Research Project: Genetic and Genomic Characterization of Soybean and Other Legumes

Location: Corn Insects and Crop Genetics Research

2021 Annual Report

Objectives
Objective 1: Identify and characterize genes, markers, and molecular networks contributing to yield, resistance to pathogens, and nutrient stress tolerance in soybean and other legumes, and work with researchers to use the information in crop improvement by conventional breeding and gene editing technology. Sub-objective 1.A. Identify and characterize legume gene expression and epigenetic networks that control nutrient homeostasis, generating information for improving resistance or tolerance to abiotic stress. Sub-objective 1.B. Identify and characterize soybean disease resistance loci and defense gene expression and epigenetic networks, generating information for improving resistance or tolerance to pathogens that cause economic loss in soybeans.

Approach
The United States leads world soybean production, contributing over 40 billion dollars to the economy in 2014. However, nutrient, disease and pest stresses limit agricultural production. The overarching goal of this project is to provide data and resources that will increase soybean (Glycine max (L.) Merrill) production by mitigating losses due to abiotic and biotic stresses. To study nutrient deficiency we will use iron deficiency chlorosis as a model. To study disease resistance responses we will use a variety of pathogens including Phakopsora pachyrhizi (Asian soybean rust), Phialophora gregata (brown stem rot), Phytophthora sojae (Phytophthora rot), and the insect pest Aphisglycines (soybean aphid). Regulation of abiotic and biotic stress responses requires constant signaling, likely controlled by gene expression and epigenetic changes. Further, a single stress exposure likely primes subsequent plant stress responses. To characterize the genes and networks involved in these responses we will couple RNA-seq, Methyl-seq and Virus Induced Gene Silencing. Finally, we will use RNA-seq data to characterize resistance loci and downstream defense responses. Successful completion of this project will result in genes, gene networks and validated markers that can be used to breed soybean germplasm with durable resistance to abiotic and biotic stress. This project will provide valuable resources to public and private soybean breeders, scientists and growers.

Progress Report
SubObjective 1A: Characterizing responses to iron deficiency chlorosis in the soybean germplasm collection. In plants, iron deficiency causes a reduction in photosynthesis, interveinal leaf yellowing and reduced yield. However, much or our knowledge is focused on model species, with most studies restricted to a single line of interest. In collaboration with researchers from Iowa State University, ARS scientists in Ames, Iowa, leveraged a previous genome wide association study to select 18 unique soybean lines from the USDA soybean germplasm collection, with a range of iron stress tolerance. The lines were grown in both iron sufficient and deficient conditions. Roots and shoots were collected for expression analyses, comparing responses between growth conditions. These analyses confirmed that multiple lines responded to iron stress within 60 minutes, much faster than observed in model species. While previous analyses suggest iron signaling is directed from shoot to root, this study confirms soybean uses a novel root to shoot signaling mechanism. Also, differentially expressed genes differed between lines, suggesting there are multiple novel mechanisms for conferring iron stress tolerance in the soybean germplasm collection. These findings highlight the need for conducting research in diverse, agronomically important crop species. Such studies will lead to development of soybean lines with improved stress tolerance and greater yield. The manuscript decribing this research is in final stages of preparation. Characterizing novel root to shoot iron stress signaling mechanisms in the soybean line Clark. Iron deficiency chlorosis is an important problem for soybeans, significantly impacting quality and yield. Previous gene expression experiments by ARS scientists in Ames, Iowa, suggested the soybean line Clark uses novel root to shoot signaling to initiate iron stress responses. To study this further, shoots from an iron stress susceptible line were grafted to roots from an iron stress tolerant line (and vice versa), while growing grafted plants in iron sufficient and iron deficient conditions. Phenotypic data revealed that only iron stress tolerant roots confer resistance to iron stress, supporting the root to shoot signaling hypothesis. The experiment was repeated and both roots and shoots were harvested for gene expression analyses. Whole genome expression analyses identified key genes involved in iron stress tolerance signaling networks from roots to shoots. These new genes are now being characterized using virus-induced gene silencing (VIGS). Analyses of these data will identify novel iron stress signaling mechanisms and will provide genes as markers for improving crop responses to abiotic stress conditions. In addition, this research demonstrates important differences between crop and model species. Characterizing candidate genes in the historic iron stress tolerance quantitative trait locus in soybean. Previous research by ARS and Iowa State University scientists demonstrated that a region on soybean chromosome 3, associated with iron deficiency tolerance, was actually composed of four distinct regions, each containing candidate iron stress response genes. ARS scientists in Ames, Iowa, are using VIGS technology to target these genes individually and in combination. Silenced-plants were grown in soil and in iron stress conditions to identify changes in iron stress tolerance due to gene silencing. Plants with altered responses were then selected for whole genome expression analyses of both roots and shoots. These analyses confirmed that multiple genes in this region are required for iron stress tolerance. Analyses of these data sets will identity novel iron stress response genes in soybean and the molecular networks they control. This research will provide new genes for improving crop responses to abiotic stress conditions, leading to the development of new and improved varieties. Characterizing novel regions of the soybean genome for tolerance to iron deficiency. The soybean cultivar, Fiskeby III, thrives under multiple abiotic stress conditions, including iron deficiency, which has made it a valuable addition to Northern US and Canadian breeding programs. Mandarin (Ottawa) has heavily contributed to the genetic base of Northern genotypes, but is highly susceptible to abiotic stresses. Collaborators at the University of Minnesota identified a small region on chromosome 5 responsible for a significant portion of iron deficiency stress tolerance. In a project funded by the United Soybean Board, and working with collaborators at the University of Minnesota, ARS scientists in Ames, Iowa, used whole genome expression analyses to compare the Fiskeby III and Mandarin (Ottawa) responses to iron deficient conditions and have coupled virus induced gene silencing of the high priority candidate genes in Fiskeby III with whole genome expression analyses to identify stress tolerance networks unique to Fiskeby III. Results from these studies will provide plant breeding programs novel markers and genes associated with abiotic stress tolerance. The manuscript describing this research is in final stages of preparation. SubObjective 1B: Characterization of the Resistance to Phakospora Pachyrhizi 1 B (Rpp1b) and Rpp3 loci in soybean. Asian soybean rust is a threat to soybean production worldwide. Identification of resistance/defense genes is essential for improving commercial cultivars. In collaboration with ARS researchers in Ft. Detrick, Maryland, ARS scientists in Ames, Iowa, used a map-based cloning approach to identify candidate genes for Rpp1b. Virus-induced gene silencing approaches are being use to test the function of candidate genes. In contrast, the Rpp3 locus has not been amenable to a map-based cloning approach. Therefore, the team has leveraged the reference soybean genome sequence, coupled with targeted gene expression analyses and VIGS to identify candidate resistance genes for Rpp3. Breeders and scientists will be able to use the markers and genes developed by this project to incorporate resistance to ASR into improved commercial cultivars. Characterizing candidate brown stem rot resistance genes in soybean. Brown stem rot, (BSR), caused by the fungus Phialophora gregata, reduces soybean yield by 38%. Identifying new sources of resistance has been complicated by time-consuming phenotyping methods and conflicting genetic studies. In a previous study, ARS scientists in Ames, Iowa, combined historical mapping data with genotype expression differences to identify clusters of receptor like proteins (RLPs), similar to known fungal resistance genes, associated with each of the previously identified resistance genes (Rbs1, Rbs2, and Rbs3). In work funded by the National Institute of Food and Agriculture, we developed VIGS constructs to knock down the activity of these clusters individually and in combination. Already, we have determined that members of two clusters are required for Rbs1-mediated resistance. Whole genome expression analyses confirm silencing of two RLP clusters suppresses defense responses leading to disease susceptibility. This novel research demonstrates the utility of combining contrasting genotypes, classical genetic studies and whole genome expression analyses to characterize complex disease resistance traits. Breeders and scientists will be able to use markers and genes identified by this project to incorporate BSR resistance into commercial cultivars. The manuscript describing this research is in preparation. Characterizing the Resistance to Phytopthora sojae 2 (Rps2) locus in soybean. Phytophthora is the second most damaging disease of soybean. In research funded by the United Soybean Board and in collaboration with The Ohio State University, ARS scientists in Ames, Iowa, previously used map-based cloning to sequence the Rps2 locus in the resistant parent L76-1988. Of particular interest were two novel classes of resistance genes, containing portions of classical disease resistance genes fused to other genes (a calmodulin gene or a defense-related protein). While such fused resistance genes can be found in plant genomes, their contribution to disease resistance remains largely unknown. Therefore, the research team used VIGS to turn off the activity of these genes in both resistant and susceptible lines. Surprisingly, all silenced plants appeared to issue a strong resistance response, a response confirmed by whole genome expression analyses. Characterizing these novel resistance genes will provide new avenues for generating new disease resistance specificities for crop improvement. Using genomics to determine the molecular effects of stacking Resistance to Aphis glycines 1 and 2 (Rag1 and Rag2).Throughout history, plant breeders have stacked multiple resistance genes targeting specific pathogens or pests within a single line. Plants containing the Rag1/Rag2 double stack are significantly more resistant to aphid attack then plants containing either Rag1 or Rag2 alone. To understand how stacking resistance genes enhances resistance, researchers from Iowa State University and the USDA-ARS conducted whole genome expression analyses of four soybean lines (aphid-susceptible line, Rag1, Rag2 and the Rag1/Rag2 double stack), collecting samples six and 12 hours after infestation or mock infestation with soybean aphids. We identified thousands of genes specifically expressed in response to aphids in the double stack line, but not in either of the single resistance gene lines. Many of the genes unique to the double stack were related to defense, detection of pests and pathogens and defense signaling. This data identified a strong candidate for the Rag1 resistance gene. Understanding how stacking resistance genes results in enhanced resistance is essential for future crop improvement. The manuscript describing this research is in the final stages of preparation.

Accomplishments
1. Iron deficiency followed by phosphate deficiency induces novel gene expression changes in soybean. Plants grown in the field experience many abiotic stresses throughout the growing season. The cumulative effect of these stresses is a loss of yield. To understand the genes and networks underlying stress tolerance, ARS scientists in Ames, Iowa, measured gene expression in soybean in response to iron deficiency stress followed by exposure to phosphate deficiency stress (sequential stresses), to more closely mimic field grown plant stress exposure. These analyses determined that sequential stress induces a unique suite of genes not differentially expressed under repeated stress conditions. These novel genes are usually involved in highly specialized processes such as pollen development. However, after sequential stress exposure these same genes are recruited to function in novel tissues, performing basic processes such as cell wall modifications. These findings improve our understanding of soybean response to complex nutrient deficiency stress exposure and will improve our understanding of the genes and networks underlying plant stress tolerance which can be leveraged by researchers to improve stress tolerance in soybean and other important crop species.

2. Molecular responses to iron stress are conserved across time and tissues. Iron is a micronutrient essential for the proper growth and development of all organisms. In plants, lack of usable iron can result in iron deficiency chlorosis (IDC), which is characterized by yellowing of the leaves, reduced plant growth and lower yield. To understand the molecular mechanisms contributing to iron stress responses, ARS scientists in Ames, Iowa, studied two nearly genetically identical lines, one line was iron stress tolerant, while the other line was susceptible to iron stress. The two lines were then grown in three different conditions: normal growth conditions for ten days (no stress control), normal growth conditions for eight days followed by two days of iron stress, or for ten days in iron stress conditions. We compared gene expression differences between the different growing conditions and between lines. Whole genome analyses of root and shoot tissue identified thousands of genes with altered expression patterns, with functions related to the cell cycle, gene silencing, iron acquisition and defense. Comparing these results with previous studies from our group suggest early gene expression changes are initiated in the root, then extend to the leaves. In this study, with later timepoints, the leaves signal back to the roots when iron needs have been met or if additional iron is needed. These novel signaling mechanisms have not been described in model species. These findings improve our understanding of the genes and networks underlying plant stress tolerance which can be leveraged by researchers to improve iron stress tolerance in soybean and other important crop species.

3. Utilizing the whole plant to understand disease resistance signaling from root to shoot. Brown stem rot (BSR) reduces soybean yield by up to 38%. The causal agent of BSR is Phialophora gregata, a slow growing necrotrophic fungus whose life-cycle includes inactive and active disease causing phases, each lasting several weeks and spreading from root to shoot. BSR leaf symptoms are often misdiagnosed as other soybean diseases or nutrient stress, making BSR resistance especially difficult to phenotype and utilize in breeding programs. To shed light on the genes and networks contributing to resistance, ARS scientists in Ames, Iowa, funded by the National Institute of Food and Agriculture, conducted whole genome expression analyses of infected and mock-infected root, stem, and leaf tissues of a BSR resistant soybean at 12, 24 and 36 hours. Comparing infected and mock-infected plants revealed that leaves, stems and roots use the same defense pathways. Gene networks associated with defense, photosynthesis, nutrient regulation, DNA replication and growth are the hallmarks of BSR resistance. These same resistance pathways were identified in an earlier study comparing resistant and susceptible responses seven days after infection. Since P. gregata is a slow growing pathogen, with disease symptoms taking several weeks to appear, breeders must wait for five weeks to score plants for resistance or susceptibility, in largely destructive assays. Our data suggests resistance and susceptibility can be detected molecularly, hours after infection. The genes and networks described here will be used to develop novel diagnostic tools to facilitate expedited breeding and screening for BSR resistance. In addition, candidate disease resistance genes can be used for introducing resistance to elite soybean lines using traditional breeding or transgenic approaches.

4. Genotypic characterization of the U.S. peanut core collection. Cultivated peanut (Arachis hypogaea) is an important oil, food, and feed crop worldwide. In work funded by the National Institute of Food and Agriculture, ARS scientist in Ames, Iowa, and a broad team of scientists collected tissue and extracted and sequenced DNA from 812 select peanut lines representing differences in phenotype and country of origin in the U.S. peanut collection. Analyses identified 14,430 high-quality, informative markers across the collection. Analysis of the data divided 812 lines into five distinct genotypic clusters, largely corresponding with botanical variety and market type, but not country of origin. A genetic cluster, with accessions coming primarily from Bolivia, Peru, and Ecuador, is consistent with these having been the earliest lines cultivated by ancient farmers. Comparing these lines with their predicted parents, suggests subgenome exchanges are an important source of diversity. These diverse regions are likely novel sources of resistance and phenotypic variation. Markers associated with these regions can be used by peanut breeders and growers to develop improved peanut varieties.

Review Publications
O'Rourke, J.A., Graham, M.A. 2021. Gene expression responses to sequential nutrient deficiency stresses in soybean. International Journal of Molecular Sciences. 22(3). Article 1252. https://doi.org/10.3390/ijms22031252.
Atencio, L., Salazar, J., Moran Lauter, A., Gonzales, M.D., O'Rourke, J.A., Graham, M.A. 2021. Characterizing short and long term iron stress responses in iron deficiency tolerant and susceptible soybean (Glycine max L. Merr.). Plant Stress. 2.Article 100012. https://doi.org/10.1016/j.stress.2021.100012.
Brown, A.V., Connors, S., Huang, W., Wilkey, A., Grant, D.M., Weeks, N.T., Cannon, S.B., Graham, M.A., Nelson, R. 2020. A new decade and new data at SoyBase, the USDA-ARS soybean genetics and genomics database. Nucleic Acids Research. 49(D1):D1496-D1501. https://doi.org/10.1093/nar/gkaa1107.
Otyama, P.I., Kulkarni, R., Chamberlin, K., Ozias-Akins, P., Chu, Y., Lincoln, L.M., MacDonald, G.E., Anglin, N.L., Dash, S., Bertioli, D.J., Fernandez-Baca, D., Graham, M.A., Cannon, S.B., Cannon, E.K.S. 2020. Genotypic characterization of the U.S. peanut core collection. G3, Genes/Genomes/Genetics. 10(11):4013-4026. https://doi.org/10.1534/g3.120.401306.
McCabe, C.E., Graham, M.A. 2020. New tools for characterizing early brown stem rot disease resistance signaling in soybean. The Plant Genome. 13(3). Article e20037. https://doi.org/10.1002/tpg2.20037.