress and combine the high oleic (HO) high oleic low linolenic (HOLL) and low raffinose family oligosaccharide (RFO) traits into improved Midwestern germplasm. Using Polymerase Chain Reaction-based markers, we are selecting BC5F2 from populations segregating for two genes conferring the HO trait, and performing a sixth introgression cross. For the HOLL, HO-loRFO, and HO-low-sats traits we have selected two genes from each segregating population (BC2F2) and will cross these to the advanced HO line (BC3) (so that the two oleic genes will be fixed in the cross). The progeny of these crosses will be propagated and genotyped in the greenhouse during the winter of 2024-2025. We will evaluate seed composition in the BC2F3 seed after harvest in 2024. For Objective 2.A, soybean leaf samples showing frogeye leaf spot symptoms were collected from 29 counties in Indiana. The leaf samples were placed in moisture chambers for 48 hours to produce conidia. The conidia were transferred to water agar plates amended with antibiotics and single germinating conidia were transferred to potato dextrose agar plates. A total of 593 single-conidial isolates of Cercospora sojina were obtained from 234 lesions. One thousand and fifty-seven isolates were collected from 21 soybean producing states in the United States through collaborations. We also collected eight isolates from China , 32 isolates from Brazil and 2 isolates from Nigeria. All isolates were purified by generating single-conidial isolates. DNA extraction is in progress. For Objective 2.B, soybean differentials for frogeye leaf spot in maturity groups I-V (Blackhawk, Kent, Lincoln, Peking, Richland, and S-100) were grown in four-row plots in West Lafayette, Indiana. The center two rows of each differential line were harvested using a plot thresher. Differentials in maturity group VI and later (CNS, Davis, Hood, Lee, Palmetto, PI 594774, PI 594891, and Tracy) were planted in Stoneville, Mississippi, through collaboration and harvested as described above. For Objective 3, 514 soybean breeding lines and checks were evaluated at 44 locations in 10 states in the United States and three provinces in Canada in 2023. These lines were evaluated for yield, disease resistance (Phytophthora root rot and soybean cyst nematode), seed composition and other agronomic traits. We conducted field trials in three locations in Indiana. Data were collected from our own field trials as well as from 17 collaborators. The data were analyzed and the results were published in the report book “The Uniform Soybean Test Northern Region 2023”. Hard copies were sent to all collaborators. Electronic version is freely available online (https://www.ars.usda.gov/ARSUSERFILES/50200500/UST/2023.PDF). In 2023, 18 soybean varieties evaluated in the Uniform Soybean Test Northern Region program were publicly released or licensed to private companies. For 2024 test, we organized the coordination meeting in February 2024, finalized the experimental design, collected seeds from all collaborators, packaged and distributed seeds to all test locations. In West Lafayette, Indiana, we are growing field trials in three locations in Indiana. Planting was completed on time and lines are growing well.

Accomplishments
1. Discovered a gene responsible for stem elongation in soybean. Modern soybean plants are a result of thousands of years of selection for valuable agronomic traits such as yield, nutritional value of seeds, and also architectural traits that make the plant better suited for row-crop farming environments. Soybean breeders continue to try to understand and identify genetic variation for morphological traits that could contribute to more efficient plants. ARS scientists in West Lafayette, Indiana, isolated a soybean plant of very short stature (named “Compact”) from a mutant population, and found that they overexpress a gene involved in the metabolism of the plant hormone gibberellic acid (GA). While GA has long been known to affect cell elongation (understanding plant response to gibberellin was a major contributor to the “green revolution”, to make high yielding plants shorter and more resistant to lodging) few genes controlling soybean plant height are currently known. The compact soybean plants discovered will be a valuable tool for plant biologists to further investigate the effect of endogenous and environmental factors on soybean growth and stem architecture. Furthermore, this trait may also be used by soybean geneticists in breeding semi-dwarf soybean for dense cultivation or in environments where short stature allows dense planting with high yield.

2. Identified soybean germplasm and associated molecular markers for resistance to Fusarium graminearum. Seedling diseases caused estimated yield loss of $3.35 billion dollars in the United States in the past decade (2014-2023). The role of Fusarium graminearum as an aggressive member of the pathogens that cause soybean seedling diseases was unknown until relatively recently and, consequently, publicly and commercially available varieties with resistance to this pathogen are unavailable. To address the need for resistant germplasm and to improve our understanding of the genetic basis underlying the resistance, an ARS scientist in West Lafayette, Indiana, screened a set of 208 accessions of soybean from the USDA Soybean Germplasm Collection. Eight soybean accessions (PI 548311, PI 438500, PI 561318 A, PI 547690, PI 391577, PI 157484, PI 632418, and PI 70466-3) were found to carry significantly higher resistance than the population mean. A genome-wide association study identified five DNA markers that are linked to the resistance. This study provided germplasm sources and associated molecular markers that are valuable for breeding soybean varieties with resistance to F. graminearum.

3. Release of soybean germplasm DS1260-2. Mature seed damage in soybean results in price discounts when grain is sold and caused estimated yield loss of over one billion dollars in the United States during the past decade (2014-2023). An ARS scientist in West Lafayette, Indiana, in collaboration with fellow ARS scientists, developed and released soybean germplasm DS1260-2 (PI 705148). This is a germplasm line with reduced mature seed damage when subjected to hot humid conditions during plant maturation and dry down. DS1260-2 showed improved tolerance to mature seed damage compared to public soybean cultivars, which is manifest as reduced visual mold (fungal growth), discoloration, wrinkling, purple seed stain, and green seed, and with improved germination. This germplasm will be valuable for improving mature seed quality and increase income for soybean growers in hot humid environments. It has been used by soybean breeding programs in University of Arkansas and University of Missouri to develop commercially-competitive soybean cultivars with tolerance to mature seed damage.

4. Sequenced and characterized the genome of Anisogramma anomala. The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut trees. It is a minor disease on its native host, the American hazelnut (Corylus americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB limits commercial production of hazelnut to west of the Rocky Mountains. To better understand the pathogen responsible for EFB, an ARS scientist in West Lafayette, Indiana, and collaborators, sequenced, annotated, and characterized its genome. This study identified a large suite of genes likely involved in pathogenicity, including carbohydrate active enzymes, secreted proteins and effectors that the fungus secretes into the host to aid infection and trigger disease. This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of its life cycle and a solid foundation for plant pathologists, geneticists, and hazelnut breeders studying EFB.

Review Publications
Da Silva, L., Tian, H., Schemerhorn, B.J., Xu, J., Cai, G. 2023. Genome-wide informative microsatellite markers and population structure of Fusarium virguliforme from Argentina and the USA. The Journal of Fungi. https://doi.org/10.3390/jof9111109.
Detranaltes, C.E., Ma, J., Cai, G. 2023. Identification of soybean germplasm and associated molecular markers with resistance to Fusarium graminearum. Agronomy. 13. Article 2376. https://doi.org/10.3390/agronomy13092376.
Cohen, A., Cai, G., Price, D.C., Molnar, T., Zhang, N., Hillman, B. 2024. The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut. BMC Genomics. https://doi.org/10.1186/s12864-024-10198-1.
Devereaux, R., Carrero-Colon, M., Hudson, K.A. 2024. Mutations in KASIIB result in increased levels of palmitic acid in soybean seeds. Journal of the American Oil Chemists' Society. https://doi.org/10.1002/aocs.12827.
Escamilla, D., Dietz, N., Bilyeu, K.D., Hudson, K.A., Rainey, K. 2024. Genome-wide association study reveals GmFulb as candidate gene for maturity time and reproductive length in soybeans (Glycine max). PLOS ONE. 19(1). Article e0294123. https://doi.org/10.1371/journal.pone.0294123.
Ranjan, R., Srijan, S., Balekuttira, S., Agarwal, T., Ramey, M., Dobbins, M., Kuhn, R., Wang, X., Hudson, K.A., Li, Y., Varala, K. 2024. Organ-delimited gene regulatory networks provide high accuracy in candidate transcription factor selection across diverse processes. Proceedings of the National Academy of Sciences (PNAS). https://doi.org/10.1073/pnas.2322751121.
Liu, X., Wickland, D., Lin, Z., Liu, Q., Borges Dos Santos, L., Hudson, K.A., Hudson, M.E. 2023. Promoter deletion in the soybean Compact mutant leads to overexpression of a gene with homology to the C20- gibberellin 2-oxidase family. Journal of Experimental Botany. https://doi.org/10.1093/jxb/erad267.