Project Details:

Title:
An integrated approach to enhance durability of SCN resistance for long-term, strategic SCN management (Phase III)

Parent Project: An integrated approach to enhance durability of SCN resistance for long term strategic SCN management
Checkoff Organization:North Central Soybean Research Program
Categories:Insects and pests, Insects and pests, Nematodes
Organization Project Code:
Project Year:2022
Lead Principal Investigator:Andrew Scaboo (University of Missouri)
Co-Principal Investigators:
Thomas Baum (Iowa State University)
Gregory Tylka (Iowa State University)
Melissa Mitchum (University of Georgia)
Brian Diers (University of Illinois at Urbana-Champaign)
Matthew Hudson (University of Illinois at Urbana-Champaign)
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Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

The soybean cyst nematode (SCN), or Heterodera glycines, is the most damaging pathogen to soybean production in North America and current annual yield losses are estimated at more than $1.2 billion. Though SCN-resistant soybean varieties are available to minimize yield loss, producers are faced with limited options for rotation once virulent SCN populations have developed in their fields. The widespread lack of diversity for SCN resistance genes utilized and available for farmers in commercial soybean varieties has significantly increased the prevalence of virulent SCN populations across the mid-west (mainly HG 1.2.5.7), and reduced the effectiveness of current sources of resistance. Thus, we have two major research challenges that, when successfully achieved, will enable us to develop more efficient management practices for this pest in the future.
• Nematologists need to 1) identify the SCN genes, and their functionality, required for the adaptation to reproduce on resistant varieties, 2) use these as molecular markers to monitor nematode population shifts in the field in real time, and 3) exploit this knowledge to help plant breeders identify the best resistance gene combinations for short- and long-term nematode management.
• Breeders need to increase the availability of genetically diverse SCN resistance in commercial soybean varieties and work with nematologists to determine the most effective rotation practices that preserve the efficacy of the known sources of SCN resistance.
To address these issues we are proposing the first year of Phase III of an integrated, collaborative, and multi-state project among plant breeders, molecular biologists, bioinformaticians, and nematologists. Our proposed objectives specifically address the following key research area in the current RFP - Basic and applied research directed at soybean disease, nematode, insect pest and abiotic stress biology, management and yield loss mitigation, including new and emerging threats, of consistent or potentially significant economic impact across the North Central Region - and this proposed research complements funding from federal agencies, as well as respective state and United Soybean Board check-off support. The genetic resources developed and knowledge gained from this project will provide immediate and long-term benefit to soybean producers and researchers in both the private and public sector.

Project Objectives

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Sub-objective 1.1: Combine, compare, and catalogue the genomes that compromise the SCN pan-genome. (Hudson, Baum, Mitchum)
• Continuously update SCNBase with novel sequence data and also with a complete database of all known SCN effectors and variants
• Finish, annotate and publish all SCN genome sequencing projects started in phase II and analyze gene variants between HG types
• Establish the proximity labeling approach for use in our nematode effector studies and identify comprehensive interactomes of SCN effectors in planta
• Understand on a molecular level how SCN is able to inactivate certain soybean defense mechanisms
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
• Sequence populations of SCN as virulence changes and analyze for selected genes
• Complete the analyses of early gland-expressed gene differences between virulent and avirulent SCN populations
• Generate later-stage gland transcriptomes of virulent and avirulent SCN populations
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
• Successfully perform the Pool-seq strategy and identify SCN genome regions conditioning virulence phenotypes
• Validate potential candidates for a correlation with virulence in field populations of known HG types using molecular-based assays
Objective 2: Complete the evaluation of how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles. (Diers, Scaboo, Tylka, Mitchum)
• Upon completion of this project in 2024, we will have the ability to recommend specific rotation strategies to reduce SCN populations densities and combat shifting virulence in SCN populations due to the continuous use of PI 88788 type varieties
• These data are unique only to this project and will likely be a foundation for the long term management of SCN for farmers by precisely using genetic resistance in a rotation program beyond the traditional crop rotation
Objective 3: Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers and inform growers on effective rotation schemes designed to protect our resistant sources. (Tylka, Mitchum)
• The project will be described during interviews conducted by Mitchum and Tylka and the information will appear in print media, on the radio, and in presentations given at large farmer-oriented events such as Commodity Classic and the Farm Progress Show. Also, information and results from this project will be distributed to mass farmer audiences through the communication vehicles used by the SCN Coalition (videos on YouTube, videos on TheSCNCoalition.com, press releases, etc.)
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Diers)
• The data generated form this portion of the project is crucial for evaluation and release of new SCN resistant soybean varieties, and it is utilized by both public and private soybean breeders to request material for incorporation into their respective programs
Objective 5: Diversify the genetic base of SCN resistance in soybean by developing and evaluating germplasm and varieties with new combinations of resistance genes in high-yielding backgrounds. (Diers, Scaboo)
• Development and release of new germplasm and varieties with unique SCN resistance that will be transferred to private companies as well as other public and private soybean breeders and researchers
• These new germplasm and varieties will lead to improved productivity and profitability for farmers by maintaining yield potential in heavily infested fields

Project Deliverables

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Sub-objective 1.1: Combine, compare, and catalogue the genomes that compromise the SCN pan-genome. (Hudson, Baum, Mitchum)
• Continuously update SCNBase with novel sequence data and also with a complete database of all known SCN effectors and variants
• Finish, annotate and publish all SCN genome sequencing projects started in phase II and analyze gene variants between HG types
• Establish the proximity labeling approach for use in our nematode effector studies and identify comprehensive interactomes of SCN effectors in planta
• Understand on a molecular level how SCN is able to inactivate certain soybean defense mechanisms
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
• Sequence populations of SCN as virulence changes and analyze for selected genes
• Complete the analyses of early gland-expressed gene differences between virulent and avirulent SCN populations
• Generate later-stage gland transcriptomes of virulent and avirulent SCN populations
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
• Successfully perform the Pool-seq strategy and identify SCN genome regions conditioning virulence phenotypes
• Validate potential candidates for a correlation with virulence in field populations of known HG types using molecular-based assays
Objective 2: Complete the evaluation of how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles. (Diers, Scaboo, Tylka, Mitchum)
• Upon completion of this project in 2024, we will have the ability to recommend specific rotation strategies to reduce SCN populations densities and combat shifting virulence in SCN populations due to the continuous use of PI 88788 type varieties
• These data are unique only to this project and will likely be a foundation for the long term management of SCN for farmers by precisely using genetic resistance in a rotation program beyond the traditional crop rotation
Objective 3: Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers and inform growers on effective rotation schemes designed to protect our resistant sources. (Tylka, Mitchum)
• The project will be described during interviews conducted by Mitchum and Tylka and the information will appear in print media, on the radio, and in presentations given at large farmer-oriented events such as Commodity Classic and the Farm Progress Show. Also, information and results from this project will be distributed to mass farmer audiences through the communication vehicles used by the SCN Coalition (videos on YouTube, videos on TheSCNCoalition.com, press releases, etc.)
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Diers)
• The data generated form this portion of the project is crucial for evaluation and release of new SCN resistant soybean varieties, and it is utilized by both public and private soybean breeders to request material for incorporation into their respective programs
Objective 5: Diversify the genetic base of SCN resistance in soybean by developing and evaluating germplasm and varieties with new combinations of resistance genes in high-yielding backgrounds. (Diers, Scaboo)
• Development and release of new germplasm and varieties with unique SCN resistance that will be transferred to private companies as well as other public and private soybean breeders and researchers
• These new germplasm and varieties will lead to improved productivity and profitability for farmers by maintaining yield potential in heavily infested fields

Progress of Work

Updated April 6, 2022:
A description of relevant progress for principle and co-principle investigators is below for each objective and sub objective in our proposal.

Objective 1.1: The Baum group has accomplished an analysis of gene expansions and contractions, as well as the presence and absence of gene families in H. glycines compared to 13 related species in the Tylenchomorpha. We are functionally characterizing these gene families and their association with previously published H. glycines effectors, as well as their secretory status, expression, nuclear localization, and impact of their variability across 15 populations of H. glycines. We have shown that 551 gene expansions in sedentary nematodes differentiate them from migratory nematodes, 124 gene expansions in cyst nematodes differentiate them from root-knot nematodes, and 1,100 gene expansions in H. glycines differentiates it from Globodera species. We have also shown a number of gene gain and loss events that have directly impacted the H. glycines genome, many of which have shown an atypical inheritance pattern across related species. In one instance, we show 175 gene families that are only shared between H. glycines and Meloidogyne species, 15 of which are targeted to the secretory pathway, including two predicted H. glycines effectors. Using effectors from relatively closely related species, we have identified 405 putative effector families present in H. glycines, though only 137 of these families contain a gene that produces a protein with a signal peptide for secretion. Of these 137 putative effectors, 11 were associated with gene expansions in sedentary nematodes compared to migratory nematodes, 6 were associated with the expansions of gene families in cyst nematodes versus root-knot nematodes, and 24 were associated with the expansion of Heterodera gene families in comparison to Globodera. Using population variation from 15 distinct populations of H. glycines, we were able to show which genes typically produce secreted proteins across the assayed SCN lines. In this respect, we show that 59 gene expansion families have consistent signals for secretion across multiple populations, even though they were not predicted to be secreted in the previously published TN10 genome. These results warrant further research into gene evolution. We also have developed plans to update SCNBase with new RNAseq from the recently published J2 gland RNAseq and will integrate gene family criteria. To complement the methods of identifying genes involved in the parasitism of H. glycines, we have undertaken a genome assembly and annotation project of male and female specimens. Males and females dramatically change their parasitic behavior with the onset of adulthood and, thus, studying their genomes and transcriptomes will reveal insights into gene functions, particularly for effectors. We sequenced male and female genomes using nanopore long reads and added male and female specific RNA-seq to this analysis to better predict the gene variation among the sexes. Since the last report we have scaffolded these male and female genomes with HiC to obtain 9 pseudomolecules for each genome. Interestingly, we found large differences in genome size between these nanopore genomes (male: 115.5Mb, female: 112.6Mb) and our previously published TN10 genome (158Mb), which is likely attributable to the differences in sequencing technology and assembly software. Subsequently, we used male and female RNAseq with Braker to annotate genes in each genome, finding that the disparity continues between these nanopore genomes and TN10 genome at the genic level. In the male and female gene annotations, we found 16,421 genes and 16,530 genes, respectively. These gene annotation totals are substantially reduced from our previous annotation of the TN10 genome at 22,465 genes, though the gene copy number reduction is in proportion with the genome size reduction. We have begun to compare the genomes at the level of gene structure and expression using Orthofinder and differential expression analyses. Thus far we have found that 9-10,000 of differentially expressed genes cluster to the same orthologue family, leaving slightly more than half of the genes with significant divergence between the sexes. The Mitchum group submitted the following manuscript during the past quarter - Verma A, Lin M, Smith D, Lee C, Walker JC, Hewezi T, Davis EL, Hussey RS, Baum TJ, Mitchum MG. A novel cyst nematode effector (2D01) targets the Arabidopsis HAESA receptor-like kinase. Mol. Plant-Microbe Interact.

Objective 1.2: Phase III of this project sees the Baum group developing further resources to expand the toolbox that will aid us in understanding SCN virulence. Focusing once again on the three gland cells that SCN uses to produce the tools (effectors) required for establishment of successful infection, as well as defense suppression, we have improved on the technology used in phase II. Taking advantage of recent developments toward single cell sequencing, and specifically new technology available for the generation of single cell RNA-seq libraries, we have applied these technologies to our work with gland cell isolation and transcriptomics in SCN. We successfully generated single cell RNA-seq libraries for our avirulent (PA3) and virulent (MM10) populations. These libraries represented four biological replications of 5 dorsal and subventral gland cells per rep. This was our group’s first attempt at applying single cell sequencing technologies to SCN transcriptomics and required a fair amount of optimization. Through this process, we developed a technique for live gland cell collection, versus fixed tissue collection, which seems to have improved the quality of RNA collected and allowed us to collect picogram quantities of RNA from individual gland cells. The analysis of the libraries generated via the single cell sequencing approach show that these libraries are as good, if not slightly better, than the libraries previously generated for this project and previously in our lab group. The overall coverage of genes identified within these new J3 single cell libraries, with reference to the SCN TN10 genome is slightly greater than the coverage of genes identified with our previous pooled gland cell parasitic J2 libraries. We identified a total of 14,667 and 14,000 genes at a normalized read count of at least 5 counts from the SCN PA3 J3 and MM10 J3 libraries, respectively. This is compared to a total of 12,495 and 12,289 genes at the same read count cutoff for the respective SCN populations with our older technology. While we cannot rule out that this could be due to life stage differences, it certainly points to the fact that this new technology can yield similar, if not better numbers of identified genes from lesser amounts of material. We are currently working on the transcriptomics to generate both broad and specific comparisons of both the life stages and virulence differences that exist in our two SCN reference populations. This will provide an extensive and novel look at SCN virulence at this level.

All seven of the genomes of the additional Hg types are now assembled, and the assemblies are completed and frozen. They are ready to distribute or submit to NCBI, however we need to annotate and analyze them before publishing a paper on the genomes, and to make them useful to more people in the group.

The Hudson group has been proceeding with analysis in several ways. Firstly, genome synteny for the 7 newly assembled SCN strains was compared with mummer (dotplots) and circos (BLAST hits) to the chromosome level SCN assembly TN10 (Masonbrink et al 2021). These data showed that the assembled 9 chromosomes were in similar structure with the previously reported TN10, although not identical. We started the genome functional and structural annotation. Repetitive elements modeled with RepeatModeler and quantified repetitive content with RepeatMasker. The repeat analysis showed repeat content was similar across strains with respect to repeat class and quantity. Subsequently, we began gene prediction using RNAseq evidence (previously reported Gardner et al 2018 & Lian et al 2019) and protein models (from previously reported TN10 Masonbrink et al 2021) and other cyst nematodes including Globodera pallida (Cotton et al 2014) and Globodera rostochiensis (Eves-van der Akker 2016). Preliminary gene models show similar structure to the previous assemblies gene models. Our future work will include refinement of gene models and subsequent functional annotation of the gene models.

Objective 1.3: The Baum group has indicated that characterization of the function of 28B03 effector family has been advanced to a natural stopping point to publish the first extensive report on this effector’s function during parasitism. A complete manuscript has been written and all data compiled. We are waiting on last additions and reviews from co-authors and then will submit the manuscript to start the publication process. Given the thorough study of this effector, this manuscript is a very extensive and thorough functional assessment of this effector and its ability to interfere with a plant signal transduction pathway. In short, 28B03 targets a novel plant kinase protein, which in turn cooperatively with another plant kinase leads to the initiation of signal transduction processes that initiate a subset of plant defense responses. We have shown that the 28B03 effector interferes with this signal transduction, thus, compromising plant defenses and leading to increased host susceptibility. Through our confocal experiments utilizing co-localization studies of 28B03 and the identified kinases, we can infer that our proposed cascade model is valid, as the effector and kinases are co-localized together in these assays. This work identifies a potential plant target to increase plant resistance (i.e., one can now devise mechanisms to interfere with the inhibitory function of 28B03 to prevent the nematode from inactivating a plant defense signal transduction kinase).

The Mitchum lab collected sufficient genetic material of two pairs of SCN populations (unadapted or adapted to reproduce on resistant soybeans) and optimized their DNA extraction procedure to meet the stringent requirements necessary for the Pool-Seq strategy. We have completed the sequencing and bioinformatic analysis is currently underway. The Pool-Seq approach should help guide us toward the candidate virulence regions important for breaking the Peking-type (Rhg4-mediated) resistance.

Objective 2: The Diers group is preparing and distributing seed for all collaborators for the 2022 SCN resistance source rotation study. This will be the fourth year of the rotation study and we will be rotating the plots back to what was grown in them during 2020. We now have egg numbers from the plots grown in 2019-2021 and HG type values from 2019-2020. These results show that continuous planting of PI 90763 had the lowest egg number increases in plots across the three years. However, the continuous production of this source of resistance is selecting nematodes that can overcome this resistance and the female index (FI) in plots grown with PI 90763 was 41 after 2020, which indicates that the nematode population in these plots may start increasing. The rotation that had the lowest increase in egg numbers over the three years is rhg1b+soja+ch10 in 2019 followed by PI 90763 in 2020 and then rhg1b+soja+ch10 in 2021. This rotation also showed a low increase in egg numbers in other states and did not increase the FI on PI 88788 or Peking. The study will be repeated in 2022 to provide further insight into these resistance sources.

The Scaboo group processed soil samples for determining egg density and HG types for each microplot. The egg density results showed that there is an increasing trend in SCN population density in the third year of rotation except for plots with continuous PI 90763 and rhg1-b+G. soja+10 rotated with PI 90763. There was also a significant reduction in the percentage change in the egg count for these two treatments. The HG type data also showed that the continuous rhg1-a + rhg4 and continuous PI 90763 treatments facilitated the development of more virulent nematode populations with HG type (HG type: 1.2.3-; race 4), where nematode had adapted on the Peking type resistance sources (PI 90763, Peking and Pickett). Similarly, nematode populations from treatments involving rotations of rhg1-b (and/or stacked resistances) with PI 90763 showed to have reduced female index on PI 88788, PI 90763, and Peking indicator lines (Figure 2B). We look forward to the upcoming planting season where we plan on rotating an additional cycle of continuous and rotated schemes as conducted previously in 2020.

The Mitchum group was sent SCN material recovered from the continuous and rotation microplots and they were increased, processed for eggs, and archived as part of a wormplasm collection for future sequencing efforts to pinpoint virulence genes.

At the conclusion of the 2021 growing season, The Tylka group collected two separate multi-core soil samples from each microplot in the experiments conducted in central Iowa and north central Iowa. One set of soil samples from each experiment were processed at Iowa State University to determine the end-of-season SCN egg population density in each microplot. The second set of soil samples were sent to the University of Missouri for HG Type testing to determine how the soybean genotypes grown in the microplots in 2021 have affected or shifted the virulence profiles (HG types) of the SCN populations from the 2020 growing season and from the initial SCN populations that were added to the microplots in the spring of 2019.

Preliminary data analysis show some trends in changes SCN population densities. In both experiments, the greatest SCN population densities occurred in microplots in which the susceptible soybean variety was grown. Most of the microplots that had continuous cropping of the same resistance in 2019, 2020, and 2021 had greater SCN population densities than microplots in which resistant genotypes were rotated in 2019, 2020, and 2021. The lowest population densities occurred in microplots where soybeans with SCN resistance from PI 90763 were grown. The microplots in which soybeans with rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 SCN resistance were grown (collectively referred to as genotypes having “PI88788-type” resistance) in 2019, rotated to soybeans with PI 90763 SCN resistance in 2020, and then rotated back to the same resistance as in 2019 had increased SCN population densities in 2021 compared to population densities at the end of both previous years. Even though SCN population densities declined after rotating from genotypes with PI88788-type resistance in 2019 to rhg1-a + rhg4 (or “Peking-type”) resistance in 2020, population densities increased to levels greater than in 2019 and 2020 when genotypes with PI88788-type resistance were again grown in the microplots in 2021. Similarly, plots in which rhg1-a + rhg4 or Peking-type resistance was grown in 2019 then rotated to rhg1-b, rhg1-b + soja, or rhg1-b + soja + ch10 SCN (PI88788-type resistance) resistance in 2020, and then back to rhg1-a + rhg4 resistance in 2021 had greater SCN population densities at the end of the 2021 growing season than in the previous two years.

The results of the HG Type tests on the SCN populations in the microplots at harvest in 2020 revealed that almost all of the SCN populations at both experimental locations had increased virulence from 2019, with the SCN populations in each plot having elevated female indices (FI) on Peking and most having increased FIs on PI 88788. SCN populations in the two experiments were HG Type 1.2 or 1.2.3 and the FIs ranged from 9-33% on Peking in Kanawha, 18-53% on Peking in Ames, 40-64% on PI 88788 in Kanawha and 33-64% on PI88788 in Ames. In both experiments, the SCN populations in microplots in which PI 90763 or rhg1-a + rhg4 (Peking-type) resistance was grown had elevated FIs on PI 90763, with a range of 0%- 23% in Kanawha and 2%-32% in Ames. The SCN populations in microplots that were rotated from rhg1-a + rhg4 resistance to the three different genotypes containing rhg1-b (namely rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10) had decreased FIs on PI 90763 from 2019 to 2020. Changes in virulence (FIs) on PI437654 were not detected in SCN populations in any of the microplots with any of the cropping sequences. HG Type test results of the SCN populations in soil samples collected from the microplots at harvest in 2021 are not yet available.

Objective 3: Greg Tylka conducted 19 interviews with radio and newspaper/magazine journalists and gave 12 presentations (in person and virtual) from October 2021 through March 2022. The loss of effectiveness of PI88788 SCN resistance was discussed in every interview and presentation, and this current NCSRP-funded research project was mentioned and described whenever time/space permitted.

Melissa Mitchum is serving as the Chair of the organizing committee for the 2022 National Soybean Nematode Conference (NSNC). The location, venue, and draft schedule was developed during this reporting period. Save the date flyers and the scientific program will be developed and announced in the next reporting period.

Objective 4: The Diers group has continued to organize these tests. The results from the 2021 SCN Regional Test were received from cooperators and summarized in a report. The initial version of the report was sent to cooperators on December 16th and the final version was delivered on January 13th. These timely deliveries of results are important so cooperators can make decisions on selections in time for winter crosses and nurseries. Plans have been made for the 2022 SCN Regional Test. This test will include 225 entries that range from MG 0 to IV. The tests have been organized, the seed has been received from cooperators, repackaged, and is being shipped to cooperators. Arrangements also have been made to test the lines for SCN resistance in a greenhouse at the University of Missouri. We are moving the tests to the University of Missouri because the nematology lab at the University of Illinois has been slow in providing test results.

Objective 5: The Diers and Scaboo groups have continued to advance breeding efforts towards the development of cultivars with novel SCN resistance. For this reporting period, we are excited to report that we have now completed successful crossing attempts (3 back-crosses) using PI 90763 as a donor parent, and LD11-2170 and SA13-1385 as recurrent parents, for three major genes associated with resistance to virulent nematode populations (rhg1-a, rhg2, and Rhg4). For each crossing attempt, we have identified desirable F1 plants using marker assisted selection, and we have sped up the process by utilizing our winter nurseries in Hawaii and Puerto Rico for the last two years. As I type this report, our staff and student are in Kekaha Kauai Hawaii tissue sampling BC3F2 plants to identify homozygous individuals with desired combinations of our target genes. During the summer of 2022, we will grow plant rows derived from selected plants, and our first yield trials of this material will be in the summer of 2023.

Final Project Results

Benefit to Soybean Farmers

The soybean cyst nematode (SCN), or Heterodera glycines, is the most damaging pathogen to soybean production in North America and current annual yield losses are estimated at more than $1.2 billion. Though SCN-resistant soybean varieties are available to minimize yield loss, producers are faced with limited options for rotation once virulent SCN populations have developed in their fields. The widespread lack of diversity for SCN resistance genes utilized and available for farmers in commercial soybean varieties has significantly increased the prevalence of virulent SCN populations across the mid-west (mainly HG 1.2.5.7), and reduced the effectiveness of current sources of resistance. Thus, we have two major research challenges that, when successfully achieved, will enable us to develop more efficient management practices for this pest in the future.
• Nematologists need to 1) identify the SCN genes, and their functionality, required for the adaptation to reproduce on resistant varieties, 2) use these as molecular markers to monitor nematode population shifts in the field in real time, and 3) exploit this knowledge to help plant breeders identify the best resistance gene combinations for short- and long-term nematode management.
• Breeders need to increase the availability of genetically diverse SCN resistance in commercial soybean varieties and work with nematologists to determine the most effective rotation practices that preserve the efficacy of the known sources of SCN resistance.
To address these issues we are proposing the first year of Phase III of an integrated, collaborative, and multi-state project among plant breeders, molecular biologists, bioinformaticians, and nematologists. Our proposed objectives specifically address the following key research area in the current RFP - Basic and applied research directed at soybean disease, nematode, insect pest and abiotic stress biology, management and yield loss mitigation, including new and emerging threats, of consistent or potentially significant economic impact across the North Central Region - and this proposed research complements funding from federal agencies, as well as respective state and United Soybean Board check-off support. The genetic resources developed and knowledge gained from this project will provide immediate and long-term benefit to soybean producers and researchers in both the private and public sector.

Performance Metrics

For each objective below, we have listed the respective KPI’s and deliverables. The economic impact of these deliverables will be in the form of both increased knowledge, as well as direct profitability for farmers through improved management strategies and the availability of novel soybean germplasm and varieties.

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Sub-objective 1.1: Combine, compare, and catalogue the genomes that compromise the SCN pan-genome. (Hudson, Baum, Mitchum)
• Continuously update SCNBase with novel sequence data and also with a complete database of all known SCN effectors and variants
• Finish, annotate and publish all SCN genome sequencing projects started in phase II and analyze gene variants between HG types
• Establish the proximity labeling approach for use in our nematode effector studies and identify comprehensive interactomes of SCN effectors in planta
• Understand on a molecular level how SCN is able to inactivate certain soybean defense mechanisms
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
• Sequence populations of SCN as virulence changes and analyze for selected genes
• Complete the analyses of early gland-expressed gene differences between virulent and avirulent SCN populations
• Generate later-stage gland transcriptomes of virulent and avirulent SCN populations
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
• Successfully perform the Pool-seq strategy and identify SCN genome regions conditioning virulence phenotypes
• Validate potential candidates for a correlation with virulence in field populations of known HG types using molecular-based assays
Objective 2: Complete the evaluation of how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles. (Diers, Scaboo, Tylka, Mitchum)
• Upon completion of this project in 2024, we will have the ability to recommend specific rotation strategies to reduce SCN populations densities and combat shifting virulence in SCN populations due to the continuous use of PI 88788 type varieties
• These data are unique only to this project and will likely be a foundation for the long term management of SCN for farmers by precisely using genetic resistance in a rotation program beyond the traditional crop rotation
Objective 3: Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers and inform growers on effective rotation schemes designed to protect our resistant sources. (Tylka, Mitchum)
• The project will be described during interviews conducted by Mitchum and Tylka and the information will appear in print media, on the radio, and in presentations given at large farmer-oriented events such as Commodity Classic and the Farm Progress Show. Also, information and results from this project will be distributed to mass farmer audiences through the communication vehicles used by the SCN Coalition (videos on YouTube, videos on TheSCNCoalition.com, press releases, etc.)
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Diers)
• The data generated form this portion of the project is crucial for evaluation and release of new SCN resistant soybean varieties, and it is utilized by both public and private soybean breeders to request material for incorporation into their respective programs
Objective 5: Diversify the genetic base of SCN resistance in soybean by developing and evaluating germplasm and varieties with new combinations of resistance genes in high-yielding backgrounds. (Diers, Scaboo)
• Development and release of new germplasm and varieties with unique SCN resistance that will be transferred to private companies as well as other public and private soybean breeders and researchers
• These new germplasm and varieties will lead to improved productivity and profitability for farmers by maintaining yield potential in heavily infested fields

Project Years