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ARS Home » Midwest Area » Ames, Iowa » Corn Insects and Crop Genetics Research » Research » Research Project #434493

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

Location: Corn Insects and Crop Genetics Research

2020 Annual Report

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.

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
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. In collaboration with researchers from Iowa State University, ARS scientists in Ames, Iowa, previously demonstrated that a region on soybean chromosome 3 associated with iron deficiency tolerance was actually four distinct regions, each contributing to the trait. The team has now selected 18 unique lines from the ARS soybean germplasm collection representing eight of the possible combinations of these four regions. 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. Also, differentially expressed genes differed between lines, suggesting there are multiple novel mechanisms for conferring 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. 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. 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 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. 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, developed virus induced gene silencing for all 11 genes within this region. In soil-grown plants, silencing of three unique genes was associated with altered phenotypes. Gene expression analyses of silenced plants is ongoing. In addition, whole genome expression analyses of Fiskeby III grown in control and iron deficient conditions identified few differentially expressed genes in leaves or roots, suggesting a novel iron stress response compared to other soybean lines. Results from these studies will provide plant breeding programs novel markers and genes associated with abiotic stress tolerance. Investigating gene expression changes in response to sequential nutrient deficiencies in soybean. Throughout a growing season, plants experience a multitude of short periods of different abiotic stresses which have long-term impacts on plant performance and yield. The majority of studies examining transcriptional changes induced by stress focus on single stress events. Few studies have examined the transcriptional response of plants exposed to sequential stresses. ARS scientists in Ames, Iowa, examined the transcriptional profiles of soybean plants exposed to either two rounds of iron deficiency stress, two rounds of phosphate deficiency stress, or iron deficiency stress followed by phosphate deficiency stress. Scientists identified a core suite of genes modified by both iron and phosphate stress, genes whose expression profile was unique to each stress, and genes that are uniquely induced by the novel sequential stress exposure. These findings improve our understanding of the genes and networks underlying plant stress tolerance which can be leveraged to mitigate end of season yield loss. Characterization of the resistance to Phakospora Pachyrhizi 1 B (Rpp1b) locus in soybean. Asian soybean rust is a threat to soybean production worldwide. Identification of resistance/defense genes is essential for improving commercial cultivars. Rpp1 is a gene that confers complete immunity, while Rpp1b confers resistance. Rpp1b and Rpp1 map to the same region on soybean chromosome 18, but are present in different soybean lines. Previously, in collaboration with ARS researchers in Ft. Detrick, Maryland, ARS scientists in Ames, Iowa identified eight candidate genes for Rpp1 using map-based cloning, gene expression analyses and gene silencing. Knocking down the expression of the Rpp1 immunity gene resulted in resistance, not susceptibility. To better understand the differences between immunity, resistance and susceptibility, the same approach is being used to identify and characterize Rpp1b from soybean line PI594538A. Breeders and scientists can 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, caused by the fungus Phialophora gregata, reduces soybean yield by 38%. Though an agronomically important pathogen, 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, 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, virus induced gene silencing constructs were developed to knock down the activity of these clusters. Already, we have determined that members of two clusters are required for Rbs1-mediated resistance. 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 can use markers and genes identified by this project to incorporate disease resistance into commercial cultivars. 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, used map-based cloning to sequence the Rps2 locus in the resistant parent L76-1988. Bioinformatic analyses identified 26 predicted resistance genes within the region. To determine which genes confer resistance, silencing constructs were developed to target specific subgroups within the candidate genes. These constructs are being tested with different races of P. sojae to determine the impact of gene silencing on disease resistance. We identified two constructs that appear to enhance disease resistance through unknown mechanisms. Identifying and characterizing these candidate resistance genes will provide new avenues for generating new disease resistance specificities for crop improvement. Comparing abiotic stress gene expression changes across major crop species. Studies in soybean have identified gene expression signatures of abiotic stress tolerance. A cross disciplinary team of ARS scientists in Ames, Iowa, conducted a flooding and heat tolerance experiment in maize to determine if stress responses are conserved across crop species. For this project, maize was grown in both plots and in small soil pillars where water tables could be altered to generate flooding conditions. Experimental plants were grown in multiple growth chambers to control heat stress regimes. A subset of plants were harvested for whole genome expression analyses, while the remaining plants were left growing under different flood and heat stress conditions for phenotypic analysis throughout development. Data analysis is ongoing. In Phase 2, the experiment will be repeated using soybean, allowing the identification of conserved stress tolerance mechanisms in crops. Characterizing the diversity of soybean root architecture. Root architecture regulates water and nutrient acquisition, nutrient storage, and plant stability. To investigate the genetic and phenotypic diversity of the root systems represented in the USDA National Soybean Germplasm collection, collaborators at Iowa State University performed high throughput phenotyping of 300 soybean lines representing the breadth and depth of the collection. The phenotypic data were paired with single nucleotide polymorphism data in a genome wide association study to identify novel genomic regions associated with root architecture. ARS scientists in Ames, Iowa, in collaboration with colleagues from Iowa State University, analyzed these regions to identify novel high priority candidate genes potentially responsible for root architecture traits. Virus-induced gene silencing constructs have been developed to target these genes and experiments are currently being conducted to determine their role in root architecture. The genomic regions, markers, and genes identified in this study can all be used to tailor root architecture to maximize yield.

1. Identifying gene expression changes in response to micro- and macro- nutrient deficiencies in soybean. Preserving crop yield is critical for US soybean production and the global economy. Crop species have been selected for increased yield for thousands of years with individual lines selected for improved performance in unique environments, constraints not experienced by non-crop model species such as Arabidopsis. This selection likely resulted in novel stress adaptations unique to crop species. Given that iron deficiency is a perennial problem in the soybean growing regions of the US and phosphate deficiency looms as a limitation to global agricultural production, nutrient stress studies in crop species are critically important. ARS scientists in Ames, Iowa, directly compared whole-genome expression responses of leaves and roots to iron (a micronutrient) and phosphate (a macronutrient) deficiency. Conducting experiments side by side allowed direct comparison of nutrient stress responses, examined 24 hours after the onset of stress. While soybean responds largely to iron deficiency and not resupply, it responds strongly to phosphate resupply and not deficiency. Though the timing of responses was different, both nutrient stress signals used the same molecular pathways. The study was also used to examine gene expression changes in response to multiple stress events. It identified 865 genes whose gene expression changed between first and second stress exposure and 3,375 genes only differentially expressed after a second stress event. This study demonstrates the speed and diversity of the soybean stress response to multiple nutrient deficiencies. Understanding the molecular underpinnings of these responses in crop species could have major implications for improving stress tolerance and preserving yield.

2. Using genotypic, phenotypic and shape based clusters to understand soybean root system architecture. Root system architecture is difficult to phenotype and past studies suffer from limited scale and scope, as well as variability in measuring techniques. In collaboration with researchers at Iowa State University, a ARS scientist in Ames, Iowa, studied the root system architecture of 292 soybean lines from the soybean germplasm collection. This phenotypic data was combined with the soybean genotyping chip to explore the genetic diversity of soybean root system architecture traits. Genomic locations of interest were identified for traits such as root shape, length, number, mass, angle. Phenotypic and genotypic data were used to group lines into eight genotype- and phenotype-based clusters. Genotype-based clusters correlated with geographic origins and demonstrated that much of US elite germplasm lacks genetic diversity for root system architecture traits. Combining genetic and phenotypic analyses results provides opportunities for targeted breeding efforts to maximize the beneficial genetic diversity for future genetic gains.

Review Publications
O'Rourke, J.A., Mccabe, C.E., Graham, M.A. 2020. Dynamic gene expression changes in response to micronutrient, macronutrient, and multiple stress exposures in soybean. Functional and Integrative Genomics. 20:321-341.
Assefa, T., Zhang, J., Chowda-Reddy, R.V., Moran Lauter, A.N., Singh, A., O'Rourke, J.A., Graham, M.A., Singh, A.K. 2020. Deconstructing the genetic architecture of iron deficiency chlorosis in soybean using genome-wide approaches. Biomed Central (BMC) Plant Biology. 20:42.
Falk, K.G., Juberi, T.Z., O'Rourke, J.A., Singh, A., Sarkar, S., Ganapathysubramanian, B., Singh, A.K. 2020. Soybean root system architecture trait study through genotypic, phenotypic and shape-based clusters. Plant Phenomics.
Moran Lauter, A., Rutter, L., Cook, D., O'Rourke, J.A., Graham, M.A. 2020. Examining short-term responses to a long-term problem: RNA-seq analyses of iron deficiency chlorosis tolerant soybean. International Journal of Molecular Sciences. 21:3591.