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 and analyze 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 from 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
Updated April 4, 2023:
A description of relevant progress for principal and co-principal investigators is below for each objective and sub objective in our proposal. Our team has made tremendous progress in accomplishing our research goals, conducting field experiments, publishing refereed journal articles, and communicating our results to scientists and soybean producers. We attended a wonderful SCN conference in Savannah Georgia in December of 2022 where many project members and their respective staff and students presented research supported by this project including Dr.’s Mitchum, Tylka, Diers, and Scaboo. We are planning our next group meeting for the first week of April 2023 to discuss current research progress and goals, and we are on track to continue our cutting-edge research in soybean cyst nematode biology, management, and breeding for novel resistance.
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)
The Baum lab finished the analysis of gene expansion in the Tylenchomorpha and have identified a number of interesting gene candidates for further analysis. Multiple genes were found to be involved in host defense suppression, host cell remodeling, manipulation of host ion concentrations, redox, and manipulating host metabolism. SCNBase has undergone some extensive updates in the past months. We clustered the predicted proteins from each of the available SCN genome assemblies (TN10 draft, TN10 pseudomolecule, and X12), so that users can more easily infer how genes are related between assemblies. In addition, we’ve aligned genes from each of the three assemblies to each other to provide better spatial interpretation of different gene predictions. We’ve also integrated BLAST databases for each of these genomes, their transcripts, and predicted proteins. There were several updates to genomic tracks in JBROWSE: transposon annotations, genes that overlap with repeats, tandem repeats, large structural variants, SNPs for all available short read data from other populations, noncoding RNAs, alignments of all H. glycines ESTs, NCBI nucleotide entries, all long read transcript sequences available, and the transcriptome from Gardner et al 2018. We combined the repetitions of all individual RNAseq samples to allow users to better evaluate expression of a gene in JBROWSE. Many of the information pages on SCNBase were updated or added. We summarized the statistics for each of the three genome assemblies and compiled these stats on a single page that is linked to SCNBase and included an explanation for better interpretation. All data that was provided with the pseudomolecule assembly of TN10 now has an improved explanation of abbreviations associated with gene conditions in the Features Database. We better defined the data types that can be found on SCNBase and have provided a list of all public SCN data. We also converted the Research page to display all papers that have been published through our NCSRP funding.
Previously, the Mitchum lab employed a dual effector prediction strategy that coupled the traditional secreted protein prediction strategy with a newly developed nematode effector prediction tool, N-Preffector, to identify novel effector candidates in a de novo transcriptome assembly of the pre-parasitic and parasitic life stages of H. glycines with potential roles in virulence. From this analysis, eight novel candidate effectors with high to moderate expression in the gland RNA-seq dataset were identified. We mined the SCN pseudomolecule genome assembly to determine the gene structures and genomic organization of these sequences and have moved forward for further analysis in sub-objective 1.3 below.
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
Previously, the Hudson group developed a successful method to extract genomic DNA from a single J2 SCN with sufficient quality and quantity for whole genome sequencing. Different DNA library construction kits were tested at Roy J. Carver Biotechnology Center on campus and the best performing one for our purposes was selected. We have now accomplished collecting DNA samples from a large number of individuals from two different SCN populations, MM1 and MM2. From the virulence perspective, MM1 and MM2 SCN populations are significantly different from each other. Although both are originally driven from a common source, they have been adapted and multiplied separately in two different ways for more than ten years to make a virulent and an avirulent population. Therefore, these data can help us to perform a comparative population genomic analysis to find SNPs and genetic regions under selection for SCN virulence. 192 samples per population were sent to the Roy J. Carver Biotechnology Center for DNA library construction and sequenced on a NovaSeq 6000. The sequence data were recently received and we are currently checking the data quality. We will then align them to our newly-generated reference-quality genome for this population and begin population genetic analysis.
The Baum group continued to build resources in the form of gland cell-specific libraries. We have continued to develop libraries for the pre-infective J2 life stage, and now have the avirulent (PA3) population library constructed. Notably, we have some encouraging preliminary data (unpublished) from a collaboration in another plant-parasitic nematode, in which we prepared an analogous library from the same pre-infective life stage. What is significant is that this data represents the first evidence that this new live single-cell library approach works very well to generate a substantial list of candidate effectors from very limited starting material. There are also other very intriguing results from this specific life stage that we look forward to confirming in SCN. Additionally, using our approach to separate gland cell types, coupled with our ability to generate single-cell libraries, we have constructed our first subventral gland-specific libraries from pre-infective J2 stages of SCN and have recently submitted this for sequencing. We are hopeful that this will yield a useful dataset from which we can perform the subtraction strategy described previously and that this strategy will yield information on which effectors are present and active in each gland type in the pre-infective J2 stage of SCN.
The Mitchum lab has completed the Pool-Seq analysis of the two pairs of SCN populations un-adapted or adapted to reproduce on resistant soybeans. Previously, we reported the identification of five genomic regions spanning four chromosomes showing strong signatures of selection, as determined by the population differentiation (FST) estimates from the entire single nucleotide polymorphism (SNP) dataset containing approximately 800 K SNPs. Although FST analyses show strongly differentiated genomic regions, they are generally known to be prone to false positives, which is why we needed to gain further confidence in these candidate regions. For this reporting period, we confirmed extra evidence of selection in these regions by conducting the Fisher’s Exact test as well as principal component analysis (PCA)- and XtX-based approaches. After filtering for the overlapping SNPs identified from multiple outlier detection methods, we discovered a total of 316 significantly overly differentiated SNPs from both pairs of SCN populations. These SNP-harboring candidate genes are now being tested for their correlation to virulence. We have also been preparing a manuscript for our Pool-Seq findings which we hope to publish this year.
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
During this most recent phase of the project, the Baum group has focused on developing tools for the scientific community to employ when conducting in planta SCN studies. Most notably, we have constructed a set of GATEWAY-compatible vectors to facilitate cloning of a gene of interest in frame with different epitopes to perform functional analysis on soybean roots. Those vectors allow the selection of transgenic roots via the mCherry fluorescence or via the expression of the novel non-invasive reporter gene RUBY (which produces a red pigmentation). To confirm functionality of those vectors, we successfully express different subcellular markers in soybean roots. Along with those vectors, we are establishing different approaches to determine and study the interacting soybean proteins for SCN effectors such as immunoprecipitation or proximity-labelling followed by mass-spectrometry.
The Mitchum group has continued characterization of the 8 novel effector candidates identified under objective 1.1 is underway. We have profiled the expression of these genes in SCN throughout the life cycle and initiated studies to confirm where they effectors potentially localize within host cells after secretion by the nematode.
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)
The Scaboo group completed processing of the fall 2022 soil samples from the Missouri location. SCN egg density and HG type test results were recently distributed for data analysis. SCN from each microplot were increased and sent to the Mitchum group for archiving. We have a group meeting in April of 2023 to review the data analysis in preparation for manuscript submission.
In October 2022 the Tylka group collected two separate multi-core soil samples from all microplots in each of the two microplot experiments conducted in Iowa. One set of samples were processed at Iowa State University to determine the end-of-season SCN egg population density in each microplot. The second set of samples were sent to the University of Missouri for HG type testing to determine how soybean genotypes grown in the microplots in 2022 affected or shifted the virulence profiles of the SCN populations and how the virulence phenotypes differed from the virulence phenotype of the SCN populations used to infest the microplots initially. Overall, SCN population densities increased in all microplots over four years and year-to-year differences occurred among plots with rotated soybean genotypes, but results varied somewhat between locations. For most microplots, rotated treatments had lower SCN population densities than treatments planted with the same genotypes continuously. Gene pyramid 2 (rhg1-b+G. soja+Chr.10) rotated with PI 90763 (rhg1-a, Rhg4, rhg2) had the lowest SCN egg population density at both locations. However, this rotation caused the virulence of the SCN population to increase, as reflected by elevated female index (FI) values. In the Ames experiment, the initial SCN population used to infest the microplots had a FI of 7 on PI 90763, and the continuous PI 90763 treatment and the rotation of pyramid 2 with PI 90763 caused the FI to increase to 27 and 12.5, respectively, over three field seasons. The FI on PI 88788 remained well above 10 across all microplots, even in SCN populations not exposed to the rhg1-b gene, which PI 88788 possesses. Additional shifts in virulence were observed but were less substantial in comparison to those described above. HG Type test results of the SCN populations in samples collected from the 2022 microplots have not been received yet.
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)
Monica Pennewitt, PhD student with Greg Tylka at Iowa State University, was selected to give a presentation at the National Soybean Nematode Conference in Savannah, Georgia in December 2022. About 200 attendees were present for Pennewitt’s presentation, which described the overall scope and organization of the experiments in objective 2 of this project. Pennewitt also presented and discussed some of the results of the microplot experiments. And between October 2022 through March 2023, Greg Tylka conducted 6 interviews with radio and newspaper/magazine journalists and gave 10 presentations to agribusiness groups (in person and virtual) about SCN. The loss of effectiveness of PI 88788 SCN resistance was discussed in every interview and presentation, and Objective 2 of our NCSRP-funded research project was mentioned and described whenever time/space permitted.
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Diers)
The Diers group sent seed to cooperators for the 2022 SCN Regional Test, received the collected data during the fall of 2022, and disseminated the complete report in February of 2023. This 2022 test includes 225 entries that range from MG 0-IV. The regional test cooperators and the Diers group have also organized this trial for 2023, and seed shipment for packaging and planting is currently underway.
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)
The Scaboo group has now completed successful crossing attempts (3 backcrosses) 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. During the summer of 2022, we grew over 100 plant rows derived from selected plants, and our first yield trials of this material will be in the summer of 2023. We have also identified several lines in our breeding program with the desirable three gene stack, confirmed phenotype of resistance, and selected them for advanced testing in 2023. Additionally, we are actively identifying and introgressing new and novel QTL/genes into our breeding program for cultivar development.
The Diers group has continued to work to diversify SCN resistance away from PI 88788 based resistance in Midwestern adapted cultivars. To do this, we have continued to select for the major SCN resistance genes rhg1-a and Rhg4. During the past summer, we tested over 5000 plants to select these genes and selected over 950. These selected plants will be advanced to plant rows during the 2023 growing season. In addition, in advanced yield tests we evaluated 35 experimental lines that carried both rhg1-a and Rhg4 and 22 lines with two SCN resistance genes from G. soja.
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 and analyze 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 from 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