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

Location: Corn Insects and Crop Genetics Research

2019 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
Characterizing novel gene networks regulating tolerance to iron deficiency chlorosis. In plants, iron deficiency chlorosis causes a reduction in photosynthesis, interveinal yellowing of the leaves and reduced yield. In previous work with collaborators from Iowa State University, we demonstrated that a region on soybean chromosome 3 previously associated with iron deficiency tolerance, was actually made up of four distinct regions. We identified 18 lines from the USDA soybean germplasm collection that represent the eight possible combinations of these distinct regions. For each combination, we selected an iron stress tolerant line and an iron stress susceptible line. We then conducted whole genome expression analyses (RNA-sequencing) of root and shoots grown in iron sufficient and deficient conditions for 60 minutes. The analyses confirmed the speed and diversity of the soybean iron stress response and suggest multiple novel mechanisms for iron stress tolerance are present within the soybean germplasm collection. The contribution of candidate genes from within these regions to iron stress tolerance are currently being examined using gene silencing technology. Understanding and characterizing novel iron stress mechanisms will lead to the development of soybean lines with improved stress tolerance and greater yield. Investigating gene expression changes in response to micro- and macro- nutrient deficiencies in soybean. Iron is an essential micronutrient for plants. However, environmental conditions can render iron insoluble and unavailable for plant use. Phosphorous, one of the most limiting nutrients in agricultural production, is often plentiful in the soils, but slow diffusion and high fixation rates within the soils leaves little available for plant use. Understanding nutrient uptake and utilization in crops is critical to improving agricultural systems. We conducted genome-wide expression analyses (RNA-seq) of soybean shoots and roots exposed to multiple nutrient stress treatments of single or mixed nutrient deficiencies. Analyses of these data identified the same genes and pathways responding to iron and phosphate deficiency. However, the pathways are activated in response to iron deficiency and phosphate resupply. We also identified a subset of genes involved in plant memory. Combining these data with historical mapping data allowed us to identify candidate genes for virus induced gene silencing. Understanding how crops respond to multiple and diverse stress exposures, as occurs in the field, could have major implications in improving stress tolerance and preserving yield. Characterization of the resistance to Phakospora Pachyrhizi 1 B (Rpp1b) locus in soybean. Asian soybean rust (ASR) has spread to every major soybean producing country. Identification of resistance/defense genes is essential for improving commercial cultivars. 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, we identified candidates for Rpp1 using map-based cloning, gene expression analyses, gene silencing and whole genome expression. Now the same approach is being used to identify and characterize Rpp1b from PI594538A. Breeders and scientists can use the markers and genes developed by this project to incorporate resistance to ASR into improved commercial cultivars. Identification and characterization of plant proteins targeted by Phakopsora pachyrhizi, the cause of ASR. Outbreaks of ASR have been reported in all major soybean-producing countries, with yield losses as high as 80%. In collaboration with the ARS Foreign Disease-Weed Science Research Unit, we used a combination of map-based cloning and gene expression analyses to identify a candidate gene for resistance to Phakopsora pachyrhizi 1 (Rpp1) in PI200492. Unlike other Rpp genes, Rpp1 contained a novel integrated plant protein domain. It is hypothesized that integrated domains in resistance genes act as bait to catch would be pathogens. This suggests that pathogens would normally target proteins structurally related to the integrated domains to cause disease. Preventing the production of these proteins might limit pathogen infection. To test this hypothesis, we have developed gene silencing constructs for ten different genes that are structurally similar to the integrated protein in Rpp1. These constructs will be tested in susceptible lines to see if silencing slows or prevents infection by P. pachyrhizi. Identifying markers and genes linked to ASR resistance will help in developing improved soybean germplasm with enhanced disease resistance. Characterizing early resistance responses to brown stem rot in soybean. Brown stem rot, caused by the fungus Phialophora gregata, reduces soybean yields by 38%. Identifying and developing resistant lines is complicated by difficult phenotyping methods and misdiagnosis with other soybean diseases. To facilitate breeding for brown stem rot resistance, additional information on early defense responses is needed. In work funded by the National Institute of Food and Agriculture, we are using whole genome expression analyses to characterize gene expression changes in resistant and susceptible lines, at 12, 24 and 36 hours after infection in leaves, stems and roots. Resistant lines in the study contain the three known disease resistance genes Rbs1, Rbs2 and Rbs3. Breeders and scientists can use markers and genes identified by this project to incorporate resistance to 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, we have used map-based cloning to sequence across 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 subgroups within the R-genes. Two of the resistance genes contained a novel sequence domain similar to a defense protein from tobacco (NtPRP27). Silencing constructs developed for these two genes resulted in plants with localized lesions of dead cells, even in the absence of the pathogen. To understand this response further, we conducted whole genome expression analyses of root tissue from silenced plants. Bioinformatic analysis revealed that silencing these two candidate resistance genes induced a strong resistance response, not the susceptible phenotype that was expected. These results suggest resistance genes with novel domains may play a role in regulating resistance responses. Understanding the function of these novel proteins will provide new avenues for generating new disease resistance specificities for crop improvement. Characterizing the diversity of soybean root architecture. In plants, root architecture regulates water and nutrient acquisition, nutrient storage, microbe interaction in the rhizosphere, and how well the plant is anchored in the soil. 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 a high throughput phenotyping of 300 soybean lines representing the breadth and depth of the collection. The resulting phenotype data were paired with available single nucleotide polymorphism (SNP) data to perform a genome wide association study (GWAS) on multiple phenotypic traits. This study identified a large number of novel genomic regions associated with root architecture. In collaboration with Iowa State University colleagues, genes within these regions were analyzed and novel high priority candidate genes potentially responsible for these root architecture traits were identified. We have developed virus-induced gene silencing constructs to target these genes to determine if silencing of these genes alters root architecture. The genomic regions, markers, and genes identified by this study can all be used to tailor roots to maximize yield. Characterizing novel regions of the soybean genome for tolerance to iron deficiency. The soybean cultivar, Fiskeby III, exhibits tolerance to iron deficiency. Collaborators at the University of Minnesota conducted a genome-wide association of Fiskeby III that identified a small region on chromosome 5 responsible for a significant portion of the stress tolerance. In a project funded by the United Soybean Board and working with collaborators at the University of Minnesota, we confirmed progeny with the Fiskeby III background exhibited higher iron deficiency tolerance and have developed virus induced gene silencing constructs to silence six genes within this region. Silencing these genes in plants grown in soils leads to stunted phenotypes in Williams 82 soybean plants. Future work will determine if silencing these genes alters stress tolerance both in Williams 82 and in Fiskeby III. The findings of this study will provide plant breeding programs novel markers and genes associated with tolerance to iron deficiency stress. Characterizing the USDA core peanut collection by generating genotype information. Peanut production in the U.S. ranks third in the world. However, pests, disease and stress challenge peanut production. In a project funded by the National Institute of Food and Agriculture, and with collaborators from Iowa State University, we have isolated DNA from 1200 peanut lines in the USDA Core Peanut Germplasm Collection. Genotyping the collection will provide peanut researchers with valuable marker information that can be used by researchers to associate markers with traits of interests that can be used for peanut improvement.

Accomplishments
1. Characterizing the speed and diversity of the soybean iron stress response. Iron deficiency chlorosis (IDC) is a global crop production problem, significantly impacting yield. Since IDC-prone fields are not uniform and IDC tolerant lines tend to have low yield on non-IDC prone soil, farmers prefer to run the risk of using high yielding, IDC susceptible lines. However, in the North Central United States IDC yield loss estimates exceed $150 million per year. While various genetic approaches have been used to identify genes involved in iron stress tolerance from model species, few studies have focused on agronomically important crop species. Therefore, ARS researchers in Ames, Iowa designed a whole genome expression study to explore the speed and diversity of the soybean iron stress response and find new avenues for crop improvement. The researchers identified over 10,000 genes whose activity was changed in response to iron stress at 30, 60 or 120 minutes after the onset of iron stress. These analyses revealed that unlike model species, the genes involved in iron uptake, defense and regulation of cell replication are hallmarks of the soybean iron stress response. Further, the findings suggest soybean uses a novel root to shoot signal to initiate the iron stress response. These findings suggest a novel approach for soybean improvement and highlight the need for conducting additional IDC studies in diverse, agronomically important crop species.

2. Deconstructing the genetic architecture of iron deficiency chlorosis (IDC) in soybean. Iron is an essential micronutrient for plant growth and development. IDC, caused by calcareous soils or high soil pH, can limit iron availability, negatively affecting soybean yield. ARS researchers in Ames, Iowa with collaborators from Iowa State University used a genome-wide study of 460 diverse soybean plant introduction lines to identify significant markers, genomic regions, and novel genes associated with or responding to tolerance to iron deficiency. Sixty-nine genomic regions associated with IDC tolerance were identified including the historical quantitative trait locus on chromosome Gm03. Cluster analysis of significant markers in this region revealed four distinct linkage blocks, enabling the identification of multiple candidate genes for iron chlorosis tolerance. This study demonstrates that integrating cutting edge genomic approaches is a powerful strategy to identify novel iron tolerance genes and networks from diverse germplasm. Our results suggest that crops, unlike model species, have undergone selection for thousands of years, constraining and/or enhancing stress responses. This emphasizes the need to understand stress responses in economically valuable crop species, highlighting an important gap in research.

Review Publications
Xu, H., Zhang, C., Li, Z., Wang, Z., Jiang, X., Shi, Y., Braun, E., Mei, Y., Qiu, W., Li, S., Wang, B., Xu, J., Navarre, D.A., Ren, D., Cheng, N., Nakata, P.A., Graham, M.A., Whitman, S., Liu, J. 2018. The MAPK kinase kinase GmMEKK1 regulates cell death and defense responses. Plant Physiology. doi:10.1104/pp.18.00903.
Pedley, K.F., Pandey, A.K., Ruck, A.L., Lincoln, L.M., Whitham, S.A., Graham, M.A. 2018. Rpp1 encodes a ULP1-NBS-LRR protein that controls immunity to Phakopsora pachyrhizi in soybean. Molecular Plant-Microbe Interactions. 32:120-133.
Rutter, L., Vanderplas, S., Cook, D., Graham, M.A. 2019. ggenealogy: An R package for visualizing genealogical data. Journal of Statistical Software. 89(13)1-31. https://doi.org/10.18637/jss.v089.i13.
McCabe, C.E., Cianzio, S.R., O'Rourke, J.A., Graham, M.A. 2018. Leveraging RNA-Seq to characterize resistance to brown stem rot and the Rbs3 locus in soybean. Molecular Plant-Microbe Interactions. 31(10):1083-1094. https://doi.org/10.1094/MPMI-01-18-0009-R.