P–1. Unraveling the Initial Signaling Cascades in Soybean Nodulation: Insights from Proteomic and Transcriptomic Analyses
Shin-ichiro Agake, Visiting Scholar, University of Missouri / Tokyo University of Agriculture and Technology, sagake@missouri.edu
Nod-factor receptor complexes, comprising NFR1, NFR5, and SymRK, play pivotal roles as essential kinase proteins in nodulation. Rhizobia employ a type III secretion system (T3SS) to enhance nodulation through the regulation of the Nod-factor triggered pathway. Notably, soybeans display delayed nodulation during the early growth stage when inoculated with T3SS knockout mutants. However, the specific initial signaling pathways governing these processes remain unclear. In this study, we sought to uncover the early signaling cascades using proteomic and transcriptomic analyses. To identify key components involved in the Nod-factor triggered pathway, we used proximal labeling with TurboID fused to the receptor kinases in the hairy root system. Subsequent LC-MS/MS analysis revealed 31 enriched proteins associated with NFR1, 11 proteins with NFR5, and 17 proteins with SymRK. Additionally, we conducted a kinase-client assay utilizing purified SymRK, which resulted in the enrichment of 69 phosphopeptides. These findings present promising candidates that may interact with Nod-factor receptors during the initial plant response. Furthermore, we investigated the T3SS triggered pathway by analyzing RNA-seq data obtained from isolated root hairs inoculated with T3SS mutant. At 12 hours post-inoculation, we identified 471 differentially expressed genes upregulated in response to T3SS, indicating a strong activation of crucial T3SS effectors at this time point. In conclusion, our comprehensive approach using proteomic and transcriptomic analyses has shed light on the early signaling events in nodulation. These findings deepen our understanding of the complex interplay between Nod-factor receptors and the T3SS machinery, providing valuable insights into the regulatory mechanisms underlying soybean-rhizobia symbiosis.
P–2. Uncovering the genomic diversity of the Soybean Cyst Nematode (Heterodera glycines) through pangenome analysis
Lucas Borges dos Santos, Doctoral Student, University of Illinois at Urbana-Champaign
Authors: Santos, L.B., Showmaker K., Walden K.O.K., Masonbrink R., Hernandez A., Severin A., Mitchum M., Hudson M.
The soybean cyst nematode (SCN) poses a significant threat to soybean yield globally. Despite its harmful impact, the mechanisms driving its virulence and adaptability largely remain a mystery. Previous investigations have employed single reference genomes to decode SCN genetic diversity, but this method risks missing or misinterpreting vital genetic variants absent in the reference. Pangenome analysis emerges as a solution, capturing the entire range of genomic diversity within a species, thus circumventing the bias tied to singular reference genomes.In this ongoing research, we aim to shed light on the structure and genomic diversity of SCN populations, which may potentially expose new directions for developing control measures. By leveraging high-quality metagenome assemblies from seven distinct SCN populations, we have gained a deeper understanding of the SCN genomic landscape. Our analysis identified 27,987 ortholog gene clusters, divided into 42% core and 58% non-core gene families. This distinction between core and non-core gene families elucidates the separation between fundamental genes found in all populations and those potentially contributing to adaptability and variation.Our functional annotation of these gene clusters has revealed critical cellular processes that core SCN genes engage in, offering insights into the nematode’s survival strategies and biology. By laying the groundwork for an SCN pangenomic framework, we enable a more comprehensive understanding of the genetic mechanisms behind its pathogenicity. This enhanced understanding is a pivotal step towards the development of effective and targeted nematode control strategies.
P–3. Phenotyping and Yield of Soybean Treated with an Archaeal Antioxidant
Jeremy Brown, Graduate Student, Biochemistry, University of Nebraska–Lincoln, jbrown353@huskers.unl.edu
ArA is an antioxidant from archaeal origin with a redox potential similar to Glutathione (GSH). Previous studies have found that application of antioxidants have the potential to increase growth parameters and ameliorate abiotic stresses in plants. Growth trials with Glycine max (Thorne) at three weeks of growth showed increased biomass vs. untreated controls in a greenhouse setting. To determine if ArA has potential for agricultural applications, we grew G. max to seed at the UNL greenhouse phenotyping facility with and without application of ArA to do phenotyping image analysis and collect yield data. Using the phenotyping facility allows for daily imaging with RGB, NIR, Fluorescence, and hyperspectral cameras for an accurate growth estimation and phenotype while the plants grow. With these images we have extracted information of plant height and approximate leaf area over the course of the plant growth to determine growth over time. Results of one growth trial showed an increase in yield and growth rate with application of 1mM ArA. Control plants between trials showed differences in yield due to seasonal differences. We also found that application of ArA changed light harvesting dynamics and treated plants displayed a smaller degree of photo protection but a faster response. We are currently extracting information from the hyperspectral images and managing a field experiment for application of ArA.
P–4. Soybeans Engineered for Enhanced Vitamin E and Effects on Oil Antioxidant Properties in Polyunsaturated Fatty Acid-Rich Gerplasm
Edgar Cahoon, Professor, University of Nebraska–Lincoln, ecahoon2@unl.edu
Engineered soybean oils enriched in nutritionally valuable omega-3 PUFAs have lower oxidative stability than conventional soybean oil, resulting in increased rancidity and associated off-flavors for food products. Oxidative stability of PUFA-enriched oils is typically enhanced by adding chemical antioxidants following extraction. This study examined biofortification of PUFA-enriched seeds with lipid-soluble vitamin E antioxidants to increase oil stability. We engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene, resulting in an 8- to 10-fold increase in total vitamin E, principally as tocotrienols. With γ-TMT co-expression, δ- and γ-tocochromanols were shifted largely to the more nutritionally valuable α- and β-tocochromanols. To test whether the high vitamin E trait improves oxidative stability of conventional and PUFA-enhanced seed oils, selected high vitamin E lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, oxidative stability index of the oil was ~15% lower than that of oil from non-engineered seeds and only modestly increased in oil from the GLA, ALA and SDA backgrounds. We show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.
P–5. SoyBase.org: Integrate genetics, genomics and breeding data to advance soybean research
Jacqueline Campbell, Faculty, Corn Insects and Crop Genetics Research Unit, USDA-ARS, Jacqueline.Campbell@usda.gov
SoyBase (https://soybase.org) is the USDA-ARS soybean resource hub for the soybean community, by providing a comprehensive collection of data, analysis tools, and links to external resources of interest to soybean researchers. SoyBase partners with other plant data resources (eg. LegumeInfo, PhyloGenes, Phytozome and SoybeanBase) to provide links to data sets not housed at SoyBase. Currently, SoyBase hosts seven annotated genomes from multiple Glycine max and Glycine soja cultivars, including the most recent Williams 82 genome (Wm82_ISU01.a2.v1). The soybean reference genome (Glyma.Wm82) GBrowse has numerous data tracks available to view including genome organization, gene annotation and expression, markers, methylation and sequence variants (SoySNP50K and GmHapMap projects). SoyBase is an actively curated database, with new data regularly being incorporated, including parentage information for Uniform Soybean Tests from both Northern and Southern regions, gene expression, GWAS QTLs, and genome sequences and annotations. The latest set of tools added to SoyBase include the Pan-Genome Sequence Search and the SequenceSever for both nucleotide and protein BLAST. The SequenceSever includes more output options and the ability to download all the hits as FASTA files and alignment of the hit and tabular reports of the hits. SoyBase provides easy access to download data including SoySNP50K marker data, QTL positions, pan-genome datasets, genome, CDS and protein sequences. A major goal of SoyBase is to assist researchers in discovering important trait, genomic, and genetic information within the vast amount of data available.
P–6. *Tabula Glycine: The Glycine max single-cell resolution transcriptome atlas
Sergio Alan Cervantes-Perez, Postdoctoral Research Associate, Department of Agronomy and Horticulture, University of Nebraska–Lincoln, alan.cervantes@unl.edu
Soybean (Glycine max), one of the most important crops in the world, is an essential source of protein and oil with high nutritional value for human and animal consumption. It also has the capability to establish symbiotic interactions with nitrogen-fixing soil bacteria. Synthetic biology offers an opportunity to improve important soybean agronomic traits. However, the development of well-sounded synthetic biology strategies requires a deep understanding of the gene expression and their associated regulatory mechanisms in each cell/cell type composing the plant. Here, we present “Tabula Glycine” the Glycine max single-cell resolution transcriptome atlas. This atlas is composed of ~133,000 nuclei isolated from 11 organs using the single-nuclei RNA-sequencing approach. Our analysis revealed 172 different groups of nuclei clustered based on their transcriptomic profile. Using spatial transcriptomic technology and comparative genomic approaches, we functionally annotated ~80% of the clusters composing the Tabula Glycine. Focusing on the transcriptional patterns of the soybean transcription factor (TFs) genes, we observed that their activity is sufficient to define a cell type, supporting the idea that TFs genes are key regulators of cell identity. Among the soybean epidermal cells, the “root hair cell” cluster is characterized by its unique transcriptome, likely a reflection of its biological specialization and polar elongation. Notably, we found 93 TFs co-expressed in the soybean root hair including orthologs to Arabidopsis TFs controlling root hair cell development and to Medicago TFs controlling the early stages of infection by rhizobia. The Tabula Glycine is a high-resolution functional genomic resource for the soybean community.
*Poster selected for oral presentation.
P–7. * Facilitating gene discovery in soybean through mutagenesis: Identification of novel genes controlling the production of four-seeded pods
Cuong X. Nguyen1, Vikranth K. Chandrasekaran1, Manh V. Nguyen1, Gary Stacey1, Minviluz G. Stacey1
1Division of Plant Sciences, University of Missouri, vkc4kf@missouri.edu
The most important soybean agronomic trait targeted for crop improvement is increased yield, which can be achieved by increasing seed number and/or seed weight. Phenotypic screening of soybean fast neutron mutants identified a mutant line, designated MO27, that produced an increased number of four-seeded pods (4-SP), producing ~30% 4-SPs per plant compared to ~4% in wildtype. Genetic mapping and co-segregation analyses showed that the 4-SP phenotype in MO27 is controlled by the additive effects of at least two alleles located in Chr02 and Chr06. Based on the functional annotations of the deleted genes, we hypothesized that deletions of GmGATA1 on Chr. 2 and GmULT1-3 and GmULT1-5 on chr.6 are the putative causative mutations underlying the 4-SP trait. These genes encode putative transcription factors. Knock-out deletions in these genes via CRISPR/Cas9 confirmed their role in controlling the number of seeds per pod in soybean, likely through the CLAVATA3 (CLV3)-WUSCHEL (WUS) signalling pathway that coordinates stem cell proliferation with differentiation in floral meristems. Knock-out mutations of orthologous genes in Arabidopsis and tomato resulted in increased production of locules in siliques and fruits, respectively, suggesting functional conservation of GmGATA1-1 and GmULT1 in plants.
*Poster selected for oral presentation.
P–8. Phosphorylation-mediated signalling at high temporal resolution in cold-stressed soybean seedlings
Ive De Smet, Group Leader, VIB-UGent Center for Plant Systems Biology, Technologiepark-Zwijnaarde 71 - 9052 Ghent - Belgium, ive.desmet@psb.vib-ugent.be
Low temperature stress limits the overall growth of soybean and leads to yield reduction. Therefore, unravelling the mechanisms associated with low temperature perception, signaling and response can provide valuable information for breeding and improving soybean yield. However, very little is known about this in soybean. To address this knowledge gap, we focused on protein phosphorylation cascades, which are responsible for transducing environmental and cellular signals. To assess how signaling pathways dynamically rewire upon environmental cues we exposed cold-sensitive and tolerant soybean seedlings to control and cold conditions, harvested leaves of five-day-old seedlings every six minutes for one hour, and performed phosphoproteomics. Next, to shed light on phosphorylation cascades in soybean seedlings upon cold treatment, we used functional annotations from our newly developed PF-NET in combination with Bayesian network principles (NetPhorce) to predict kinase and phosphatase-mediated signaling cascades using time series phosphoproteomics. Our novel network inference approach allowed us to predict key temperature response regulators in soybean, that could be checked against a range of field-grown soybean at two temperatures (10 °C and 20 °C). These response regulators are ideally suited as breeding markers or targets.
P–9. Virulence Diversity of Soybean Cyst Nematode in Minnesota
Lauren Docherty, Master’s Student, University of Minnesota, doche014@umn.edu
Soybean cyst nematode (SCN) (Heterodera glycines) is the most damaging pest affecting soybean crops, causing an estimated $1 billion in yield loss annually. The most effective method for managing SCN is the use of resistant soybean cultivars. SCN populations, however, hold the potential to overcome soybean resistance in what is known as a virulence shift. This project examines the virulence diversity of SCN in Minnesota to better understand the potential for virulence shifts. This will be done by testing the virulence of 180 inbred lines of SCN on six indicator soybean lines with diverse resistance genes. Many of these soybean lines are not part of the standard virulence test, thus their effectiveness is unknown and may include highly effective resistance to a broad sample of SCN populations. Results obtained so far indicate that many of the SCN lines can overcome the PI 88788 source of resistance. PI 90763 and PI 438489B displayed the broadest resistance, with very few SCN lines overcoming its resistance. The results also show that there is little overlap in virulence among different soybean lines, suggesting different virulence genes within SCN are required to overcome specific sources of host plant resistance. The information provided by this project will help farmers make management decisions and help soybean breeders develop cultivars with durable SCN resistance. In future research, the data from this project will be combined with genomic data to identify regions of the SCN genome associated with virulence.
P–10. SNP and small INDEL genomic variants underlying adaptive traits in soybean
Gezahegn Girma, Research Scientist, and Yong-qiang Charles, Research Molecular Biologist, Donald Danforth Plant Science Center, GTessema@danforthcenter.org
Significant efforts have been made to discover and validate genes and alleles important to soybean improvement. However, the complex nature of quantitative traits and limitations of discovery tools impede identification of loci associated with soybean adaptation to diverse environments. Our laboratory has been developing integrative data-driven technologies to discover genes and genetic alleles for soybean improvement. As part of the efforts, more than 12,000 accessions with whole genome sequence data have been retrieved from public database. A subset of 1402 accessions, including both Glycine soja and Glycine max, was selected. Variant calling analysis identified 11,101,803 SNPs and small indels. We have implemented both single and multi-locus GWAS models to identify significant loci underlying soybean adaptation based on geographic information. A total of 26 genomic regions representing 12 indels and 14 SNPs were identified for their significant association with soybean adaptation to wider environments. Annotation of the significant genomic variants resulted in nine unique genes. Population differentiation was also measured based on latitudinal information to identify new beneficial alleles that becomes fixed in each population. We will further utilize available tools such as Meta-GWAS r-package and SoyBase to discover novel genes by comparing with previous studies. Understanding the molecular basis underlying soybean adaptation to diverse environments is crucial for designing effective strategies to develop new, climate-resilient soybean varieties.
P–11. Studying iron deficiency chlorosis: using soybean to turn the models right side up
Michelle Graham, Faculty, Corn Insects and Crop Genetics Research Unit, USDA-ARS, Michelle.Graham@usda.gov
Iron deficiency chlorosis (IDC) negatively affects crop quality and yield. Studies from model species demonstrate shoots control or influence of iron uptake in roots. In this study, grafting of near-isogenic soybean lines Clark and IsoClark (iron stress tolerant and susceptible, respectively), demonstrated the Clark rootstock drives phenotypic responses in IsoClark leaves two weeks after iron stress. RNA-seq analyses from homo- and hetero-grafted plants 30 and 120 minutes after iron stress identified 518 and 846 differentially expressed genes (DEGs) in leaves and roots, respectively. Grafts with a Clark rootstock induced genes involved in iron uptake and utilization at 30 minutes in the root and by 120 minutes in the leaves, regardless of the leaf genotype. This suggests a mobile signal, initiated in Clark roots, regulates iron stress responses in soybean leaves. Better understanding of the complex differences between crop and model species will aid in the development of crops with improved IDC tolerance.
P–12. Dissecting function of the gene model that governs the soybean QTL cqSeed protein-003
Ming Guo, Research Assistant Professor, Department of Agronomy and Horticulture, University of Nebraska–Lincoln, mguo2@unl.edu
A major seed protein quantitative trait locus (QTL) in soybean that was mapped to chromosome 20 over 30 years ago, cqSeed protein-003, was fine mapped to gene model Glyma.20G085100 (Gm20P). Gm20P contains a transposon footprint in most of the domesticated soybean genotypes that interrupts the CONSTANS, CONSTANS-like (CO-like), and timing of CAB expression1 (TOC1) (CCT) domain present in the non-domesticated genotypes. The Gm20P paralog gene model resides on chromosome 10, Glyma.10G134400 (Gm10P). Using genome editing reagents, two lines were developed that harbor INDELs in both Gm10P and Gm20P alleles in the soybean genotype Thorne. One edited line carries a 200 bp deletion in the exon2 of Gm20 and small INDEL in the 5’ UTR of GM10P. The second edited line carries INDELs in Gm10P and Gm20P, a 59 base pair insertion in the Gm20P exon2 and a 4 bp deletion in the Gm10P exon2. Both edited lines display delayed maturity relative to control Thorne, with the second edited line displaying a more prominent delayed maturity under field conditions. NIR analysis from the field plots revealed seed protein content is significantly decreased in the Gm10P and Gm20P double mutant, while oil content remains similar among both mutants and wild type Thorne. Wet bench analyses on seed suggested fiber content is significantly reduced in the mutants, while starch content is increased.
P–13. Enhanced Bioactivity of Day-4 Soybean Sprouts: A Potential Functional Food for Inflammation-Associated Diseases
Rajnee Hasan, Graduate Student, Biochemistry, University of Nebraska–Lincoln, rhasan2@huskers.unl.edu
Soybean, a prominent source of proteins and phytochemicals, has sparked interest in germination as a means to enhance health-beneficial effects through increased peptides and phytochemicals. In the context of health-beneficial activities, gastrointestinal digestion plays a crucial role in releasing bioactive compounds from ingested foods that can exert anti-inflammatory effects in the body. This study aimed to investigate how 0, 2, and 4 days of sprouting influence the release of bioactive compounds and anti-inflammatory capacity of soybean in gastrointestinal tract. Major monosaccharides and amino acids significantly accumulated in Day-4 sprouts and those subjected to in vitro gastrointestinal digestion. While the digestibility of soybean sprouts was reduced over 4 days of sprouting, the peptide content was significantly increased in these digests. In vitro anti-inflammatory assays demonstrated that treatment with Day-4 sprout digests significantly reduced the induced inflammatory responses in Caco-2 intestinal cell line, showing their anti-inflammatory capacity. In a mouse model of chronic inflammation, Day-4 sprout supplementation significantly reduced body weight and fasting blood glucose levels in a dose-dependent manner. Moreover, Day-4 sprouts supplementation reduced the expression of IL-6, suggesting its potential therapeutic value in treating inflammation-associated diseases. Di- and tripeptides with hydrophobic amino acids in N-terminal and polar amino acids in C-terminal including IP, ILR, IVR, IVK, LIK, LLR, LYK and MQ accumulated in Day-4 sprout digests, indicating their potential as anti-inflammatory molecules. In conclusion, Day-4 soybean sprouts likely possess a combination of bioactive compounds with anti-inflammatory potential, making them a promising candidate of functional food to prevent inflammation-related disorders.
P–14. *An integrated single-cell comparative transcriptomic and evolutionary analysis of the legume membrane microdomain-associated protein-coding genes during the nodulation process
Md Sabbir Hossain, Doctoral Student, Department of Agronomy and Horticulture, University of Nebraska–Lincoln, mhossain8@huskers.unl.edu Legumes have the unique ability to fix atmospheric dinitrogen through a mutualistic symbiosis with rhizobia. This symbiosis starts with the infection of the legume root hair cell and ultimately leads to the development of a new root organ, the nodule. The rearrangement of the plasma membrane is a pre-requirement for rhizobial infection. For instance, membrane microdomain-associated proteins (MMAPs), including FW2.2-LIKEs (FWLs), flotillins (FLOTs), prohibitins (PHBs), and remorins (REMs), play a crucial role in the initiation and development of the infection threads, a tube-like structure that allow rhizobia to infect the plant cells. In this poster, using state-of-the-art single-cell transcriptomics technology, we evaluate the co-expression pattern of the MMAPs in each cell type composing the Glycine max and Medicago truncatula nodules. As a result, we identified two GmFWLs, one GmFLOT, two GmPHBs, and three GmREMs, and, and two MtFWLs, two MtFLOTs, three MtPHBs, and one MtREM genes preferentially expressed in the infected cells of the soybean and Medicago, respectively. Expanding our analysis to the entire soybean single-cell transcriptomic atlas, we confirmed the specific expression of these soybean MMAPs in the infected cells of the nodule. The phylogenetic analysis of the legume MMAPs revealed that most of the nodule-infected cell-specific MMAPs co-cluster together suggesting their early functional allocation to regulate the nodulation process.
*Poster selected for oral presentation.
P–15. Bolstering virus-induced gene silencing and foreign protein expression in soybean with a cowpea severe mosaic virus-based vector
Feng Qu, Seung Hyun Yang, Fides Angeli Zaulda, Junping Han, Anne Dorrance, Department of Plant Pathology, The Ohio State University, han.393@osu.edu
Soybean gene functions cannot be easily interrogated through transgenic disruption (knock-out) of genes-of-interest, or overexpression of proteins-of-interest, because soybean transformation is time-consuming and technically challenging. An attractive alternative is to administer transient gene silencing or overexpression with a plant virus-based vector. However, existing virus-induced gene silencing (VIGS) and/or overexpression vectors suitable for soybean have various drawbacks that hinder their widespread adoption. We describe the development of a new vector based on cowpea severe mosaic virus (CPSMV). This vector, designated FZ, incorporates a cloning site in the CPSMV RNA2 cDNA, permitting insertion of nonviral sequences. When paired with an optimized RNA1 cDNA (QUIN), FZ readily infects both Nicotiana benthamiana and soybean. As a result, FZ constructs destined for soybean can be first delivered to N. benthamiana to propagate the modified viruses to high titers. FZ-based silencing constructs induced robust silencing of phytoene desaturase genes in N. benthamiana, multiple soybean accessions, and cowpea. Moreover, FZ-mediated expression of the Arabidopsis transcription factor MYB75 caused N. benthamiana to bear brown leaves and purple, twisted flowers, indicating that MYB75 retained the function of activating anthocyanin synthesis pathways in a different plant. The new CPSMV-derived FZ vector provides a convenient and versatile soybean functional genomics tool that is expected to accelerate the characterization of soybean genes controlling crucial productivity traits.
P–16. Genetic Diversity in the North Dakota State University Soybean Breeding Program
Forrest Hanson, Master’s Student, North Dakota State University, forrest.hanson@ndsu.edu
Soybean is a relatively new crop in North Dakota; however, it has become the number one crop in the state for acres planted and production value. Public breeding efforts began in 1986 through North Dakota State University, and during this time, 40 varieties in maturity groups 00 and 0 have been released. Although yields have increased during this time, the yield gains and genetic diversity of the program have not been studied. It is important to understand these components of a breeding program to ensure yields do not become stagnant. We would like to determine the amount of genetic diversity in this program to improve yields further. This knowledge will allow our program and other breeders in the maturity group 00 and 0 environment to continue to improve yields.
P–17. Single-trait and multiple-trait QTL analyses for seed oil and protein contents of soybean populations with elite background
Tu Huynh, Doctoral Student, The Ohio State University, huynh.177@osu.edu
Soybean seed oil and protein contents are negatively correlated, posing a challenge in breeding efforts to enhance both traits. Previous studies have identified hundreds of oil and protein QTLs mainly via single-trait QTL analyses. Multiple-trait QTL methods for correlated traits have been shown to improve detection power and mapping precision compared to single-trait methods but have not been applied to seed oil and protein contents. Our study conducted both single- and multiple-trait multiple interval mapping (ST-MIM and MT-MIM, respectively) for oil and protein contents using three recombinant inbred line populations with advanced breeding line background tested in four environments. We detected seven ST-MIM QTLs on chromosomes 1, 8, 6, 15, 19, and two on 20, five of which were confirmed by MT-MIM. Our findings show that, unlike multiple-trait QTL analyses for other traits and crops, MT-MIM did not outperform the single-trait approach for our traits of interest. All loci exerted opposite effects on oil and protein contents, but the protein-to-oil additive effect ratio varied (-0.6 to -48.8). We calculated the allelic effects on estimated processing values (EPV) using the National Oilseed Processors Association (NOPA) and High Yield + Quality (HY+Q) methods. Oil-increasing alleles of QTLs on chromosomes 6, 15, 19, and 20 increased both EPVNOPA and EPVHY+Q, while oil-increasing alleles of QTLs on chromosomes 1 and 8 increased EPVNOPA and decreased EPVHY+Q, which penalizes low protein meal. With the populations’ elite pedigree, selected lines can be used to determine the allelic effects on yield and directly integrated into breeding programs.
P–18. *Development of EPA- and Astaxanthin-Enriched Soybean Germplasm for Aquaculture Feedstocks
Hyojin Kim, Postdoctoral Researcher, Center for Plant Science Innovation, University of Nebraska–Lincoln, hkim20@unl.edu
The omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and carotenoids such as astaxanthin are recognized for their health-promoting qualities. Marine fish and fish oil currently provide the main sources of EPA/DHA and astaxanthin for human consumption, but require this lipophilic compounds in their feed for aquaculture production. To provide a land-based source of these high-value feed components, we introduced the EPA and astaxanthin biosynthetic pathways in soybean by gene stacking. Our first version of aqua-soybean stacking EPA and astaxanthin biosynthetic genes showed poor seed quality such as reduced seed oil (<20% of seed weight), abnormal seed shape, low germination rate, decreased ABA level, and less EPA level (<2% of total fatty acids). From the design–build–test–learn (DBTL) cycle, the first version of aqua-soybean was crossed with high-alpha-linolenic acid soybean to improve EPA level. These crosses showed the enhanced seed quality such as high EPA level (~10% of total fatty acids) and normal seed shape, and the unexpected results such as improved germination rate. We generated only EPA soybean line or astaxanthin soybean line, separately. Our EPA soybean line accumulated up to 15% EPA of total fatty acids in seed with normal seed quality and germination rate. In addition, we developed astaxanthin-producing lines by introduction of biosynthetic genes (AaCBFD2 and AaHBFD1) with/without phytoene synthase gene (ZmPSY). Overall, our work represents a step toward viable soybean-based sources of astaxanthin-enriched fish oil for aquaculture production.
*Poster selected for oral presentation.
P–19. Characterization of soybean events with enhanced expression of the microtubule-associated protein 65-1
Panya Kim, Postdoctoral Research Associate, Center for Plant Science Innovation, University of Nebraska–Lincoln, kim3@unl.edu
Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 (AtMAP65-1) is a known virulence target of plant bacterial pathogens and thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both AtMAP65-1 and GmMAP65-1, the soybean AtMAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both AtMAP65-1 and GmMAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the Oomycete pathogen Phytophthora sojae, but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing AtMAP65-1 and GmMAP65-1 also display a tolerance to the herbicidal oryzalin, that has a mode of action that destabilizes microtubules. In addition, GmMAP65-1 expressing soybean plants show reduced cytosol leakage under freezing conditions hinting that ectopic expression of GmMAP65-1 may enhance cold tolerance in soybean.
P–20. Deciphering protein rebalancing for desired seed composition traits in soybean
Ritesh Kumar, Postdoctoral Research Scientist, Department of Agronomy and Plant Genetics, University of Minnesota, Kumar797@umn.edu
Seed protein content is the critical factor determining soybean’s nutritional and market value. Soybean varieties in the U.S. have around 36−38% protein. A higher percentage of the total soybean protein is constituted by the two main storage proteins, beta-conglycinin and glycinin. Based on their sedimentation coefficients, they are mainly named 7s (ß-conglycinin) and 11s (Glycinin) globulins. The 7S globulins are comprised of three subunits, alpha prime (α’), alpha (α), and beta (β). Meanwhile, five genes, gy1, gy2, gy3, gy4, and gy5, encode the 11s globulins. A slight increase in protein content significantly augments soybean crop value. The protein content regulation and subsequent protein composition are believed to be a multilevel process. To understand proteome plasticity at the molecular level, we need to identify and utilize seed storage machinery in a new fashion. In the current study, we used time-efficient and modern genome editing tools to create several storage protein family mutants in a single genotype and used multiple omics approaches to identify the common machinery involved in protein rebalancing. The common key regulators among different mutants will be tested to optimize the protein rebalancing and soybean seed composition traits.
P–21. Breeding for Resistance to Soybean Seedling Diseases
Feng Lin, Academic Specialist-Research, Michigan State University, fenglin@msu.edu
Soybean seedling disease is one of the most destructive diseases, causing 25-64 million bushels yield loss in the U.S. Seedling diseases is caused by multiple oomycetes and fungi, including Phytophthora spp, Pythium spp, Fusarium spp., Rhizoctonia spp., Phomopsis spp., etc. Pythium irregulare and Pythium sylvaticum are the most prevalent and aggressive Pythium species in soybean (Rojas et al 2017), causing seed rot, seedling damping off, and root rot. Partial resistance is the mostly known type of resistance to Pythium. Remarkably, pleiotropic QTLs have been identified (Scott et al. 2019, Clevinger et al. 2021). Phytophthora sansomeana E.M. Hansen & Reeser was differentiated from P. megasperma species complex, where P. sojae was once part of. P. sansomeana infects a wide range of plant species such as soybean, corn, clover, wild carrot, Douglas fir, and some weed species, causing sever symptoms of seed and root rot (Hansen et al. 2009; Hansen et al. 2012; Rojas et al. 2017). Unlike the resistance to P. sojae, little is known about the host resistance to P. sansomeana. In this poster, we are presenting our updates identifying resistance sources and mapping of quantitative and qualitative resistance genes/QTLs.
P–22. Temporal transcriptome profiling uncovers gene regulatory networks driving the progression of embryonic and post-germinative development
Jer-Young Lin, Assistant Research Fellow, Agricultural Biotechnology Research Center, Academica Sinica, jeryoung@gate.sinica.edu.tw
Morphogenesis occurs during both embryonic and post-germinative development in higher plants. These two morphogenesis events are connected by a period of developmental arrest, characterized by cessation of cell differentiation, and, simultaneously, a series of biological processes, including maturation, desiccation, dormancy, and germination. However, what genes are sequentially expressed, and how are genes coordinately regulated to progress the transitions of developmental programs between the two morphogenesis events in soybean seeds are relatively unknown. To address this, we analyzed the transcriptomes from 12 soybean seed stages bridging the two morphogenesis events. Analysis of this considerable time course enabled the identification of consecutively active gene sets and gene regulatory networks promoting the transitions of developmental processes. For instance, a predicted gene regulatory network is preferentially provoked in early desiccation and contains many transcription factors involved in the response to abiotic stress. The function of one transcription factor highly active in this desiccation-related gene network was examined to validate the predictive regulatory network. This study illustrates the genetic basis underlying the transitions of developmental programs across embryogeny and post-germination in soybean seeds.
P–23. Maintaining and Fine-Tuning a Gene Editing Pipeline for Soybean Trait Improvement
Junqi Liu, Postdoctoral Research Scientist, Department of Agronomy and Plant Genetics, University of Minnesota, liuxx162@tc.umn.edu
Gene editing technologies have proved to be a powerful tool for crop improvement in recent years. In particular, CRISPR/Cas9 mediated gene editing technology has evolved very rapidly. A wide variety of tool kits have been developed and made readily available to researchers in academia. Despite the abundant technological progress, targeting multiple genes simultaneously and/or stacking desired mutations are still a challenging task in the process of improving soybean germplasm. We have utilized a modular vector system for cloning multiple gRNAs in the same vector and integrated it into our whole plant transformation (WPT) pipeline. The virulence of the Agrobacterium strain and the authenticity of modular vectors are periodically assessed and monitored to maintain the efficiency of the soybean mutagenesis pipeline. So far we have successfully mutated several target genes related to important agronomic traits and stacked multiple mutations in individual soybean lines.
P–24. Identification and Genetic Mapping of Quantitative Trait Loci Controlling Seed Protein Content Stability in Soybean
Drew Mitchell, Doctoral Student, Michigan State University, mitch987@msu.edu
Soybean seed protein content is a complex physiological trait regulated by many small-effect genes that show significant environmental interaction. Protein content stability across diverse growth environments is therefore often low in soybean. The identification of genomic regions associated with protein content stability may contribute to the development of stable high-protein cultivars, as well as to the further elucidation of mechanisms underlying physiological stability in soybean. In this work, 218 recombinant inbred lines (RILs) were derived from the intraspecific cross of the high protein accession PI-555396 (BARC-6) and the low protein MSU breeding accession E14077 for the investigation of quantitative trait loci (QTL) associated with protein content and stability across multiple years and test locations. Indices for absolute and relative protein content stability were used to estimate phenotypic variation across soybean production regions representing 4 unique year/location environments. Composite interval mapping returned one QTL associated with protein content on chromosome Gm20 explaining approximately 17.8% of phenotypic variation, and one QTL associated with both relative and absolute protein stability on chromosome Gm18, explaining approximately 6.5-7.3% of phenotypic variation, respectively. These identified QTLs are being used to inform continued genomic investigation for the prospect of developing highly stable, high-protein content soybean cultivars.
P–25. Cryptic isoprene synthase and its regulation under wounding and burning conditions in soybean
Mohammad Golam Mostofa, Postdoctoral Researcher, Michigan State University
Isoprene emission from some plants constitutes the major hydrocarbon flux from the biosphere to the atmosphere. Isoprene contributes to the formation of ozone and aerosols. However, plants can also benefit from the signaling roles of isoprene, particularly under stressful conditions. In plants, isoprene is synthesized from MEP pathway metabolite dimethylallyl diphosphate (DMADP) by isoprene synthase (ISPS). It was thought that ISPS had been lost from soybean as a result of domestication pressure. In silico analysis revealed the presence of ISPS in soybean having high sequence similarity with the ISPS of known legume isoprene emitters. The phenylalanine (F), serine (S), and asparagine (N) residues, which are unique to ISPS, are conserved in soybean ISPS. We report that soybeans can make isoprene, but only transiently, when the same or nearby leaves are wounded or burned. Meanwhile, photosynthetic rate, stomatal conductance, and internal CO2 declined while isoprene was emitted from the undamaged part of the leaves. Metabolomics data showed that there is a surge in pyruvate and increases of most of the MEP pathway metabolites. ISPS of soybean, like kudzu, exhibited very strong cooperativity so that it is possible to have almost no isoprene emission at levels of DMADP sufficient for other required isoprenoid synthesis but significant isoprene emission at moderate to high DMADP. Jasmonic acid (JA) and JA-related metabolites were significantly upregulated in wounded leaves, suggesting a possible crosstalk between isoprene and the JA-signaling pathway. We conclude that soybean can make isoprene under certain stresses, when metabolic flux through the CBC to MEP pathway increases, highlighting the cryptic nature of GmISPS. Soybeans retained ISPS; future climate-related environmental pressure may result in isoprene emission from soybean, which may improve soybean resilience to various stresses.
P–26. Determining genetic mechanisms of maturity in North Dakota: expanding the molecular model for MG 00 and 0
Clara Mvuta, Graduate Student, Department of Plant Sciences, North Dakota State University, clara.mvuta@ndsu.edu
Soybean production has expanded significantly in North Dakota(ND), yet the state’s yield averages remain among the lowest in the Midwest. Maturity is a crucial agronomic trait that affects yield potential, and since ND experiences a short growing season due to frost, early maturing soybean varieties are necessary. The dominant maturity groups in the state are MG 00 and 0, and it is possible to “fine-tune” maturity to the region/environment in order to maximize yield potential. The genetic mechanisms behind soybean maturity, particularly the E1, E2, and E3 genes, have been well characterized. Functional alleles of these genes result in late maturity, while null/semi-functional alleles lead to early maturity. Variations of the non-functional/semi-functional alleles of these genes create the MG 00 or 0 phenotype. However, it remains unclear which combination of alleles is most favorable for breeding purposes, potentially impacting yield. Additionally, it is hypothesized that other genes play a significant role in this specific environment, even if they have a minor effect in other maturity groups with functional E genes. The aim of this research is to identify the major effect maturity alleles present in MG 00 and 0 germplasm in ND. This will be accomplished through the examination of known maturity gene alleles in the breeding program at NDSU to determine the favored alleles.
P–27. Evaluation of Rps1c candidate gene in soybean against Phytophthora sojae using cowpea severe mosaic virus-induced gene silencing
Nghi Nguyen, Doctoral Student, The Ohio State University, nguyen.1759@osu.edu
Breeding soybean lines for resistance is the most effective strategy against the oomycete pathogen Phytophthora sojae which causes yield-limiting soybean root and stem rot disease. In a previous study using the resistance gene enrichment sequencing (RenSeq) method, we discovered several novel Rps1c candidate-resistant gene sequences. We investigated the function of one of these genes using a newly developed plus-strand RNA cowpea severe mosaic virus (CPSMV) vector to silence these genes in resistant soybean backgrounds. Hypocotyl inoculation of soybean line Williams 79 (contains Rps1c) with P. sojae race 1 after virus silencing resulted in an estimated 10% increase in lesion length expansion in the resistant backgrounds compared to the non-silence plants. This indicated that this candidate gene played a significant role in the resistant mechanisms of the soybean against P. sojae. Moreover, our study demonstrated that CPSMV is an effective and versatile tool to study gene function in soybeans.
P–28. Turning complex genomic data into action for trait development
Jason Nichols, Principal Scientist, Syngenta, jason.nichols@syngenta.com
The cost of DNA and RNA sequencing has decreased substantially in recent years, and as a consequence for any trait of interest the sequencing of relevant genomes and the generation of functional genomic datasets describing the target trait has become much more accessible and commonplace. However, translating that wealth of genomic data into something actionable - i.e., a prioritized set of candidate loci that can be validated and advanced through biotechnology or breeding methods - remains a major hurdle for trait, especially complex trait, development. The output of such efforts should not simply be to hyper-describe a system, but it must predict that variation which is most likely to affect the desired trait and, ideally, in which direction must a locus be ‘pushed’ to affect that trait. Here we discuss advancements in tools and approaches that combine high precision genetic and genomic annotations with estimations of variant functional effects as a means to direct and prioritize hypothesis testing against target traits. Finally, we discuss some outstanding challenges, as well as opportunities, including combinatorial predictions and enabling selection of promising loci with genetic context in mind.
P–29. Investigating novel QTL to improve iron deficiency tolerance
Jamie O’Rourke, Faculty, Corn Insects and Crop Genetics Research Unit, USDA-ARS, Jamie.Orourke@usda.gov
Early quantitative trait loci (QTL) mapping studies identified major regions of the genome controlling traits of interest in agronomically important lines. Continued crop improvement requires the identification and utilization of alternative genomic regions, likely in novel germplasm. Fiskeby III is a soybean line with high tolerance to abiotic stresses, including iron deficiency, a perennial problem in the upper Midwest of the United States. A previous study identified a novel iron deficiency tolerance QTL on chromosome Gm05 in Fiskeby III. Using virus induced gene silencing (VIGS), we t argeted candidate genes in the Williams 82 genome sequence associated with the QTL. A single gene resulted in phenotypic changes under iron deficient and sufficient conditions. Using RNAseq we have examined gene expression patterns which have revealed Fiskeby III induces transcriptional reprogramming within 24 hours of iron stress, similar to other tolerant soybean varieties. While Fiskeby III induces all the canonical soybean iron deficiency responses, the individual genes and timing of these responses differs from other iron deficiency tolerant lines. Identifying the genes and understanding the pathways and timing utilized by Fiskeby III provides novel targets for improving abiotic stress tolerance in elite soybean lines.
P–30. Metabolomic Study of Aquatic-Feed Transgenic Soybean to Enhance Sustainability and Nutritional Value for Aquaculture Feedstock
Duyen Pham1, Research Assistant, Umesh Yadav1, Cintia L. Arias1, Leah McHale2, Edgar B. Cahoon4, Tom E. Clemente4, Truyen Quach3, Hyojin Kim4, Kiyoul Park4, Hae Jin Kim4, and Ana P. Alonso1
1BioDiscovery Institute, University of North Texas, Denton, TX, USA. 2Department of Horticulture and Crop Sciences, The Ohio State University, Columbus, OH, USA. 3Department of Agronomy and Horticulture. University of Nebraska–Lincoln, NE, USA. 4Center for Plant Science Innovation, University of Nebraska–Lincoln, Lincoln, NE, USA.
An increased consumption of fish products results in higher demands for fish feedstock. However, the current feedstocks are wild-caught fisheries that are not sustainable. Soybean has emerged as a promising alternative to replace fish meals in aquaculture. The objective of this study is to develop new soybean cultivars synthesizing novel compounds that best fit for aquaculture. Nine-gene stack assembly was introduced to enhance the production of omega-3 fatty acids, tocotrienols and astaxanthin in soybean seeds, normally provided to the aquacultures by the fish meal or additional supplements. Unfortunately, these transgenic events showed a non-desired concomitant reduction of the total oil content. For further optimizations aiming to reach the desired levels of novel compounds without altering soybean general agronomic performance, a better understanding of carbon partitioning in these embryos is required. Therefore, we dissected soybean embryos at five developmental stages and determined their chlorophyll and carotenoid content, biomass composition, and metabolome. The most significant differences in the transgenic events were: i) the successful production of new compounds, such as omega-3 fatty acids and carotenoids at the detriment of total oil content; ii) the transient accumulation of starch was reduced by approximately 50%; iii) the content of chlorophyll a and b were drastically reduced; and iv) a significant increase in amino acid production. Identifying metabolic bottlenecks in carbon distribution in developing embryos will guide future engineering efforts to tailor soybean lines more valuable for aquafeeds.
P–31. Genome editing of Glycinin and β-conglycinin family members in soybean (Glycine max Merr. (L.)
Truyen Quach, Research Assistant Professor, Center for Plant Science Innovation, University of Nebraska–Lincoln, tquach2@unl.edu
The glycinin and β-conglycinin seed storage proteins collectively account for approximately 55% of soybean seed protein content. Genome editing reagents were designed to specifically create null mutations in gene models Glyma.13G123500 and Glyma.10G246300, annotated as glycinin and ß-conglycinin family members, respectively. The goal being to investigate the phenotypic outcomes in seed storage reserves, and impact on expression of stacked transgenic alleles in the double null mutant background. Homozygous edited lines carrying INDELs that manifest predicted premature stop codons in each of the gene models have been developed. Imaging of total protein on SDA-PAGE gels revealed that the predicted translational products of the two gene models are devoid in the derived dual homozygous edited lines. The edited homozygous lines are rebalanced in protein content, with no observed changes in total oil or amino acid profile. A small-scale field study showed that the plot weights were reduced in the edited lines, with no change in 100 seed weight, relative to control plots. To assess if expression levels of transgenic alleles are impacted when stacked with the edited alleles, transgenic alleles designed to increase carbon flux to lipids, and for the accumulation of leghemoglobin were crossed into the edited lines for down-stream characterizations.
P–32. *A Genomic Selection Pipeline for Public Soybean Breeding Programs
Vishnu Ramasubramanian, Postdoctoral Associate, University of Minnesota, vramasub@umn.edu
Genomic selection has become an important part of plant and animal breeding programs to accelerate genetic gain. We’ve implemented a GS pipeline designed for soybean public breeding programs in the US Midwest using open source tools that are currently available. Herein, we describe the steps and tools in the pipeline and discuss results for the Northern Uniform Soy Trial Population. Soybeanbase, an instance of breedbase hosted by BreedingInsight is used for the storage of both genotypic and phenotypic data. Filtering of markers and lines are done in the R environment using rTASSEL and custom R scripts. Imputation using LD – K-nearest neighbors imputation (LDKNNi) implemented in rTASSEL often showed the highest imputation accuracy in our test data. An optimized training subset selected using the STPGA package demonstrated higher accuracies in 5-fold cross-validations compared to a randomly selected subset for certain combinations of candidate and training set sizes. In this version, we’ve implemented genomic prediction models using the rrBLUP, BWGR, BGLR and SOMMER packages for single trait and multiple traits across single or multiple environments. We’ve also implemented models that include GxE interactions and environmental covariates using the SOMMER and envRtype packages. Our preliminary results indicate that modeling GxE interactions significantly improved prediction accuracies for a subset of the NUST population and including environmental covariates improved prediction accuracies only for some conditions. In the future, we plan to refine these methods and optimize the GS pipeline.
*Poster selected for oral presentation.
P–33. Breeding Soybean for Intercropping with Pennycress: Genetic Variation of Target Traits
Lucas Roberts, Graduate Student, Department of Agronomy and Plant Genetics, University of Minnesota, robe2110@umn.edu
Pennycress, (Thlaspi arvense), is a newly domesticated winter oilseed adapted to the Midwest US. Due to its winter annual life history, pennycress is planted in the fall between rotations of maize and soybeans. In this intercropping system, pennycress provides the benefits of a cover crop while producing valuable oilseeds. The shorter growing season in Minnesota necessities that the following summer row crop overlap in time and space with pennycress 4 – 8 weeks. Soybean, (Glycine max) fits into this cropping system due to its high plasticity. Competition for resources during this overlap period is high and there is a need to develop adapted soybean varieties to ensure the adoption of pennycress into the cropping landscape. Our objectives include quantifying genotype-by-cropping system interactions and characterizing soybean traits relevant to winter oilseed intercropping. Towards identifying the genetic variation in soybean responses to intercropping, 40 soybean genotypes were planted into the pennycress variety MN-106 in 2020, 2021, and 2022. Field experiments in western and southern Minnesota utilized a split block design with two cropping treatments – soybeans intercropped into pennycress and monocropped soybeans. Intercropping with pennycress only reduced soybean yield in 2 of the 5 environments. Additionally, there were significant genotype-by-treatment interactions.
P–34. Enhancing plant water use efficiency under overexpression of photosystem II subunit S
Seema Sahay, Postdoctoral Research Associate, Biochemistry, University of Nebraska–Lincoln, ssahay2@unl.edu
Limited water availability is the most constraint on crop production. Improving water use efficiency (WUE), which means reducing the amount of water required for per unit carbon gain has been a key target for crop improvement. The photosystem II subunit S of protein (PsbS) is a pigment binding protein and ubiquitous in all vascular plants and plays a role in the non-photochemical quenching (NPQ), a photoprotective mechanism of chlorophyll fluorescence. Here, we hypothesized that PsbS has a potential vitality in the plants’ water use efficiency via modify the signal for stomata opening and heat dissipation in light together with drought. We generated transgenic tobacco with increased levels of PsbS protein than wildtype. Tobacco plants accumulated more PsbS protein in drought conditions than control condition and showed higher NPQ, water use efficiency, lower chloroplastic quinone A (QA) state, and less stomata opening which resulted into increased carbon gain at ~20-30 less water consumption relative to wildtypes. We are currently investigating the same proof-of-concept in soybean, since PsbS is universally present in all plants, and we are hypothesizing that manipulating PsbS expression should be effective in other crops.
P–35. Identification and evaluation of soybean lines conferring major resistance to Phytophthora sansomeana
Muhammad Salman, Graduate Student, Michigan State University, salmanm3@msu.edu
Several soybean (Glycine max) diseases caused a loss of $95.48 billion over the last two decades in the USA. P. sansomeana, an oomycete closely related to P. sojae, has been reported as causing extensive root rot and in some cases more severe than the latter. P. sansomeana has a wide host range including corn, plum, and Christmas trees. Very little is known about the genetic sources of resistance. In this study, 135 improved soybean lines from MSU and 470 germplasm lines were screened with P. sansomeana isolate MPS17_22 using the hypocotyl inoculation method. Surprisingly, none of the MSU improved lines showed complete resistance. Nevertheless, screening of the 470 germplasm lines identified 16 resistant lines, 39 intermediate resistant lines, and 415 susceptible lines. Replicated tests confirmed stable resistance from seven lines, including T-286, Colfax, L77-1727, Bergerac, Serda 231A, NE1900, and NE2701. Challenging these lines with 15 more P. sansomeana isolates revealed different interaction patterns and found that L17-1727 conferred resistance to all the isolates. To validate the resistance in the field conditions, two resistant lines, Colfax and NE2701 were field inoculated with MPS17-22 at the MSU plant pathology farm. Resistant lines protected 60–80% of yield, while the susceptible controls lost 87–93% of yield. These resistant germplasm lines can be used for breeding.
P–36. Effect of QDRL-18 for quantitative resistance to Phytophthora sojae on seed nutrient composition
Maria Santiago Padua, Master’s Student, and Christian Vargas-Garcia, Stephanie Karhoff, Sungwoo Lee, Anne Dorrance, Rouf Mian, Leah McHale The Ohio State University, santiago-Padua.1@osu.edu
Quantitative disease resistance (QDR) to Phytophthora sojae, causal agent of phytophthora root and stem rot (PRSR), is critical to maintaining yield in soybean fields with diverse populations of this pathogen. The molecular mechanisms of QDR are largely unknown and likely variable, with potential for pleiotropy impacting traits such as yield or seed composition. We used a previously identified major QDR locus in chromosome 18 (QDRL-18) to investigate pleiotropy in both disease conducive and non-conducive conditions. QDRL-18 was identified in recombinant inbred line (RIL) populations derived from crosses between OX-20-8 (S) and PI 427105B (R) or PI 427106 (R). Three sets of near-isogenic lines (NILs) varying only for the QDRL-18 allele were derived from the populations. Trials using NILs were previously conducted in three environments conducive and four environments not conducive to PRSR. Previous studies showed that the QDRL-18 resistance alleles enhanced yield by 13 to 29% under PRSR conducive field conditions, with no significant impact on yield in the absence of P. sojae pressure. However, beyond yield stability, nutritional content of soybean seed is critical for the food and feed industries. In the present work, harvested seeds were analyzed by near-infrared reflectance spectroscopy and seed nutritional composition evaluated by ANOVA. Among the variation in nutrient content observed, QDRL-18 allele affected protein content of lines grown under disease conditions. These findings suggest that QDRL-18 may directly impact seed nutrient composition through a pleiotropic response, or indirectly through biotic stress from P. sojae. Further research will confirm and extend these findings.
P–37. Mapping and characterizing oval leaflet shape in soybean associated with low number of seeds per pod
Alina Smolskaya, Graduate Student, University.of Minnesota, smols001@umn.edu
An interesting relationship has been found between leaflet shape and number of seeds per pod (NSPP) in soybean. Leaf shape in soybean is typically characterized as ovate, oval, and lanceolate (narrow). Lanceolate leaflet soybean lines have a high NSPP with small seeds, while oval leaflet lines produce low NSPP. Decoupling seed size from NSPP by improving our understanding of genetic architecture underlying these traits is promising for enhancing future yields. In this study, we are fine-mapping and characterizing loci causing oval leaflets and low NSPP and comparing them to the previously mapped lanceolate locus casual for high NSPP and narrow leaflets. Fine mapping for the oval trait is being done by genotyping plants with KASP markers in a segregating backcross population. Leaflet shapes are being differentiated using the MuLES image analysis macro in ImageJ. NILs with oval and lanceolate leaves sharing a common recurrent parent will be grown in a multi-location yield trial to test for differences in yield between lines with low and high NSPP. The goal of this study is to better understand the genes underlying leaflet shape and NSPP traits in soybean for enhancing future soybean yields.
P–38. Shoot architecture traits are important determinants of canopy coverage and light Interception in soybeans
Suma Sreekanta, Post-doctoral Research Associate, Allison Haaning, Austin Dobbels, Riley O’Neill, Anna Hofstad, Kamaleep Virdi, Fumiaki Katagiri, Robert M. Stupar, Gary J. Muehlbauer and Aaron J. Lorenz. University of Minnesota, sreek002@umn.edu
Shoot Architecture (SA) is a result of complex interplay between many traits. In crops such as maize and wheat, altering SA is associated with enhanced yield. However, study of SA has been limited to a few traits because measuring SA traits has traditionally been a slow, low throughput process. Our study aims to characterize the genetic variation in diverse soybean accessions and to understand variation in SA traits that may have accompanied soybean breeding for yield in the past several decades. We use a combination of high-throughput technologies including an unmanned aircraft system as well as inexpensive smartphone images to parameterize SA in terms of multiple individual leaf, branch and whole plant traits of field grown plants. Our studies show heritable variation for many of the SA traits and that the traits defining the distal portion of the plant encompassing the top four nodes significantly affect light interception and canopy coverage. We have identified QTLs for SA traits impacting canopy coverage and that a major QTL for branch angle coincides with a QTL identified for canopy coverage. Our current work is examining the SA traits in a panel of important MG I public and private variety releases ranging from 1944 to 2017. Varieties were selected based on pedigree and organization diversity, importance in production, and uniform distribution through the years within each decade. The results of this study will be useful in identifying SA traits that enhance the yield potential of soybean by optimizing canopy coverage and light interception.
P–39. Assembly and utilization of a single haplotype reference genome for soybean
Robert Stupar, Professor, University of Minnesota, stup0004@umn.edu
This report details the assembly of a new reference genome of the soybean genotype Williams 82 and its use in comparative genomics. The genome was derived from sampling a single plant of Williams 82, known as the sub-line ‘Williams 82-ISU-01’ (Wm82-ISU). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The Wm82-ISU genome adds 59.5 Mb of sequence and reduces contig number from 9,202 to 36 total contigs. The new annotation includes a significant amount of full-length cDNA sequencing which has reduced the gene count from 52,872 to 48,387. Williams 82 was derived from backcrossing genotype Kingwa into the background of Williams, leading to heterogeneous introgressed segments that persists in modern Williams 82 sub-lines. The Wm82-ISU assembly shows clean Kingwa introgression segments, reflecting its derivation from a single sub-line DNA source. In addition to Wm82-ISU assembly, we also assembled the genome of soybean line ‘Fiskeby III,’ a rich resource for abiotic stress resistance genes. We used these assemblies to study the genomic variation between ‘Fiskeby III’ and the Wm82-ISU reference within a fine-mapped QTL for iron deficiency chlorosis resistance, revealing candidate sequence polymorphisms that may be underlying the QTL variation.
P–40. Let the bees do the work: using biotechnology to convert soybean from a self-fertilizing to an outcrossing plant
Nicole Szeluga, Doctoral Candidate, Cornell University, nms244@cornell.edu
Developing a mass hybrid seed production system in soybean [Glycine Max (L.) Merr.] has been a consistent goal for decades since the mainstream utilization of hybrid vigor for crop improvement. However, the small size of soybean flowers and their predisposition towards self-fertilization results in a low percentage of outcrossing and hinders the large-scale production of hybrid seeds. The implementation of rescuable male sterility overcomes the barrier of self-fertilization but is not sufficient to amplify the production of hybrid seeds without the recruitment of insect vectors to facilitate outcrossing. Can soybean flowers be phenotypically altered to increase pollinator visitation and outcrossing? Combined with the barnase/barstar sterility rescue system, this study aims to use biotechnology to alter floral phenotype and transform soybean from a self-fertilizing to an outcrossing plant.
P–41. *Nuclear retention of transcripts as regulatory mechanism of protein translation in soybean root and nodule cells
Sutton Tennant, Graduate Student Researcher, Department of Agronomy and Horticulture, University of Nebraska–Lincoln, sutennant@huskers.unl.edu
The central dogma of molecular biology follows a simple path, DNA is transcribed into transcripts in the nucleus, and transcripts are then translated into proteins in the cytosol. However, many studies reported that protein production is not solely impacted by the level of expression of genes, but by many other regulatory processes. The number of studies exploring these post-transcriptional regulatory processes in plants is sparse. Here, combining the use of single-nucleus transcriptomic and high-resolution fluorescent in situ hybridization technologies, we provide a new perspective on the role of the nuclear retention of transcripts as a central mechanism to control RNA biology and the biology of plant cells. The analysis of confocal microscopic images of transcripts at the sub-cellular resolution combined with the use of a specifically designed Image J software package, clearly revealed the differential nuclear retention of transcripts between genes, cell types, and organs of the soybean root and nodule. This work reveals the influence of the sub-compartmentalization of transcripts as another regulatory mechanisms of protein translation and a new understanding of the central dogma of molecular biology.
*Poster selected for oral presentation.
P–42. Genomic prediction for sudden death syndrome (SDS) in soybean
Raju Thada Magar, Doctoral Student, Michigan State University, thadaraj@msu.edu
Sudden death syndrome caused by Fusarium virguliforme, is a major soybean disease prevalent in Northern United States. Genetic resistance is the most eco-friendly and economical approach to control this disease. Genomic selection (GS) has been successfully used to achieve genetic gain for various traits in soybean including disease resistance. GS utilizes all available molecular and phenotypic markers train models to calculate genomic estimated breeding value (GEBV), then trained models are used to predict GEBV of new individuals based on their genotypic information. In this study, the ridge regression best linear unbiased prediction (rrBLUP) model is employed to estimate the genomic estimated breeding value of 367 AYT(Advance Yield Trial) soybean lines for disease index, disease severity, and disease indices. With 10-fold cross-validation, the prediction accuracy is found to be 0.61 for disease incidence, 0.76 for disease severity, and 0.56 for disease indices. These findings demonstrate that genomic prediction is a viable option for facilitating selection against sudden death syndrome.
P–43. Ureide partitioning affects drought stress response in soybean
Sandi Win Thu, Doctoral Student, Washington State University, sandiwin.thu@wsu.edu
Legumes are able to access atmospheric di-nitrogen (N2) through a symbiotic relationship with bacteria residing in root nodules. In soybean [Glycine max (L.) Merr.], ureides are the products of N2 fixation and represent the primary long-distance nitrogen (N) transport compounds. Ureide transport is mediated by membrane-localized UPS ureide permeases and recent work in our lab has shown that UPS1 overexpression (UPS1-OE) in soybean leads to increased nodule-to-shoot N allocation with positive consequences for plant growth and seed development. In addition, it was demonstrated that enhanced N export from nodules positively affects N2 fixation and nodule metabolism. On the other hand, drought stress has an inhibitory effect on N2 fixation, probably due to decreased ureide transport out of, and their associated accumulation in, nodules. Here, we hypothesized that in UPS1-OE soybean plants down-regulation of N2 fixation under drought can be reduced and nodule-to-sink N allocation maintained. Soybean plants were exposed to medium and severe water-stress conditions, and we found that ureide movement from nodule to shoot in the xylem as well as from leaf to sink in the phloem was enhanced in water-stressed UPS1-OE versus wildtype plants. In addition, leaf chlorophyll content, photosynthetic rates, and sucrose phloem transport were higher in the transgenic plants under drought. The combined changes in N and carbon partitioning in UPS1-OE plants resulted in increased shoot and nodule biomass, and improved nodule numbers. Overall, the results support that ureide transport processes from nodules to sinks are essential for regulating ureide tissue levels and subsequently drought stress tolerance in soybean.
P–44. * Increasing Sulfur Content in Soybean Seed Protein
Trish Tully, Postdoctoral Associate, Donald Danforth Plant Science Center
TLA Tully1, D Duressa3, V Veena1, TP Durrett3, DK Allen1,2
1Donald Danforth Plant Science Center, St. Louis, MO, 63132; 2United States Department of Agriculture, Agricultural Research Science, St. Louis, MO, 63132; 3Kanas State University, Manhattan, KS, 66506
Protein is one of the most valuable biomass components of soybean seeds and accounts for ~40% of seed biomass. However, soy protein is not optimal for animal meal-based diets as it is deficient in sulfur containing amino acids (cys + met). In the past, attempts to increase sulfur content in soy protein have been focused on the protein level, including heterologous protein expression and overexpression of endogenous storage proteins with high sulfur content. Unfortunately, these approaches have had limited success. Here we illustrate the first steps in increasing sulfur content of soy protein at the metabolic level. In wildtype soybean, low molecular weight thiols downstream of cysteine (γEC and hGSH) accumulate over the course of development. This sequesters sulfur in non-proteogenic compounds rather than in the protein found in meal. To increase cysteine availability for protein synthesis, we have generated RNAi-knockdown lines targeting CGL; the enzyme responsible for diverting cysteine towards γEC and hGSH biosynthesis. RNAi-knockdown lines show decreased expression of all CGL homologues with seeds that are morphologically similar to wildtype. Observed levels of free amino acids and sulfur intermediates show an increase in free cysteine and a decrease in both γEC and hGSH in CGL-knockdown lines relative to wildtype. CGL-knockdown lines also show an increase in total protein as well as an increase in protein-bound cysteine relative to wildtype. Further work will combine the CGL-knockdown lines with protease-knockdown lines; combining a push and a protect to result in a hypothesized greater increase in both total protein and protein-bound cysteine.
*Poster selected for oral presentation.
P–45. Developing Robust and Durable Resistance to Soybean Rust
Rao Uppalapati, Biotech Disease Resistance Trait Portfolio Leader, Corteva Agriscience, rao.uppalapati@corteva.com
Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi (Pp), is one of the most economically important soybean disease. Farmers in Brazil spend US$1.7B on fungicides per year to control ASR and these chemical options are becoming less effective due to the emergence of fungicide resistance. Native soybean resistance from Rpp1-7 is identified but is reported to be overcome by one or more isolates from Brazil. To overcome these challenges, Corteva in collaboration with 2Blades identified a novel resistance gene from pigeon pea to provide robust and durable genetic resistance for ASR control. Cloning of CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) was previously reported (Kawashima et al., Nat Biotech, 2016). More recently, using a combination of cytology, transcriptomics, and metabolomics, we show that resistance is expressed rapidly, and Pp infection is arrested early, within 24-36 hours after inoculation and CcRpp1 shows excellent field level effiacy. The early resistance responses serve either to block initiation of haustoria formation or to inhibit maturation of incipient fungal feeding structures and effectively provide immune-level resistance against ASR.
P–46. Genomic diversity and engineering of soybean trypsin inhibitor gene family
Zhibo Wang, Postdoctoral Researcher, Donald Danforth Plant Science Center, zhibowang@danforthcenter.org
Trypsin inhibitors (TIs) have diverse biological functions. They accumulate in soybean seeds and are considered as anti-nutritional proteins that can severely reduce the digestibility of soybean meal. It is critical to understand genomic basis underlying production of TIs for developing soybean cultivars containing desirable seed TI activities without a negative effect on soybean performance. Genome-wide analysis showed that soybean contained 47 Kunitz TI (KTI) genes and 12 Bowman-Birk TI (BBTI) genes. We examined that each TI gene had a distinct temporal and spatial expression pattern, which is correlated with their phylogenetic relationship. A subfamily of 8 BBTI genes that were preferentially expressed in seeds over the course of seed maturation. The 47 KTI genes were clustered into three subfamilies and were expressed in both seed and non-seed tissues. We further identified KTI1 and KTI3 as two seed-specific TI genes. Mutant kti1 and kti3 alleles carrying small indels were created using CRISPR/Cas9-mediated genome editing approach. The KTI content and TI activity both remarkably reduced in the gene edited seeds. We further developed markers to co-select the mutant alleles of kti1/3 using a gel-electrophoresis-free method. The kti1/3 mutant line and associated markers will assist in accelerating the introduction of low TI trait into elite soybean cultivars in the future.
P–47. Rdm3 Locus – a Major QTL Underlying Resistance to Southern Stem Canker in Elite Soybean Germplasm
M Habib Widyawan, Doctoral Student, University of Georgia, widyawan@uga.edu
Soybean southern stem canker (SSC) caused by Diaporthe aspalathi, is an economically important disease in the southern United States. Five loci conferring resistance to SSC, namely Rdm1-5, have been named based on segregation analysis and reactions with different isolates. The Rdm3 locus carried by the SSC-resistant cultivar Crockett provides good comparable resistance to SSC when compared to the cultivars possessing multiple Rdm loci. However, the genomic location of this locus is unknown, and sources of resistance to SSC used in the breeding program are undetermined. This study aims to map the Rdm3 locus from Crockett and determine the key sources of resistance to SSC in the Georgia Soybean Breeding Program. Using a RIL population derived from a cross of G81-2057 ? Crockett, genetic mapping identified the Rdm3 locus on chromosome 14 that explained 55% of phenotypic variation. To determine the key sources of resistance to SSC in elite germplasm, a panel consisting of 485 experimental lines from the Georgia Soybean Breeding Program was selected to perform a genome-wide association analysis. The resistant allele at the Rdm3 locus provides a major source of resistance to SSC in this elite germplasm pool. GSM_975, a marker tightly linked with the Rdm3 locus, could accurately distinguish soybean lines based on their SSC resistance provided by the Rdm3 locus. The results revealed the prevalence of the Rdm3 locus resistance allele in the elite soybean germplasm. The QTL and flanking marker information will provide useful information and tools to assist breeders in developing SSC-resistant cultivars.
P–48. An Efficient and Practical Fixation for Plant Single Nucleus RNA-seq
Hengping Xu1, Sandra Thibivilliers1, Sergio Alan Cervantes-Perez, and Marc Libault1*, Center for Plant Science Innovation, Beadle Center, University of Nebraska–Lincoln,
1 These authors contribute equally to this work *Corresponding author: marc.libault@unl.edu
Single-nucleus RNA sequencing (snRNA-seq) technology is emerging as a robust alternative to single-cell RNA sequencing (scRNA-seq) in plant biology. Fixation of nuclei has been successfully tested on animal samples before conducting snRNA-seq. Unlike aldehyde-based fixatives or DSP [dithiobis (succinimidyl propionate)], methanol fixation has been validated when conducting single-cell RNA sequencing (scRNA-seq). Such fixation reduces potential RNA loss and enhances RNA quality improving the outcome of snRNA-seq experiments. However, little is known about the impact of methanol fixation on plant nuclei isolation and snRNA-seq experiments. In this study, we test the effect of a methanol fixative on snRNA-seq experiments conducted on Arabidopsis and Sorghum root samples. We found that methanol fixation is an efficient and convenient option to conduct with success plant snRNA-seq in transcriptomic studies. Key words: Methanal fixation, nuclei, single nucleus RNA sequencing (snRNA-seq)
P–49. Mapping Active Pathways in Developing Thorne Embryos Using 13C-Labeling
Umesh Yadav, Research Scientist, University of North Texas, umeshprasad.yadav@unt.edu
13C-metabolic flux analysis aims to elucidate the distribution of carbon in a living organism, and ultimately, identify potential bottleneck(s) that can be targeted through metabolic engineering. Glycine max cv. Thorne variety was chosen because it is less recalcitrant for transformation than others with similar seed composition, which allows identified metabolic targets to be evaluated in vivo in the same cultivar. The construction of a flux map requires: i) the establishment of in vivo embryo culture conditions that mimic the in planta ones, and ii) the utilization of isotope tracers to map the flow of carbon through metabolic pathways. For this purpose, funiculi and endosperm tissues were collected, and the most abundant carbon and nitrogen substrates were identified to establish a suitable culture medium for developing soybean embryos. The efficiency with which Thorne embryos convert carbon substrates into biomass was found to be lower than other soybean cultivars. Then, carbon and nitrogen sources from the media were replaced with 13C-labeled ones, and the incorporation of 13C-labeling in intracellular compounds was monitored using LC-MS/MS. Our results demonstrate that there is no significant action of the phosphoenolpyruvate carboxykinase, and no significant gluconeogenesis neither. However, there is labeling evidence for a flow of carbon through the plastidic malic enzyme to support de novo fatty acid synthesis. We anticipate that the construction of embryo´s flux map will allow the identification of critical control points in central metabolism that govern protein and oil content. Ultimately, those bottlenecks will be tested using plant metabolic engineering by generating transgenic lines.
P–50. Understanding the roles of soybean aphid effectors in soybean and soybean aphid interaction
Dandan Zhang, Graduate Student, and Gustavo MacIntosh, Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, ddzhang@iastate.edu
The production of soybean, an economically important crop, has been threatened by both direct and indirect damages caused by soybean aphid, which is a specialist colonizing only soybean plants. Our current soybean aphid management strategies rely heavily on the application of insecticides. However, the emergence of aphid populations with insecticide resistance has made development of novel aphid control strategies an urgent need. Undoubtedly, understanding the mechanisms underlying host and pest interaction is key to provide insights for the achievement of this goal. In this study, we examined soybean responses induced by aphid infestation including the activation of soybean mitogen-activated protein kinases (MAPKs) and the production of reactive oxygen species (ROS), which are two of the most conserved plant immune responses to pathogen attack. Our study found that soybean aphid infestation activated MPK4 and MPK6 in soybean dynamically at different time points post aphid infestation. Colonization by aphids also inhibited chitin-induced ROS production in soybean but failed to suppress flg22-dependent induction. Three soybean aphid effector candidates predicted by our transcriptome analyses-based pipeline were selected and tested for their potential to influence aphid-triggered MAPKs activation and ROS production. C002, one of the putative effectors, was found to be able to inhibit both chitin and flg22 induced ROS production, and another putative effector, MP10 showed the ability to enhance both chitin and flg22 induced ROS production. These findings provide a deeper understanding of how soybean responds to aphid infestation and the potential roles of two effectors in soybean and soybean aphid interaction.