Progress 04/01/24 to 03/31/25

Outputs
Target Audience:Farmers, Breeders, Researchers, Public (information on advancing crops with yield protection) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two post-doc fellows and one graduate student were directly involved in this project. They were trained to manage the project and coordinate the experimental activities to meet the project objectives. They were trained to be familiar with the procedures of how to run a scientific research project. Part-time undergraduate students received training in genotyping using molecular markers, DNA extractions, and greenhouse phenotyping. How have the results been disseminated to communities of interest?Two refereed publications are being prepared as follows: Genetic architecture of waterlogging tolerance in soybean during seed germination and seedling emergence stages (Under preparation). Physiological and Transcriptomic Responses of Soybean (Glycinemax) Varieties to Combined Cold and Waterlogging(Under preparation). What do you plan to do during the next reporting period to accomplish the goals?We are preparing two manuscripts to publish the results generated from Yr. 1 to report identification of genetic architecture of early season waterlogging tolerance in soybean and molecular mechanisms underlying soybean responses to waterlogging and cold at the seedling stage through bulk transcriptomics and metabolomics. Objective 1: Characterize the genetic architecture of soybean germplasm lines in response to early-season waterlogging, cold, and their combination. 1) Characterize a set of soybean lines for cold tolerance during the seedling establishment stage: We worked with the USDA germplasm collection team to identify a core set (300 lines) of early maturity soybeans for cold tolerance investigation. We plan to screen the cold tolerance performance of this set of lines at the seedling stage. 2) Identify the genetic architecture and candidate genes associated with tolerance to early-season waterlogging and/or cold: We will finalize GWAS on waterlogging tolerance in the soybean set, aiming to identify candidate genes. We will also start GWAS on cold tolerance traits in soybean lines, once phenotyping results are available. ?Objective 2: Reveal the molecular mechanisms and regulatory networks underlying their responses to the early season waterlogging, cold, and their combination through transcriptomics and metabolomics analyses. 1) Elucidation of molecular mechanisms underlying soybean responses to waterlogging, cold, and their combination at the seedling stage through bulk transcriptomics analysis: Sequencing reads are being aligned to the Glycine max reference genome Wm82.a6.v1 with STAR. Differentially expressed genes (DEGs) will be called with DESeq2 ( log?FC ≥ 1, FDR < 0.05). Gene Ontology (GO) enrichment and KEGG pathway analyses will be carried out in the 'clusterProfiler' R package. Weighted gene-co-expression network analysis (WGCNA) will be then applied to the full DEG set to infer stress-responsive regulatory modules. To validate the transcriptome data, a minimum of 20 DEGs from 'combined cold and waterlogging-tolerant vs. cold and waterlogging-sensitive' comparison will be selected, and their expression profiles will be determined by RT-qPCR analysis. 2) Comparative analysis of metabolic responses of soybean to waterlogging, cold and their combination at VE stage through bulk metabolomics: LC-MS/MS experiment was performed, and data is being analyzed. Compound discovery will be used for peak selection and compound annotation of the MS signals. The obtained data matrix will be normalized using cross-contribution, compensating for multiple standard normalization. Differentially produced metabolites (DPMs) between treatments and genotype groups will be identified using MetaboAnalyst v5.0. Pathway enrichment analysis will be conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG) Mapper to identify significantly perturbed metabolic pathways. Hormonal profiles (top 5 tolerant and top5 sensitive soybean lines,) will be conducted (through paid service of Novogene, MA, U.S.) using the radicle and hypocotyl samples collected in parallel as those for transcriptomics using LC-MS/MS. KEGG enrichment pathway analysis will be performed using 'clusterProfiler' package in R. Gene-metabolite correlation analysis of the data obtained from different treatment combinations will also be performed using the statistical package R. Objective 3: Develop breeding and genetic engineering strategies for improving soybean yield in responses to the early-season waterlogging and/or cold. 1) Marker-assisted breeding to incorporate the stress tolerance traits/genes into elite soybean germplasm: We will develop DNA markers once the significant genetic loci have been confirmed. We will use these markers to select soybean progenies carrying the tolerant alleles in the developed breeding populations.

Impacts
What was accomplished under these goals? Objective 1:Characterize the genetic architecture of soybean germplasm lines in response to early-season waterlogging, cold, and their combination. 1) Confirm waterlogging tolerance in a set of soybean lines in the greenhouse: Sixty contrasting lines (30 vs 30) in waterlogging tolerance level during germination and seedling emergence were selected to confirm soil pot-derived results in soil-bed (greenhouse). Seeds were sown in the soil bed and treated with waterlogging for 4 days. Seedling standing was counted 10-day after removing water. Significant correlation (r = 0.92) was observed in seedling emergence/standing rates between the previous pot experiment and the current soil-bed experiment. We successfully confirmed waterlogging tolerance results in this soybean set. 2) Characterize a set of soybean lines for cold tolerance during the seedling establishment stage: Thirty two out of 60 lines from Objective 1.1 were selected to screen for plant performance at the seedling stage in response to cold (low temperature). Seeds were sown in pots and grown under normal conditions (growth chamber) for four days, followed by six days of stress treatments, including waterlogging, cold, and a combination of both. Plant phenotypes were assessed after the stress period to identify lines with contrasting responses. Compared to waterlogging alone, cold and combined waterlogging/cold had more pronounced effects on seedling growth. Therefore, tolerant and sensitive lines were identified based on their phenotypes under the combined cold and waterlogging. In total, five tolerant and five sensitive lines were selected as candidates for transcriptomic and metabolomic analyses in Obj 2. 3) Identify the genetic architecture and candidate genes associated with tolerance to early-season waterlogging and/or cold stresses: We finished screening waterlogging tolerance during seedling emergence of 300 diverse soybean lines. Waterlogging treatment was found to significantly delay seedling establishment by seven days, and the establishment rates were reduced by waterlogging by approximately 50% on average. Significant genetic variabilities in WTI were identified, and WTI was found to be an inheritable trait with a broad-sense heritability of 0.68 across the four repetitions. Seven loci were detected across the soybean genome through a GWAS, in which one QTL identified on Chr. 08 is the major locus for the highest association [-log10 (p) = 6.0] (Fig. 2B). This major locus could be our focus in marker-assisted breeding and gene function characterization in the proposed project. We will use our newly developed soybean pan-genome resources to assist GWAS and candidate gene identification in Yr. 2. Objective 2: Reveal the molecular mechanisms and regulatory networks underlying their responses to the early season waterlogging, cold, and their combination through transcriptomics and metabolomics analyses. 1) Elucidation of molecular mechanisms underlying soybean responses to waterlogging, cold, and their combination at the seedling stage through bulk transcriptomics analysis: Comparative bulk transcriptome profiling of radicle and hypocotyl samples (stress vs control) from top 5 tolerant and top 5 sensitive lines identified in Obj 1.2 were performed using bulk RNA-seq analysis. The selected lines were grown under growth chamber conditions in parallel to Obj 1.2. For each of the 5 top tolerant and 5 top sensitive genotypes, equal amounts of radicle and hypocotyl tissues were harvested and pooled from three individual seedlings exposed either to single or combined cold and waterlogging stress, as well as from matched non-stressed controls (3 seedlings per biological replica × 5 biological replicates × 2 genotypes). In total, 8 biological bulks were generated [2 genotype groups (5 tolerant and 5 sensitive) × 4 treatments (control, cold, waterlogging, and combined cold + waterlogging)]. Total RNA was extracted for RNA-seq experiment to identify differentially expressed genes and identify potential candidate genes for stress tolerance. and verified by NanoDrop spectrophotometry and agarose gel electrophoresis. Sequencing (150 bp paired-end) has been performed by Novogene and sequencing quality has been examined to be good. Currently, data analysis is ongoing and results will be reported in the next annual report. 2) Comparative analysis of metabolic responses of soybean to waterlogging, cold, and their combination at the VE stage through bulk metabolomics: The same radicle and hypocotyl tissues used for RNA-seq were also submitted for untargeted metabolomic profiling (through paid service of Novogene, MA, U.S.) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Currently, LC-MS/MS has been finished, and data is being analyzed. Results will updated in the next annual report. Objective 3: Develop breeding and genetic engineering strategies for improving soybean yield in responses to the early-season waterlogging and/or cold. 1) Marker-assisted breeding to incorporate the favorable traits/genes into elite soybean germplasm: As the promising soybean lines were identified in our Yr. 1 experiment for waterlogging and cold tolerance (early season), we are working with the soybean breeder at the University of Arkansas to make cross between the elite soybean lines and promising exotic lines.

Publications