Updated April 28, 2025:
This semi-annual report outlines the progress made for the FY25 project, which commenced on October 1, 2024. The FY25 project marks the 3rd year of a three-year project aiming to explore, apply, and optimize RenSeq technology for accelerated identification of candidate R genes conferring resistance to various soybean pathogens that are prevalent in the Midwest region, and to expedite the development of disease-resistant soybean cultivars through precise R gene selection. We proposed five specific objectives to achieve these goals, some of which are consequential and others ongoing. Objectives 1 and 2 were fully achieved in FY 23 and FY24. In this report, we focus on progresses made on Objectives 3-5, which are continuous and proposed to be completed by the end of the whole project.
Objective 3: Analysis of R Gene Expression and Responses to Various Soybean Pathogens:
We have expanded gene expression analysis by including more soybean varieties with major QTLs conferring resistance to soybean pathogens including Phytophthora sojae and Phytophthora sojae, Phytophthora sansomeana, and frogeye leaf spot. These QTLs have been mapped to typical NLR (or R)-gene clusters and are likely to each be underlain by an NLR gene. Candidate Rps and Rpsan genes showing responses to respective pathogens have been pinpointed based on gene expression data. Some of these candidate genes have been further challenged by multiple pathogen races/isolates. Currently we are mapping the RNA-seq data to 26 reference soybean genomes representing genetic diversity as well as the assembled genomes from the donor lines of the resistance QTLs. We hope to be able to pinpoint the candidate genes for Rpsan1, Rps12, 13, 14, 15 and 16 through efforts from the Ma, Bhattacharyya, Wang, and Lin labs. A major QTL underlying resistance to Fusarium graminearum has been identified by Dr. Cai lab. Nevertheless, this QTL was not located in an NLR-gene cluster and thus we have decided to identify non-NLR genes responsive to infection with the pathogen, hoping to be able to find candidate gene (s) responsible for the resistance or genes involved in genetic pathways underlying the resistance. Dr. Lorenz lab initiated a gene expression experiment using Minsoy and Noir1 to help guide us towards candidate genes in a previously mapped QTL region on chromosome 7. Four biological replicates of each soybean genotype in a white mold inoculation treatment versus control were set up. The plants are currently growing and awaiting inoculation, followed by tissued collection for RNA extraction and analysis. PI Ma continues coordinating the efforts on analyses of gene expression and identification of candidate genes.
Objective 4: Evaluation of Resistance to Various Pathogens and Mapping of Major R Genes and QTL:
These are continuous efforts being made by all participating labs in this project. Different labs started evaluation of resistance in parental lines, development of QTL mapping populations, and QTL mapping and fine mapping at different times; nevertheless, all have been mapping satisfactory progresses.
- Co-PIs Dechun Wang at Michigan State University and Feng Lin at University of Missouri have fine-mapped Rpsan1 and identified candidate genes for Rpssan1 using RNA-seq data. To pinpoint Rpsan1, larger mapping population composed of ~1,300 lines has veen developed in the field in the 2024 field season and ~70 markers were developed for finer mapping of the candidate gene. Dr. Wang lab has also developed a mapping population for identification of gene(s) conferring resistance to frogeye leaf spot (Cercospora sojina). - Co-PD Feng Lin developed F4-derived mapping populations each composed of >180 progeny lines for dissecting Stink Bug resistance and Cercospora Leaf Blight resistance, and has started gene mapping in 2025. In addition, Dr. Lin lab found that PI 594527 shows resistance to both P. sojae and P. sansomeana and made numerous attempts to cross this line with several breeding lines, but was only successful with only one breeding lines due to the late maturity group of this PI line (MG IX).
- Co-PI Madan Bhattacharyya at Iowa State has made significant progress on mapping novel Rps genes. The two Rps genes, Rps12 and Rps13 were mapped to the Rps4/6 region located in the southern arm of Chromosome 18. The Rps12 gene was mapped to a locus, close to Rps 4, 6 and 13 loci. It is not established whether the Rps 4, 6 and 13 genes are allelic or tightly linked genes. To determine if Rps6 and Rps13 are allelic, we have been investigating the segregants generated from the cross between L89-1581 (Rps6) and PI399036 (Rps12Rps13). We studied 1,074 segregants for the recombination events between two indel markers that include the Rps6/12/13 region; and identified 358 recombinants. We identified 73 genotypes with single breakpoint and 33 recombinants with double breakpoints. We observed 182 recombinants with more than two break points which most likely originated from experimental artifacts. We are currently revisiting the SNP panel to understand the reason behind such an anomaly. We have reported earlier the development of the Rps6 contig by generating long read sequences through the Oxford Nanopore Technologies. This contig is expected to shed light on the structural variation in the Rps6/12/13 region. We observed considerable variation in seed yield (seed number/plant) among the heterozygous plants and among the recombinant individuals. We have gained evidence suggesting that recombination in the Rps6/12/13 region affects seed yield due to novel gene actions.
- Dr. Aaron Lorenz has made four crosses for mapping of wild mold resistance QTLs and five crosses for mapping of Brown Stem Rot resistance QTLs during this reporting period. Additionally, Dr. Lorenz lab screened our latest advanced breeding lines for resistance to two races of brown stem rot and identified several advanced breeding lines show some strong resistance to both races. These lines can be used as future breeding parents to create new cultivars, and new genetic mapping resources.
- Co-PI Guohong Cai at USDA-ARS has identified major QTL underlying Fusarium graminearum resistance and evaluated the levels of resistance of the patental lines and some of the progeny lines.
- Co-PI Carrie Miranda has mapping populations for white mold and brown stem rot, and obtained F3 seeds. It is anticipated to receive F5 seeds in the fall of 2025 and then start phenotyping in collaboration with the NDSU soybean pathologist immediately.
Objective 5: Development of Candidate R-Gene-Based Molecular Markers for Precision Breeding
This is an ongoing and continuous effort at different stages of breeding for various diseases in different states. Past efforts focused on known genes. Our goal is to integrate novel, more effective R genes into elite soybean varieties in public breeding programs in these collaborating states. Lorenz lab has created and advanced four new breeding populations with the Rps6 gene, known to confer good resistance to most of the isolates in Minnesota and are developing resistant soybean lines with markers linked to this gene. Despite the effectiveness of this gene in resistance to Phytophthora, it is seldomly used in commercial breeding and mostly unavailable in current commercial cultivars. The four new breeding populations developed by the Lorenz lab were advanced to F2 and F4 populations in the winter nursery, and will be planted for another generation of advancement this coming summer. In addition, integration of Rps11 in elite soybean breeding lines has been initiated by all participating breeding programs. The females used for mapping population development by the Miranda lab are high yield North Dakota lines, and it is thus very likely that highly-yielding and resistant progeny lines will be developed during the gene discovery process.
Publications:
C Detranaltes, C Quigley, Q Song, J Ma, G Cai, 2025. A Novel Quantitative Trait Locus (QTL) Reduces Fusarium graminearum Infection in Glycine max Seedlings. Phytopathology.