The soybean cyst nematode (SCN), or Heterodera glycines, is the most damaging pathogen to soybean production in North America. 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 develop in their fields. The widespread lack of genetic diversity for SCN resistance genes in commercial soybean varieties has significantly increased the prevalence of virulent SCN populations 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.
1. Plant breeders need to increase the genetic diversity of SCN resistance in commercially available soybean varieties and work with nematologists to determine the most effective rotation practices that preserve the efficacy of the known sources of SCN resistance.
2. Nematologists need to identify the SCN genes required for the adaptation to reproduce on resistant varieties, use these as markers to monitor nematode population shifts in the field, and exploit this knowledge to help plant breeders identify the best resistance gene combinations for long-term nematode management.

To address these issues we are proposing Phase II of an integrated, collaborative, and multi-state project among plant breeders, molecular biologists, bioinformaticians, and nematologists. Our proposed objectives specifically address performance measures under Goals 1 (1.1, 1.2, 1.3), 3 (3.1, 3.2), 4 (4.6), and 5 (5.1) of the 2015-2020 SCN-Soybean North Central Research Program Strategic Plan and complement funding from federal agencies and the United Soybean Board. The genetic resources developed and knowledge gained from this project will provide immediate benefit to soybean producers and researchers in both the private and public sector.

Objective 1. Diversify the genetic base of SCN resistance in soybean
• 1.1: Develop and evaluate germplasm with new combinations of resistance genes in high yielding backgrounds (Diers, Scaboo).

Objective 2. Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties
• 2.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum).
• 2.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum, Baum).
• 2.3: Re-sequence the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses (Severin, Hudson, Mitchum, Baum).
• 2.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum).

Objective 3. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
• 3.1: Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum).

Objective 4. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers
• 4.1: Inform growers on effective rotation schemes designed to protect our current sources of resistance (Tylka, Mitchum).

Objective 5. Coordinate the testing of publicly developed SCN resistant experimental lines
• 5.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario.

YEAR 1
Objective 1:
1.1 A population segregating for Rhg1 and Rhg4 from PI 437654, the genes on chromosomes 15 and 18 from G. soja, and a gene on chromosome 10 from PI 567516C will be yield evaluated in two locations to test for the impact of these resistance genes on yield. Breeding will be done to incorporate new combinations of resistance genes into high yielding genetic backgrounds.

Objective 2:
2.1 Create and release to collaborators draft genomes of at least five nematode HG types. Further develop SCN-Base cyber-infrastructure, GBrowse, tracks, using existing draft assembly and available SNP data/transcript data.
2.2 Further inbreed nematodes on soybean with different resistance genes and generate sufficient material for sequencing.
2.3 Extract DNA and RNA, begin genome re-sequencing and transcriptome analysis of virulent SCN populations selected on different resistance genes.

Objective 3:
3.1 Year 1 field trials to evaluate different rotation schemes for effectiveness at reducing population densities and the selection pressure on the nematode population.

Objective 5:
5.1 Tests of SCN resistant experimental lines will be coordinated.

YEAR 2
Objective 1:
1.1 A population segregating for Rhg1 and Rhg4 from PI 437654, the genes on chromosomes 15 and 18 from G. soja, and a gene on chromosome 10 from PI 567516C will be yield evaluated in two locations to test for the impact of these resistance genes on yield. Breeding will be done to incorporate new combinations of resistance genes into high yielding genetic backgrounds.

Objective 2:
2.3 Finish genome re-sequencing and transcriptome analysis of virulent SCN populations selected on different resistance genes and initiate comparative analyses. Deposit data to the SCN-Base website, and use to mine for candidate genes involved in SCN virulence.
2.4 Molecular, biochemical and/or genetic studies initiated on genes identified as putative virulence genes.

Objective 3:
3.1 Year 2 field trials to evaluate different rotation schemes for effectiveness at reducing population densities and the selection pressure on the nematode population.

Objective 5:
5.1 Tests of SCN resistant experimental lines will be coordinated.

YEAR 3
Objective 1:
1.1 Breeding will be done to incorporate new combinations of resistance genes into high yielding genetic backgrounds.

Objective 2:
2.1 Complete all sequencing and genome assemblies. SCN-Base will be updated with the final assembly and annotation of all genome sequences.
2.3. Continue comparative analysis using genomic sequences to identify genes under selection.
2.4 Genes identified as putative virulence genes will be further characterized using molecular, biochemical and/or genetic analysis.

Objective 3:
3.1 Year 3 field trials to evaluate different rotation schemes for effectiveness at reducing population densities and the selection pressure on the nematode population.

Objective 4:
4.1 Results communicated to SCN Coalition

Objective 5:
5.1 Tests of SCN resistant experimental lines will be coordinated.

Updated September 14, 2020:
Objective 1: Diversify the genetic base of SCN resistance in soybean
1.1: Develop and evaluate germplasm with new combinations of resistance genes in high-yielding backgrounds

Diers – Our research is continuing to develop new varieties with non-PI 88788 sources of resistance. New, high yielding lines with SCN resistance from non-PI 88788 sources were selected in 2019. Five new high yielding lines that have Rhg1-b from PI 88788 combined with two resistance genes from G. soja were selected for testing in uniform or regional test this summer.

Scaboo – We are developing germplasm and varieties in the relative maturities 3.5 to 4.2 with resistance to SCN HG 1.2.5.7 (Race 2), which is currently the predominate HG type in Missouri. We are using novel SCN resistant sources including PI 437654, PI 90763 and PI 468916, with most support from USB and MSMC for these efforts. We will continue population development by forward and backcrossing, using phenotypic screening and molecular markers for SCN resistance selection, and by yield testing elite germplasm in both state and regional tests (run by Diers). Our first yield tests of novel soybean material with Race 2 resistance was during the summer of 2019, and we plan on having this material in various stages of yield testing every year thereafter.

Objective 2: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
2.1: Sequence, curate and annotate SCN reference genomes for each common HG type

Baum/Severin – One of the major milestones in SCN biology is developing a completely annotated SCN reference genome, which we have recently accomplished (Masonbrink et al., 2019). Using NCSRP funds we continue to refine this resource further. We have made considerable progress in that direction and now have a chromosome level genome assembly ready and annotated. Currently, we are preparing a manuscript describing these results to be submitted shortly.
Simultaneously, we have developed a centralized, web-based repository called SCNBase (SCNBase.org). We have developed this web portal de novo and published it in November 2019 (https://doi.org/10.1093/database/baz111). This web portal now is the home to all bioinformatic, genetic, genomic, and molecular data generated for SCN, most of it through soybean check-off funding. Using this web portal, researchers and breeders from all over the world can access and analyze all public bioinformatic data generated in this proposal and curated from previous research at the nucleotide level. We are already seeing significant “web traffic” to this web portal suggesting that it is generating considerable interest in the SCN community from all over the world. For example, since SCNBase has been open to the public earlier in 2019 it has been accessed by 1,792 unique users. During the period of January 1 to March 26, 2020, alone, this web portal had 307 unique users, almost half of which were located outside of USA. As such, SCNBase provides prime visibility and impact of the collective data procured through farmer investments in research, which and will enable others in a coordinated fight against this pathogen. This current NCSRP project is already generating and will continue to generate thousands of gigabytes of data, which will be incorporated into SCNBase as we continue to develop this web portal.

Hudson – Our project has now generated high quality genome sequences for five SCN isolates: OP50, TN 7, 8, 20, and 22. These sequences are all more complete than the TN10 sequence published by our consortium last year. The TN10 reference has now been improved to comparable or better quality by the Iowa group. We thus have six complete genomes of different SCN strains with assemblies each with N50 > 10MB, plus the Chinese group’s assembly of the X1 strain, which is less reliable than those from our own project. The five completed sequences have been annotated and effector sequences have been identified using sequence similarity to a set of 80 curated plant parasitic nematode effectors. So far, the variability of size in the seven different assemblies, and the sequencing and assembly methods used, seem to be affecting the overall number of genes and effector genes more than the likely biological differences. We are in the process of curating the assemblies to make them more comparable. However, we are able to identify the presence of between 63 and 69 of the 80 effector families in each genome, giving each isolate a substantial arsenal of effector proteins. The correlation between effector families in the genome and virulence on specific hosts is an ongoing analysis expected to generate preliminary data by September 2020. We are in the process of repeating and enhancing these methods on two more isolates, MM26 and PA3. We are using the new PacBio sequencer which should produce even better data than the nanopore sequencing used previously. Sequencing is completed for PA3 and in progress for MM26. Assembly for PA3 has begun. Both genomes are expected to be completed by September 2020, giving us high quality whole-genome sequences of all the key HG types.

Mitchum – Continuously maintained live cultures, prepped and provided all of the genetic material for each common HG type in sufficient quantities for various sequencing platforms used to generate the reference genomes described above.

2.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance

Mitchum – In Phase I of this project (under Obj 2.3 of the previous project) we identified a HG type 1.2.5.7 field population and continuously selected this population in the greenhouse on either a susceptible soybean line (SCN inbred population MM-BD1), a soybean line containing the Rhg1 resistance gene from PI 88788 (SCN inbred population MM-BD2), a soybean line containing the Rhg1 and Rhg4 resistance genes from PI 437654 (Hartwig) (SCN inbred population MM-BD3), and a soybean line containing resistance genes on chromosomes 15 and 18 from wild soybean G. soja (inbred population MM-BD4) for more than 12 generations. The original SCN field population and this series of SCN populations selected for virulence on each set of resistance genes has been continuously reared to generate sufficient genetic material for sequencing. The populations were subjected to another 6 months of selection in this project period, HG typed, and frozen.

2.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses

Baum/Severin – Our group is actively involved in developing, analyzing and comparing gene expression in from virulent (i.e., able to infect resistant soybean cultivars) and avirulent (i.e., unable to infect resistant soybean cultivars) SCN populations with the goal to identify the genetic determinants of virulence. For that purpose, we are focusing on three individual gland cells, in which the nematode produces the effectors/tools required for infection and defense suppression, using a method we previously had developed. Toward that end, we have completed three independent biological replications of mRNA purification, library construction and sequencing (which is essentially an identification of all genes expressed in the gland cells at a given time) from each of the virulent (MM10) and avirulent (PA3) SCN populations. Furthermore, gene identities from all sequencing experiments have been generated utilizing the SCN TN10 genome produced by us (Masonbrink et al., 2019). We identified 13,617 unique transcripts from the PA3 population and 8,820 unique transcripts from the MM10 population, which represent a snapshot of gene activity during the early parasitic stage of SCN infection (parasitic J2). As a point of validation, we have identified all previously discovered SCN effectors expressed during this early parasitic stage within our gland cell-specific sequence data. Initial analysis of these sequence data has revealed intriguing gene expression differences between these virulent and avirulent SCN populations. Specifically, there are 32 genes up-regulated in the SCN PA3 libraries versus the MM10 libraries. Protein products of 13 of these genes are predicted to be nuclear localized and likely have gene regulatory functions. This includes a diverse group of proteins involved in functions such as structural maintenance of chromosomes, splicing factors, a glycosyltransferase involved in increased virulence in other parasitic nematodes, DNA-binding proteins, and a ‘ran’-binding protein, among others. Conversely, we discovered 17 genes that are up-regulated in SCN MM10 versus PA3. Protein products of eight of these genes are predicted to be nuclear localized. Among this group are previously reported effectors, a microtubule binding protein homolog from an animal-parasitic nematode, signal transduction proteins, and several unknown proteins. We are still at the initial stages of analysis but it is already clear that we have generated a rich source of critical data needed to understand SCN virulence.

Mitchum – The MM26 parental population for the adapted populations outlined in 2.2 above was prepared and sent for genome sequencing; the others will follow. RNA-seq data generated from early parasitic life stages of a virulent and avirulent population of SCN were used to conduct a reference-based transcriptomic analysis with the aid of the SCN genome. We identified 207 genes unique to the virulent SCN that could be turned on to overcome resistance mechanisms; conversely, 92 genes that could be turned off to evade triggering resistance mechanisms.
Hired a new postdoctoral research associate with expertise in advanced bioinformatics, population genetics, and genomics. He will focus on extending our work on the molecular genetic and comparative genomics analysis of soybean cyst nematode population structure as it relates to virulence on resistant host plants. Unfortunately, we are now faced with a significant delay in his joining our team due to COVID-19.
We continued our collaborative effort with the Baum lab to generate nematode gland-specific RNA-seq datasets of a virulent and avirulent population of SCN; several joint meetings were held to discuss comparative analyses which are underway to identify differentially expressed gene candidates with a potential role in virulence using the newly annotated pseudomolecule SCN genome assembly.

2.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets

Mitchum – As candidate SCN virulence genes are identified, gene function will be confirmed through biochemical and/or genetic analysis to not only better understand the mechanisms of virulence, but to also evaluate these gene targets as vulnerable points of disruption in the SCN life cycle as a means to enhance resistance in soybean. We focused our efforts on validating the genes identified in 2.3 above to prioritize candidate virulence genes for further molecular functional studies.

Baum – Being a sedentary endoparasite that relies exclusively on its host for survival, SCN has to suppress host defense responses for a significant duration in order to survive. The SCN achieves this by producing a large number of effector molecules and delivering them into the soybean cells via its mouth spear. These effectors specifically target host factors and modulate their functions, thereby also altering soybean defense responses. Generating an in-depth understanding of how individual effectors help SCN establish and maintain infection is a very difficult but necessary task that will reveal vulnerable “nodes” in host defense pathways that can be strengthened via either breeding or molecular approaches.
As a part of this project, we are actively involved in conducting in-depth molecular characterization of SCN effectors specifically involved in host defense suppression. For this reporting period, we specifically focused on the effector named 28B03. We have identified that this particular effector is a robust host defense suppressor. We have observed that due to its function, plants become more susceptible to cyst nematodes. We have also identified that this effector specifically targets a previously uncharacterized plant protein kinase. By physically interacting with this novel protein kinase, the 28B03 effector suppresses phosphorylation of its substrate protein and completely alters the associated signal transduction pathway. This discovery and an in-depth molecular characterization of a novel defense response related kinase cascade in plants are breakthrough discoveries of this study. We are currently writing a manuscript describing these results. Simultaneously, we are gearing up to conduct another high-impact protein interaction experiment in order to identify the complete ‘interactome’ of the proteins associated with this kinase cascade.

Objective 3. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.

3.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles

Diers – The Illinois location of the rotation trial was harvested and fall soil samples were taken to evaluate SCN population changes over the summer. Samples from susceptible checks show that the SCN population was an HG type 2 (Race 5) in two plots and a HG type 1.2 (Race 2) in one plot. The analysis of the change in egg number between the spring to the fall show that there was a 5 X increase in eggs through the growing season for the susceptible check, a 3.8 X increase for lines with only Rhg1-b, a 1.6 X increase for lines with Rhg1-b plus two QTL from G. soja, a 3 X increase for Rhg1-b plus two QTL from G. soja and a fourth QTL on chromosome 10, a 2 X increase for lines with Rhg1a + Rhg4 from Peking, and finally only a 0.3 X change for PI 90763. These results are close to what we expected and show that the different sources influenced SCN reproduction in the field. Preparations are being made to plant the second year of the test in the field.

Scaboo/Mitchum – Microplot field experiments were established to evaluate how rotations of SCN resistance gene combinations impact SCN field population densities and virulence profiles. Treatments with resistance genes: Rhg1-a, Rhg1-b, Rhg4, cqSCN-006/cqSCN-007, and the chromosome 10 QTL, along with susceptible and resistant checks were designed to form 12 treatments with 3 replications in a randomized complete block design. Hand planting and inoculation were done on May 31st, 2019. Soil samples from all 36 plots were obtained directly after inoculation to determine the baseline of SCN egg density. A moderate level of infestation was achieved. At 60 days post-inoculation (DPI), soil samples from the three susceptible plots and one field sample collected between the microplots were collected to determine the initial HG type. SCN HG type at 60 DPI was the same for all three susceptible plots. The HG type for these plots was HG type 2.5.7, which was similar to the SCN population present in the field location between plots. At the end of the growing season, soil samples from all plots were obtained to determine egg density and SCN HG type at harvest. All resistance genotypes had a reduction in egg densities and the susceptible treatment had an significant increase in SCN egg density, while PI 90763 had the greatest percentage of egg count reduction followed by genotypes with resistance genes, Rhg1a + Rhg4. SCN HG type results at the end of the first growing season show no specific trend.

Tylka – At the conclusion of the 2019 growing season, two separate soil samples were taken from each microplot in both experiments conducted in Iowa. The first set of soil samples were used to obtain an end-of-season population density of SCN in each plot, and the second set of samples were sent to the University of Missouri for HG Type testing to determine if and how the soybean genotypes grown in the microplots in 2019 might have shifted the virulence phenotypes (HG types) of the SCN populations added to the microplots in the spring of 2019. Preliminary data analysis was completed for each of the experiments in Iowa, and some trends in SCN population density were observed. In both experiments, the highest SCN population densities were found in microplots in which the susceptible soybean variety was grown. The second highest SCN population densities were found in microplots in which soybeans with only Rhg1-b resistance were grown. The lowest population densities were found in plots where PI 90763 and Rhg1-b+soja+ch10 resistance were grown. The results of the HG Type test results on the SCN populations in the soil samples collected from the microplots at harvest in 2019 are not yet available. Preparation and planning for the second growing season has begun with the arrival of seed.

Objective 4. Translate the results of Objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.

4.1: Inform growers on effective rotation schemes designed to protect our resistant sources

Tylka/Mitchum – A news release about stacking SCN genetic resistance and rotation strategies was created and disseminated in 2019 and it led to interviews with Melissa Mitchum on Brownfield Ag Network and Brian Diers on Adams on Agriculture radio programs, and an interview by Melissa Mitchum with Successful Farming/Agriculture.com (print/digital media). Also, the February 2020 issue of Progressive Farmer magazine had a special section titled “Crop Invaders”, and it contained four pages devoted to SCN resistance (see Appendix) and an article titled “A New Movement: The Push for SCN Varietal Resistance Broadens” in which Brian Diers discusses novel SCN resistance genes and rotating their use to preserve their effectiveness. Also, our NCSRP-funded research was mentioned in radio and print interviews with Greg Tylka by the following media at the Commodity Classic meeting in San Antonio, February 27-28, 2020: Emily Unglesbee, DTN; Gil Gullickson, Successful Farming; Mike Perrine, MP Ag Radio; Michelle Rook, WNAX; Mike Adams, Adams on Ag; Anna Hastert, Iowa Agribusiness Radio; DeLoss Janke, Illinois FB radio; Clinton Griffiths, Farm Journal; George Bower, KICD radio, Spencer; Ashley Davenport, Michigan Ag Today radio; and Mick Kjar, AgNews 890.

Objective 5. Coordinate the testing of publicly developed SCN resistant experimental lines.

5.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario

Diers – The report from the 2019 SCN Regional Test was completed and the first version of the results was sent to all test collaborators on 19 December 2019. This version included the agronomic, composition and resistance test results. This version was followed by the distribution of the final version on 6 January 2019 which included the results of testing the egg number and HG type of nematode populations in field environments. This quick reporting of the 2019 results is important for breeders so this information can be used in release and crossing decisions. Planning has taken place for the 2020 tests and entry lists for the trials have been made and seed has been distributed to cooperators. The 2020 tests will include 194 entries that range from maturity groups 00 to IV. The test will be grown in 32 environments in 10 states and one Canadian province. Because of the Covid 19 outbreak, it is not known how many locations will be planted.

Please see attachment for addition information and Appendix.

View uploaded report

Updated October 31, 2020:
A description of relevant progress for principle and co-principle 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 even during the challenges faced due to the current COVID-19 pandemic. We had our last group research meeting in April of 2020, and our next group meeting is scheduled for December 4th of 2020 for research planning and group discussions.

1.1: Develop and evaluate germplasm with new combinations of resistance genes in high-yielding backgrounds (Diers, Scaboo)

During the summer of 2020, field tests of lines with new combinations of SCN resistance genes were grown in both IL and MO. New lines with unique resistance gene combinations were selected in preliminary tests and we are still waiting for results from advanced and uniform tests to make selections in these tests. In IL, marker-assisted selection was conducted to select plants with new combinations of SCN resistance genes with an emphasis on combining the two genes from Glycine soja with resistance genes from PI 88788 and to select plants with Hartwig type resistance in high yielding backgrounds. In MO, we have focused on utilizing novel resistance genes Rhg1-a and a QTL on Chr 11 to incorporate race 2 type resistance.

2.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum, Mitchum)

The Baum and Severin groups have made major achievements in exploring SCN biology by publishing a quality annotated SCN reference genome (Masonbrink et al., 2019), followed by publishing a web-based repository called SCNBase (SCNBase.org) (https://doi.org/10.1093/database/baz111) (Masonbrink et al 2019). SCNBase is the home of all bioinformatic, genetic, genomic, and molecular data generated for SCN, the traffic of which has had 1,112 new users since the beginning of 2020, with over 1,500 sessions. This project will continue to produce data and analyses, which will be incorporated into SCNBase as they becomes available. We have prepared a manuscript to publish the chromosome-scale assembly of the TN10 genome. Extra steps were taken to ensure the quality of the assembly, and annotations were much higher than the published X12 genome assembly. Extensive annotations and bioinformatic resources were created for this measure, all of which will be publicly available in SCNBase upon the manuscript’s submission. Of these, previouslyThe Baum ans published RNA-seq from SCN was combined with RNA-seq from SCN gland cells, currently unpublished, but in preparation for release via a Resource Announcement, to identify differential expression between SCN gene expression in pre-parasitic stages, parasitic stages on a susceptible soybean root, and parasitic stages on a resistant root. We further refined the SCN gland expression analyses to include a contrast between the aforementioned RNA-seq derived from whole worms and a clearer representation of gland expression between virulent and avirulent nematodes. Additional transcriptomic resources were pursued to better annotate the expression of genes associated with different stages of the nematode, including male and female sexual differentiation. Because male and female balance can dramatically shift under different conditions, with males abandoning their feeding sites, understanding this transition may provide new targets for resistance development. At the University of Georgia, live cultures were continuously maintained for this project, prepped and provided all of the genetic material for each common HG type in sufficient quantities for various sequencing platforms used to generate the reference genomes.

The Hudson group has now generated high quality genome sequences for five SCN isolates: OP50, TN 7, 8, 20, and 22. These sequences are all more complete than the TN10 sequence published by our consortium last year. The TN10 reference has now been improved to comparable or better quality by the Iowa group. We thus have six complete genomes of different SCN strains with assemblies each with N50 > 10MB, plus the Chinese group’s assembly of the X1 strain, which is less reliable than those from our own project. The five completed sequences have been annotated and effector sequences have been identified using sequence similarity to a set of 80 curated plant parasitic nematode effectors. So far, the variability of size in the seven different assemblies, and the sequencing and assembly methods used, seem to be affecting the overall number of genes and effector genes more than the likely biological differences. We are in the process of curating the assemblies to make them more comparable. However, we are able to identify the presence of between 63 and 69 of the 80 effector families in each genome, giving each isolate a substantial arsenal of effector proteins. The correlation between effector families in the genome and virulence on specific hosts is an ongoing analysis expected to generate preliminary data by September 2020. We are in the process of repeating and enhancing these methods on two more isolates, MM26 and PA3. We are using the new PacBio sequencer which should produce even better data than the nanopore sequencing used previously. Sequencing is completed for PA3 and in progress for MM26. Assembly for PA3 has begun. Both genomes are expected to be completed by September 2020, giving us high quality whole-genome sequences of all the key HG types.

2.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum, Baum)

In Phase I of this project (under Obj 2.3 of the previous project) Mitchum identified a HG type 1.2.5.7 field population and continuously selected this population in the greenhouse on either a susceptible soybean line (SCN inbred population MM-BD1), a soybean line containing the rhg1 resistance gene from PI 88788 (SCN inbred population MM-BD2), a soybean line containing the rhg1 and Rhg4 resistance genes from PI 437654 (Hartwig) (SCN inbred population MM-BD3), and a soybean line containing resistance genes on chromosomes 15 and 18 from wild soybean G. soja (inbred population MM-BD4) for more than 12 generations. The original SCN field population and this series of SCN populations selected for virulence on each set of resistance genes has been continuously reared during this project period. A manuscript describing these populations has been drafted and shared with co-authors.

2.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)

The Mitchum group is focused on identifying SCN virulence genes used by the nematode to overcome the Peking-type (Rhg4-mediated) resistance. To accomplish this, we are conducting a comparative transcriptomic analysis using RNA-seq data generated from the early parasitic stages of virulent and avirulent SCN populations, which are adapted on soybean recombinant inbred lines (RILs) that only differ at the Rhg4 locus (i.e., a resistant RIL with a resistant Rhg4 allele and a susceptible RIL containing a susceptible Rhg4). Our preliminary analysis using the published SCN genome has identified differentially expressed genes that could be involved in overcoming the Peking-type resistance. Unique to the virulent SCN were 207 genes that could be turned on to overcome resistance, and 92 genes that could be turned off to evade triggering resistance mechanisms. We repeated this analysis, utilizing the newest pseudomolecule SCN genome and applying modified statistical cutoff criteria, to reveal 133 genes uniquely up-regulated in the virulent SCN, which includes previously known effectors and virulence factors found not only in parasitic nematodes but also in other parasites; some of these genes are related to RNA interference, while others encode secreted proteins and antioxidants that suppress or function as a “shield” from plant defense mechanisms. Conversely, 112 genes exclusively down-regulated in the virulent SCN also contain known effectors from other plant-parasitic nematodes and pathosystems. Because differentially expressed gene candidates enriched with single nucleotide polymorphisms (SNPs) or insertions and deletions (INDELs) could provide a stronger confidence that they indeed have a potential role in virulence, we also conducted a variant call analysis in addition to the differential expression studies. We discovered that the virulent SCN harbors 72,232 SNPs and 4,564 INDELs, while the avirulent SCN contains 73,026 SNPs and 4,513 INDELs, relative to the reference genome with statistical significance. While it is possible that many of these variants could be present in both virulent and avirulent SCN (i.e., variants overlapping in both populations), those that are uniquely present in one population which also happens to be uniquely differentially expressed in that population would clearly provide a stronger correlation to virulence. For this reporting period, we are currently filtering and prioritizing our virulence gene candidates based on differential expression and variant call analyses. After that, our final list of candidate genes will be cross-compared with the nematode gland-specific RNA data sets (i.e., in silico subtraction for gland-specific genes) to select for virulence effectors. We continued our collaborative effort with the Baum lab to generate nematode gland-specific RNA-seq datasets of a virulent and avirulent population of SCN; several joint meetings were held to discuss comparative analyses which are underway to identify differentially expressed gene candidates with a potential role in virulence using the newly annotated pseudomolecule SCN genome assembly.
We interviewed and hired an excellent new postdoctoral research associate with expertise in advanced bioinformatics, population genetics, and genomics who would have been an ideal fit for the project. Unfortunately, after waiting more than 6 months, he was unable get a visa to join us in the US due to COVID-19 situation.

In the hopes of further resolving the genes important to the host-parasite exchange, the Baum and Severin group leveraged the existing H. glycines RNA-seq. All possible comparisons were made between pre-parasitic (i.e., before root penetration) second-stage juvenile nematodes (PP), second-stage parasitic (i.e., after root penetration) nematodes on a susceptible host (C for compatible interaction), and second-stage parasitic nematodes on a resistant host (IC for incompatible interaction). This data were integrated into a tabular database of genes, functional annotations, sequences, and differential expression. Using this database, we filtered genes in the H. glycines genome for key traits of genes involved in parasitism, including differential expression, fold change, signal peptide presence, and the absence of a transmembrane domain. Of the 1,421 genes this discovered, 12 genes were upregulated in C vs PP and 35 genes were upregulated in IC when compared to PP. Novel comparisons between C and IC samples revealed 351 and 35 differentially expressed genes producing secreted proteins, respectively. To dissect which secreted proteins are involved in host cell reprogramming, we annotated those genes that contained predicted nuclear localization signals, revealing up to 2 (C vs PP), 6 (IC vs PP), 60 (C over IC), and 1 (IC over C) secreted proteins that may be targeted to the host nucleus, respectively. We further validated this approach by identifying previously published effectors among the secreted proteins encoded by differentially expressed genes. Of those, we found up to 5 genes in C vs PP, 5 in IC vs PP, 48 in C over IC, and 1 in IC over C.
2.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

As candidate SCN virulence genes are identified by the Mitchum group, gene function studies are being conducted using biochemical and/or genetic analysis to not only better understand the mechanisms of virulence, but to also evaluate these gene targets as vulnerable points of disruption in the SCN life cycle as a means to enhance resistance in soybean. The Mitchum group continued our characterization of novel stylet-secreted effectors of the soybean cyst nematode Heterodera glycines parasitome, 16B09 and 2D01. 16B09 and 2D01 belong to the same superfamily of effectors, highly expanded in the genome, share the same gene structure, harbor conserved protein domains, and exhibit the same spatial and temporal expression in the dorsal gland cell during parasitism. Host-induced gene silencing (RNAi) of 16B09 demonstrated a requirement of this effector protein for successful parasitism. Protein interaction studies identified a specific interaction of 2D01 with a plant plasma membrane-associated protein kinase. We further demonstrate that this protein kinase is expressed in feeding sites and plants unable to produce this kinase showed increased resistance to nematodes. At present we are validating this interaction in planta. A comparative genomics analysis of this effector family across populations of SCN differing in virulence on resistant soybean is currently under investigation.

The Baum group has been working to understand the robust defense suppression and comprehensive re-engineering of the feeding site, which are two of the hallmarks of the successful cyst nematode infection. The cyst nematode achieves this by producing a large number of effector molecules and delivering them into the host cells via its mouth spear. These effectors specifically target host factors and modulate their functions. Generating an in-depth understanding of how individual effectors help the cyst nematode establish and maintain infection is a difficult but necessary task that will reveal vulnerable “nodes” in host signal transduction pathways that can be altered via either breeding or molecular approaches. As a part of this project, we are actively involved in conducting in-depth molecular characterization of one of the effectors, 28B03. We have identified that this particular effector is a robust host defense suppressor as well as plant growth enhancer. We have observed that due to its function, plants become more susceptible to cyst nematodes, while showing accelerated root and shoot growth. We have also identified that this effector specifically targets a previously uncharacterized plant protein kinase. Our data show that this effector specifically targets this kinase to interfere with the signaling transduction pathway associated with it and suppresses phosphorylation of the substrate protein of this particular kinase. This discovery and an in-depth molecular characterization of a novel defense response-related kinase cascade in plants are breakthrough discoveries of this study. We are currently writing a manuscript describing these results. Simultaneously, we are conducting experiments to identify the complete ‘interactome’ of the proteins associated with this kinase cascade. For this purpose, we are using a state of the art “TurboID biotin proximity labeling system”. In short, we have developed multiple fusion constructs in which we have fused an unmodified kinase, a dead version of this kinase as well as a truncated version of this kinase to a highly active derivative of biotin ligase enzyme. We have developed stable transgenic Arabidopsis lines expressing these constructs. Once we obtain homozygous plants, we plan to treat them with exogenous biotin, which is readily taken up by plant cells. Proteins in the near vicinity of the biotin ligase will be biotinylated, and then can be purified using streptavidin beads. Biotinylated proteins will then identified using mass-spectrometry, which will identify the complete ‘interactome’ of this particular kinase.

3.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

In IL, the second season of the rotation test was grown in the field during 2020 and soil samples were taken at the end of the growing season for analysis. These samples will be shipped to the SCN diagnostic lab at the University of Missouri. The field experiment was also harvested in October of 2020 in MO, and soil samples were taken of each microplot, and analysis off egg counts and HG types will take place during the winter.

At the conclusion of the 2020 growing season in IA, two separate soil samples were taken from each microplot in both experiments conducted in Iowa. The first set of soil samples were used to obtain an end-of-season population density of SCN in each plot, and the second set of samples were sent to the University of Missouri for HG Type testing to determine if and how the soybean genotypes grown in the microplots in 2020 might have shifted the virulence phenotypes (HG types) of the SCN populations added to the microplots in the spring of 2019. The HG type test results from the fall 2020 samples also will be compared to the virulence of the SCN populations in the microplots at the end of the 2019 growing season.

4.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Tylka conducted 20 radio and newspaper/magazine interviews between April and September 2020. The loss of effectiveness of PI 88788 SCN resistance often was discussed and our NCSRP-funded research project was mentioned and described if time/space permitted. Also, Iowa State University was to host the SCN Coalition’s Tode Tour on August 31, 2020. The event was to be a gathering of key farmers and agricultural media from across the country to learn about SCN biology, management, and ongoing research, but plans for the event had to be changed because of the COVID pandemic. With the help of communications firm MorganMyers, the coalition developed the “Let’s Talk Todes” video collection, which can be viewed on www.TheSCNCoalition.com, instead of the originally planned event. In the videos, farmers, scientists and soybean checkoff leaders share best management practices for SCN and highlight how partnerships and a checkoff investment in SCN-related research are bringing new tools to combat SCN. Our NCSRP-funded research project is the highlighted example of checkoff-funded research developing new SCN management options for farmers. The videos feature Tylka; Kaitlyn Bissonnette, University of Missouri; and Sam Markell, North Dakota State University as well as Matt Bierman, a soybean farmer from Mills County, Iowa and Ed Anderson, Iowa Soybean Association. The videos were shot at Iowa State’s Field Extension Education Laboratory (FEEL) near Ames, IA on July 21 and August 31. Another set of videos will be produced at the University of Georgia in early November and added to the Let’s Talk Todes collection. The videos in Georgia will feature SCN research being conducted in the Mitchum lab as well as research by Wayne Parrott and Zenglu Li at the university.

5.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

Planning was completed for the 2020 SCN Regional Test and the University of Illinois received seed from breeders who entered lines in the trial. This seed was repacked and distributed to all cooperators and most locations of the trials were planted despite the Covid 19 outbreak. The final list of entries included 184 experimental lines and checks that ranged from MG 00 to MG IV. The University of Illinois will soon receive results from the trials and plans to provide an initial report to cooperating breeders in December.

View uploaded report

Updated August 4, 2021:
1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum, Mitchum)

The Baum and Severin groups would like to announce another significant milestone that we achieved during this funding period; the publication of our most detailed, chromosome-level assembly of the TN10 H. glycines genome in a high impact journal, Molecular Ecology Resources (Masonbrink RE, Maier TR, Hudson M, Severin A, Baum T. A chromosomal assembly of the soybean cyst nematode genome. Mol Ecol Resour. 2021 May 25. doi: 10.1111/1755-0998.13432. Epub ahead of print. PMID: 34036752.). As a consequence, all associated resources were publicly released on SCNBase, including: BLAST and JBROWSE capability for the genome assembly, predicted proteome, genome, transcriptome, and alignments for ribosomal genes, genome structural variation, SNPs and INDELs, alignment of X12 gene models, effector alignments, ncRNAs, multiple repeat predictions, and every putative input gene annotation. This will serve as a major informative resource to the entire SCN community worldwide.

Simultaneously, we are working on another project; we previously surveyed SNP and INDEL variation from 15 H. glycines populations. We created SNP/INDEL-modified versions of the TN10 genome to represent pseudo-genomes of these 15 populations. Using these, we identified genomic variation that affects gene structure and its potential for secretion. We have incorporated this information into a larger work that assesses the extent of gene family duplication and contraction that has occurred in SCN and related species at three phylogenetic nodes. Overall, the comparisons include sedentary vs migratory nematodes, root knot vs cyst nematodes, and Globodera species vs SCN. In our preliminary work, we identified 551 gene family expansions in sedentary nematodes vs migratory outgroups, 124 gene expansions in cyst nematodes vs root knot nematodes, and 479 gene family expansions in SCN vs Globodera species. These data are currently being prepared for submission and will result in the release of 323 columns of data describing every gene in SCN, across 15 SCN populations, and gene families among 13 related parasitic nematodes. We are also working on collecting and analyzing data to distinguish gene expression patterns and genomic structure between male and female H. glycines nematodes, which may provide new targets for resistance development.

The Hudson group, in collaboration with the Severin/Baum group, now has all seven of the target genotype assemblies completed and frozen. The Severin/Baum group assisted with manual curation using tools from their TN10 assembly workflow. Additionally, where possible, the genomes were improved by polishing with Illumina short reads using algorithms currently employed by large-scale genome projects. Alternate haplotype contigs were purged to generate seven assemblies each containing 9 pseudomolecules that correspond to the 9 SCN chromosomes. All seven assemblies have genome completeness scores that exceed those of the published TN10 assembly. Annotation is now underway to generate the predicted proteomes for comparison across the 8 SCN lines.

1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum, Baum)

Inbred populations have been maintained by the Mitchum group on a monthly turn over schedule since they were started. Thus, the original SCN field population and the series of SCN populations selected for virulence on each set of resistance genes has been continuously reared during this project period. We are also conducting a Pool-Seq strategy which pools individual nematodes from the same population to increase the amount of DNA required for sequencing. Pool-Seq will provide genome-wide polymorphic data and variant allele frequencies across populations to help pinpoint the candidate regions important for virulence. Four populations, including the aforementioned HG type 1.2.5.7 field population (MM26) and the inbred population MM-BD3, along with the avirulent (MM1) and virulent (MM2) inbred populations used for our transcriptomic analysis, will undergo Pool-Seq to help us map to candidate virulence regions important for overcoming the Peking-type (Rhg4-mediated) resistance. We have determined that sequencing virgin female nematodes which have successfully adapted on their respective hosts, as opposed to other life stages, would further bias their virulence, and provide us with a better resolution of virulence traits among different populations. Therefore, we have been harvesting virgin females from these four populations and have been optimizing DNA extraction protocols in preparation for Pool-Seq.

1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)

The Mitchum group is focused on identifying SCN virulence genes used by the nematode to overcome the Peking-type (Rhg4-mediated) resistance. Our comparative transcriptomic analysis comprises (1) differential expression and (2) variant call analysis by utilizing RNA-seq data generated from the early parasitic stages of virulent and avirulent SCN populations adapted on soybean recombinant inbred lines (RILs) that only differ at the Rhg4 locus (i.e., a resistant RIL with a resistant Rhg4 allele and a susceptible RIL containing a susceptible Rhg4). The differential expression analysis has allowed us to narrow down to 59 genes upregulated in the virulent SCN population. These genes were cross-compared with the nematode gland-specific RNA data sets to select 14 genes of special interest, categorized into three subgroups: (1) putative effectors involved in defense suppression, (2) putative enzymes related to reactive oxygen species, and (3) putative vitamin B-associated genes. These genes will be validated with qRT-PCR to confirm upregulation in the virulent population.

For this reporting period, the Baum and Severin groups would like to announce publication of the MPMI Resource Announcement, entitled “Esophageal Gland RNA-seq Resource of a Virulent and Avirulent Population of the Soybean Cyst Nematode, Heterodera glycines,” (Mol Plant Microbe Interact, 2021 Apr 26. doi: 10.1094/MPMI-03-21-0051-A.). We are exploring the RNA-seq dataset derived from a comparison of SCN gland cell transcriptomics of a virulent (MM10) and avirulent (PA3) population. This data analysis is published in the above Resource Announcement paper. Briefly, with this submission, we have announced the availability of a unique gland-specific RNA-seq dataset for the SCN community, which provides an expression snapshot of gland cell activity during early infection of a virulent and avirulent SCN population. This represents a highly valuable resource for researchers examining effector biology and nematode virulence. The data generated continue to be analyzed and is being used by the Baum lab in several avenues to progress omics research within the lab.

The Hudson group has made the finished genome assemblies available to collaborators in the group. Annotation is necessary to understand the results of transcriptome and resequencing work, and is the next step in the genomics process.

1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

The Mitchum group continued the characterization of novel stylet-secreted effectors of the soybean cyst nematode Heterodera glycines parasitome, 16B09 and 2D01. These effectors belong to a highly expanded superfamily of novel effectors, share the same gene structure, harbor conserved protein domains, and exhibit the same spatial and temporal expression in the dorsal gland cell during parasitism. This group of effectors was also identified as upregulated in the RNAseq data set as potentially involved in virulence (outlined under 1.3). Thus, we conducted a full transcriptome and genome analysis of this group of SCN genes. A search of the latest nine-scaffold, 158 Mb pseudomolecule assembly of the H. glycines TN10 genome identified 20 full-length (4 exon-3 intron gene structure) or partial copies of this effector gene superfamily (Masonbrink et al., 2021). Twelve members of this family were present on scaffold 5/Chromosome 1 and eight on scaffold 6/Chromosome 6. A sequence similarity tree constructed using the neighbor-joining method of the Clustal Omega program (Sievers et al., 2011) and including the original SCN parasitome sequences revealed three distinct subgroups. These subgroup sequences clustered together in the genome and may share functional similarities with some members involved in manipulating host developmental programs and others involved in suppressing host defenses. The variation observed across SCN populations is consistent with the highly expanded and diversified nature of this gene family.

The Baum group has been characterizing the robust defense suppression and comprehensive re-engineering of the feeding site, which are two of the hallmarks of the successful cyst nematode infection. As a part of this project, we are actively involved in conducting in-depth molecular characterization of the 28B03 effector family. Our analysis has shown that members of this effector family are robust defense suppressors. Our work has also revealed that a member of this family achieves its defense suppressive ability by interfering with a previously uncharacterized kinase cascade in plants. We continue to characterize the interactome associated with the kinases from this cascade as it will reveal signal transduction pathways that this particular effector modulates. To identify such an interactome, we are establishing a “proximity labeling assay system” in our laboratory, which is the latest and the most advanced technique to identify protein interactors in planta. In short, we have developed multiple fusion constructs in which we have fused an unmodified kinase, a dead version of this kinase as well as a truncated version of this kinase to a highly active derivative of the biotin ligase enzyme. We believe that establishing and characterizing such a system will prove pivotal for all our in planta protein interaction studies involving other effectors, particularly in soybean.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

Activities by the Tylka group for sub-objective 2.1 at Iowa State University from November 1, 2020 through July 31, 2021 were as follows. At the conclusion of the 2020 growing season, two separate multiple-core soil samples were taken from each microplot in the two experiments that were conducted in Iowa. The first set of soil samples were used to determine end-of-season SCN population densities in each plot, and the second set of samples were sent to the University of Missouri for HG type testing to determine if and how the soybean genotypes grown in the microplots in 2020 might have shifted the virulence phenotypes of the SCN populations originally added to the microplots in spring 2019.
Preliminary data analysis was completed for each of the experiments, and some trends in changes in SCN population densities were observed. In both experiments, the highest SCN population densities in fall 2020 occurred in microplots in which the susceptible soybean variety was grown. The microplots that were continuous with the same resistance from 2019 had higher SCN population densities in fall 2020 than the microplots that had rotated resistant varieties from 2019. The lowest population densities were found in plots where PI90763 resistance was grown in 2020. The SCN population densities in plots with continuous PI90763 also were lower than those that had previously had rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 in 2019 and were then rotated to PI90763 in 2020.

The results of the HG Type test results on the SCN populations in the soil samples collected from the microplots at harvest in 2020 show promising results. The SCN population in the soil in the experiment at the Kanawha, IA location had an HG type of 1.2 with a range of 35-80% female index on PI88788 and 7-40% female index on Peking. The SCN population in the soil in the experiment at the Ames, IA location had an HG type of 1.2.3 with a range of 27-80% female index on PI88788, 12-73% female index on Peking and 1-47% female index on PI 90763. The plots with rhg1-a + rhg4 and PI90763 had the highest female index on Peking and PI90763, while every plot had a female index greater than 10% on PI88788 in experiments at both locations and a female index greater than 10% on Peking at the Ames location. Increased virulence on PI437654 was not observed. After 2020 we observed increased SCN reproduction and some slight changes in female index (virulence) between microplots with rotated and with continuous cropping sequences.

In May 2021, each microplot was planted according to the deployment sequence being used in all microplot experiments in the project. This sequence included a third year of planting the same variety in half of the microplots (continuous sequence) and a rotation sequence of varieties to what was planted in 2019 in the remaining half of the microplots. At 30 days after planting, each row in each microplot was thinned to 30 plants and those with fewer than 30 plants were recorded.

During this period, the Diers group finished the 2019 rotation experiment and grew the 2020 experiment. We also recently received the HG type data from the fall 2019 plots as the results had been delayed until now because of Covid. Although the results have not been fully analyzed, a few observations can be reported. One is that the nematode population in all plots could overcome PI 88788 resistance and the female index (FI) on this source ranged from 10% to 96%. The fact that the nematodes could overcome PI 88788 resistance was expected because this was the HG type of the nematodes used to inoculate the plots. There were six plots that had nematodes that could overcome Peking resistance and the two plots with the highest FI on Peking (37-23%) were planted to a line with Peking type resistance. We are still waiting for the HG type data from the fall 2020 soil samples and these results should be available by the second week of August.

In Missouri, the Scaboo group harvested Fall 2020 soil samples for SCN egg counts, HG type tests were processed, and data was collected. The egg count data has shown that the susceptible plots have the highest egg densities followed by rhg1-b plots. The PI 90763, rhg1-a + Rhg4, and rhg1-b + G.soja + ch.10 treatments had the lowest population densities and the highest reduction in SCN egg densities compared to initial levels. In continuous treatments, the highest reduction in egg counts was observed in PI 90763 and rhg1-a + Rhg4. Similarly, the highest reduction in percentage change in the egg counts is in those treatments rotated with PI 90763. Interestingly, rhg1-a + Rhg4 treatments rotated with rhg1-b or its multiple gene stacks showed the largest reduction in egg counts that have been observed in the treatments with rhg1-b stacked with other resistance loci. The SCN HG type data has shown that the highest shifts in virulence were in continuous PI 90763 and continuous rhg1-a+Rhg4 plots. In continuous rhg1-a+Rhg4 treatment, increased virulence on PI 90763 and Peking has been observed. Similarly, in continuous PI 90763 treatment, an increase in virulence on Peking and PI 88788 has been observed. The seeds obtained for the third-year field season experiment have been checked, packaged and were planted in the microplots during June of 2021.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

The Mitchum lab helped to produce The SCN Coalition’s “Let’s Talk Todes” Research Collection video series now released online https://www.thescncoalition.com/lets-talk-todes/research-collection. In this 7 video series, soybean growers and scientists (nematologists, breeders, plant geneticists, extension pathologists) who are battling SCN explain the checkoff-funded research they are conducting that's focused on bringing new tools to soybean growers in the fight against parasitic nematodes. During this reporting period, MorganMyers deployed promotions of the “Let’s Talk Todes” Research Collection through a 7-week digital campaign (5/23/21-7/10/21). The campaign content was seen 2,656,014 times across web and social media. The digital ads and social posts generated 969,057 video views and 2,100 website page views. Mitchum assisted with content for three press releases and conducted five Ag media interviews including with Delta Farm Press, Adams on Ag Live, Brownfield Ag News, Iowa Business Radio Network, and Michigan Ag Today. Mitchum assisted with content for a 3-page article published by Gil Gulickson in Successful Farming, “It Starts With SPIT” highlighting the research directed at developing new sources of resistance to SCN. She also participated in a Webinar “Let’s Talk Todes: Actively Managing SCN” alongside SCN Coalition leaders Sam Markell and Greg Tylka and hosted by Farm Journal’s Agritalk with Chip Flory targeted at crop consultants, agronomists, extension agents, and growers.

The Tylka group conducted 24 radio and newspaper/magazine interviews from November 2020 through July 2021. The loss of effectiveness of PI88788 SCN resistance was discussed and this current NCSRP-funded research project was mentioned and described whenever time/space permitted. Also, SCN Coalition communications experts, a videographer, and Gil Gullickson, reporter from Successful Farming magazine, traveled to Athens, Georgia in November 2020 to videotape University of Georgia professors Melissa Mitchum, Richard Hussey, Zenglu Li, Bob Kemerait, and Wayne Parrott as well as a Georgia soybean farmer to create numerous additional videos for the “Let’s Talk Todes” video collection. The videos focusing on research, including this research project, can be viewed at www.thescncoalition.com/lets-talk-todes/research-collection. See https://youtu.be/0YqEM0CcvtY for the video in which Melissa Mitchum explains this current, NCSRP-funded research project.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

During the period of the grant, the results from the 2019 SCN Regional Test were sent to the University of Illinois by collaborators, summarized, and the first version of the report was provided to cooperators on the 19th of December 2020. This version included the agronomic, composition and resistance test results. This version was followed by the distribution of the final version on the 6th of January 2019 which included the results of testing the egg number and HG type of nematode populations in field environments. The 2020 SCN Regional Test was planned, seed was sent to cooperators, data were returned and the results were summarized in a report. The 2020 test included a total of 184 entries that ranged from MG 0 to IV. The initial version of the report was sent to cooperators on December 10th and the final version was delivered on January 12th. 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 2021 SCN Regional Test. This test includes 242 entries that range from MG 0 to IV. The tests were organized and the seed was shipped to the University of Illinois by cooperators and then redistributed to cooperators. The tests have been planted and are currently growing in the field.

View uploaded report

During this year of our project we have made several substantial and important discoveries noted by several important publications and presentations. The Baum and Severin groups would like to announce another significant milestone that we achieved during this funding period; the publication of our most detailed, chromosome-level assembly of the TN10 H. glycines genome in a high impact journal, Molecular Ecology Resources (Masonbrink RE, Maier TR, Hudson M, Severin A, Baum T. A chromosomal assembly of the soybean cyst nematode genome. Mol Ecol Resour. 2021 May 25. doi: 10.1111/1755-0998.13432. Epub ahead of print. PMID: 34036752.). As a consequence, all associated resources were publicly released on SCNBase, including: BLAST and JBROWSE capability for the genome assembly, predicted proteome, genome, transcriptome, and alignments for ribosomal genes, genome structural variation, SNPs and INDELs, alignment of X12 gene models, effector alignments, ncRNAs, multiple repeat predictions, and every putative input gene annotation. This will serve as a major informative resource to the entire SCN community worldwide.

The Hudson group, in collaboration with the Severin/Baum group, now has all seven of the target genotype assemblies completed. The Severin/Baum group assisted with manual curation using tools from their TN10 assembly workflow. Additionally, where possible, the genomes were improved by polishing with Illumina short reads using algorithms currently employed by large-scale genome projects. Alternate haplotype contigs were purged to generate seven assemblies each containing 9 pseudomolecules that correspond to the 9 SCN chromosomes. All seven assemblies have genome completeness scores that exceed those of the published TN10 assembly. Annotation is now underway to generate the predicted proteomes for comparison across the 8 SCN lines.

The Mitchum group is focused on identifying SCN virulence genes used by the nematode to overcome the Peking-type (Rhg4-mediated) resistance. Our comparative transcriptomic analysis comprises (1) differential expression and (2) variant call analysis by utilizing RNA-seq data generated from the early parasitic stages of virulent and avirulent SCN populations adapted on soybean recombinant inbred lines (RILs) that only differ at the Rhg4 locus (i.e., a resistant RIL with a resistant Rhg4 allele and a susceptible RIL containing a susceptible Rhg4). The differential expression analysis has allowed us to narrow down to 59 genes upregulated in the virulent SCN population. These genes were cross-compared with the nematode gland-specific RNA data sets to select 14 genes of special interest, categorized into three subgroups: (1) putative effectors involved in defense suppression, (2) putative enzymes related to reactive oxygen species, and (3) putative vitamin B-associated genes. These genes will be validated with qRT-PCR to confirm upregulation in the virulent population.

Additionally, the Baum and Severin groups would like to announce publication of the MPMI Resource Announcement, entitled “Esophageal Gland RNA-seq Resource of a Virulent and Avirulent Population of the Soybean Cyst Nematode, Heterodera glycines,” (Mol Plant Microbe Interact, 2021 Apr 26. doi: 10.1094/MPMI-03-21-0051-A.). We are exploring the RNA-seq dataset derived from a comparison of SCN gland cell transcriptomics of a virulent (MM10) and avirulent (PA3) population. This data analysis is published in the above Resource Announcement paper. Briefly, with this submission, we have announced the availability of a unique gland-specific RNA-seq dataset for the SCN community, which provides an expression snapshot of gland cell activity during early infection of a virulent and avirulent SCN population. This represents a highly valuable resource for researchers examining effector biology and nematode virulence. The data generated continue to be analyzed and is being used by the Baum lab in several avenues to progress omics research within the lab.

The Mitchum group continued the characterization of novel stylet-secreted effectors of the soybean cyst nematode Heterodera glycines parasitome, 16B09 and 2D01. These effectors belong to a highly expanded superfamily of novel effectors, share the same gene structure, harbor conserved protein domains, and exhibit the same spatial and temporal expression in the dorsal gland cell during parasitism. This group of effectors was also identified as upregulated in the RNAseq data set as potentially involved in virulence (outlined under 1.3). Thus, we conducted a full transcriptome and genome analysis of this group of SCN genes. A search of the latest nine-scaffold, 158 Mb pseudomolecule assembly of the H. glycines TN10 genome identified 20 full-length (4 exon-3 intron gene structure) or partial copies of this effector gene superfamily (Masonbrink et al., 2021). Twelve members of this family were present on scaffold 5/Chromosome 1 and eight on scaffold 6/Chromosome 6. A sequence similarity tree constructed using the neighbor-joining method of the Clustal Omega program (Sievers et al., 2011) and including the original SCN parasitome sequences revealed three distinct subgroups. These subgroup sequences clustered together in the genome and may share functional similarities with some members involved in manipulating host developmental programs and others involved in suppressing host defenses. The variation observed across SCN populations is consistent with the highly expanded and diversified nature of this gene family.

We have also now begun to collect enough data to have preliminary results from our field rotation study across Iowa, Missouri, and Illinois. At the conclusion of the 2020 growing season, two separate multiple-core soil samples were taken from each microplot in the two experiments that were conducted in Iowa. The first set of soil samples were used to determine end-of-season SCN population densities in each plot, and the second set of samples were sent to the University of Missouri for HG type testing to determine if and how the soybean genotypes grown in the microplots in 2020 might have shifted the virulence phenotypes of the SCN populations originally added to the microplots in spring 2019.
Preliminary data analysis was completed for each of the experiments, and some trends in changes in SCN population densities were observed. In both experiments, the highest SCN population densities in fall 2020 occurred in microplots in which the susceptible soybean variety was grown. The microplots that were continuous with the same resistance from 2019 had higher SCN population densities in fall 2020 than the microplots that had rotated resistant varieties from 2019. The lowest population densities were found in plots where PI90763 resistance was grown in 2020. The SCN population densities in plots with continuous PI90763 also were lower than those that had previously had rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 in 2019 and were then rotated to PI90763 in 2020.

The results of the HG Type test results on the SCN populations in the soil samples collected from the microplots at harvest in 2020 show promising results. The SCN population in the soil in the experiment at the Kanawha, IA location had an HG type of 1.2 with a range of 35-80% female index on PI88788 and 7-40% female index on Peking. The SCN population in the soil in the experiment at the Ames, IA location had an HG type of 1.2.3 with a range of 27-80% female index on PI88788, 12-73% female index on Peking and 1-47% female index on PI 90763. The plots with rhg1-a + rhg4 and PI90763 had the highest female index on Peking and PI90763, while every plot had a female index greater than 10% on PI88788 in experiments at both locations and a female index greater than 10% on Peking at the Ames location. Increased virulence on PI437654 was not observed. After 2020 we observed increased SCN reproduction and some slight changes in female index (virulence) between microplots with rotated and with continuous cropping sequences.

In May 2021, each microplot was planted according to the deployment sequence being used in all microplot experiments in the project. This sequence included a third year of planting the same variety in half of the microplots (continuous sequence) and a rotation sequence of varieties to what was planted in 2019 in the remaining half of the microplots. At 30 days after planting, each row in each microplot was thinned to 30 plants and those with fewer than 30 plants were recorded.

The Diers group has finished the 2019 rotation experiment and grew the 2020 experiment. We also recently received the HG type data from the fall 2019 plots as the results had been delayed until now because of Covid. Although the results have not been fully analyzed, a few observations can be reported. One is that the nematode population in all plots could overcome PI 88788 resistance and the female index (FI) on this source ranged from 10% to 96%. The fact that the nematodes could overcome PI 88788 resistance was expected because this was the HG type of the nematodes used to inoculate the plots. There were six plots that had nematodes that could overcome Peking resistance and the two plots with the highest FI on Peking (37-23%) were planted to a line with Peking type resistance. We are still waiting for the HG type data from the fall 2020 soil samples and these results should be available by the second week of August.

In Missouri, the Scaboo group harvested Fall 2020 soil samples for SCN egg counts, HG type tests were processed, and data was collected. The egg count data has shown that the susceptible plots have the highest egg densities followed by rhg1-b plots. The PI 90763, rhg1-a + Rhg4, and rhg1-b + G.soja + ch.10 treatments had the lowest population densities and the highest reduction in SCN egg densities compared to initial levels. In continuous treatments, the highest reduction in egg counts was observed in PI 90763 and rhg1-a + Rhg4. Similarly, the highest reduction in percentage change in the egg counts is in those treatments rotated with PI 90763. Interestingly, rhg1-a + Rhg4 treatments rotated with rhg1-b or its multiple gene stacks showed the largest reduction in egg counts that have been observed in the treatments with rhg1-b stacked with other resistance loci. The SCN HG type data has shown that the highest shifts in virulence were in continuous PI 90763 and continuous rhg1-a+Rhg4 plots. In continuous rhg1-a+Rhg4 treatment, increased virulence on PI 90763 and Peking has been observed. Similarly, in continuous PI 90763 treatment, an increase in virulence on Peking and PI 88788 has been observed. The seeds obtained for the third-year field season experiment have been checked, packaged and were planted in the microplots during June of 2021.

The Mitchum lab helped to produce The SCN Coalition’s “Let’s Talk Todes” Research Collection video series now released online https://www.thescncoalition.com/lets-talk-todes/research-collection. In this 7 video series, soybean growers and scientists (nematologists, breeders, plant geneticists, extension pathologists) who are battling SCN explain the checkoff-funded research they are conducting that's focused on bringing new tools to soybean growers in the fight against parasitic nematodes. During this reporting period, MorganMyers deployed promotions of the “Let’s Talk Todes” Research Collection through a 7-week digital campaign (5/23/21-7/10/21). The campaign content was seen 2,656,014 times across web and social media. The digital ads and social posts generated 969,057 video views and 2,100 website page views. Mitchum assisted with content for three press releases and conducted five Ag media interviews including with Delta Farm Press, Adams on Ag Live, Brownfield Ag News, Iowa Business Radio Network, and Michigan Ag Today. Mitchum assisted with content for a 3-page article published by Gil Gulickson in Successful Farming, “It Starts With SPIT” highlighting the research directed at developing new sources of resistance to SCN. She also participated in a Webinar “Let’s Talk Todes: Actively Managing SCN” alongside SCN Coalition leaders Sam Markell and Greg Tylka and hosted by Farm Journal’s Agritalk with Chip Flory targeted at crop consultants, agronomists, extension agents, and growers.

The Tylka group conducted 24 radio and newspaper/magazine interviews from November 2020 through July 2021. The loss of effectiveness of PI88788 SCN resistance was discussed and this current NCSRP-funded research project was mentioned and described whenever time/space permitted. Also, SCN Coalition communications experts, a videographer, and Gil Gullickson, reporter from Successful Farming magazine, traveled to Athens, Georgia in November 2020 to videotape University of Georgia professors Melissa Mitchum, Richard Hussey, Zenglu Li, Bob Kemerait, and Wayne Parrott as well as a Georgia soybean farmer to create numerous additional videos for the “Let’s Talk Todes” video collection. The videos focusing on research, including this research project, can be viewed at www.thescncoalition.com/lets-talk-todes/research-collection. See https://youtu.be/0YqEM0CcvtY for the video in which Melissa Mitchum explains this current, NCSRP-funded research project.

During the period of the grant, the results from the 2019 SCN Regional Test were sent to the University of Illinois by collaborators, summarized, and the first version of the report was provided to cooperators on the 19th of December 2020. This version included the agronomic, composition and resistance test results. This version was followed by the distribution of the final version on the 6th of January 2019 which included the results of testing the egg number and HG type of nematode populations in field environments. The 2020 SCN Regional Test was planned, seed was sent to cooperators, data were returned and the results were summarized in a report. The 2020 test included a total of 184 entries that ranged from MG 0 to IV. The initial version of the report was sent to cooperators on December 10th and the final version was delivered on January 12th. 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 2021 SCN Regional Test. This test includes 242 entries that range from MG 0 to IV. The tests were organized and the seed was shipped to the University of Illinois by cooperators and then redistributed to cooperators. The tests have been planted and are currently growing in the field.

This project will benefit soybean producers by creating a long term management strategy for SCN through knowledge and soybean germplasm development.

Objective 1: Diversify the genetic base of SCN resistance in soybean.
Accomplishments: In Phase I of this project, we developed experimental lines with new combinations of resistance genes. Genes that were combined included Rhg1 from PI 88788 or PI 437654 (Hartwig), Rhg4 from PI 437654, genes on chromosomes 15 and 18 from wild soybean G. soja, a gene from PI 437654 on chromosome 11, and genes on chromosomes 10 and 18 from PI 567516C. Combinations of these genes were tested for SCN resistance. Associations with yield in SCN-infested environments were made to determine whether these resistance genes are associated with greater yield. Gene combinations effective in controlling SCN are being bred into elite soybean germplasm using marker-assisted selection.

Objective 2: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Accomplishments: In Phase I of the project we used innovative methods and new sequencing technologies to enhance and complete the current SCN reference genome assembly for the TN10 population (HG type 0) and the sequence of its bacterial endosymbiont. Additionally, we used state of the art bioinformatics tools to make substantial progress to annotate and curate the SCN genome. We generated a comprehensive SCN gene expression atlas from whole nematodes, which was also used in the annotation of the SCN genome. We created a genomic toolbox for SCN (SCN-Base) that facilitates the integration of very large sequence data sets, molecular markers, QTL data and genetic maps into an easy-to-use web interface. This resource will soon be released as a website that includes a Genome Browser for easy visualization of the SCN genome. We also developed lower-cost methods to produce reference genomes of additional HG types.

Objective 3. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
Accomplishments: As described above, experimental lines with resistance gene combinations developed in Objective 1 during Phase I of this project were tested in four different rotation schemes with experimental lines containing various resistance gene combinations in a greenhouse study. SCN population increase was measured after each generation for 8 generations. Following the eighth generation of selection, the HG type of each population was determined. From this, we identified alternative resistance gene combinations that when used in rotation reduce the selection pressure on the SCN population thereby slowing nematode adaptation to resistant varieties.

Objective 4. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
Accomplishments: In Phase I of the project, an extension and outreach coordinator, advised by project Co-PI Dr. Tylka, was hired to provide farmer education and outreach for the project. A survey of extension and outreach educational materials about SCN biology and management in the NCSRP states was conducted by the coordinator. Materials from land-grant universities and private seed and chemical companies were gathered, analyzed, and compared.

Objective 5. Coordinate the testing of publicly developed SCN resistant experimental lines.
Accomplishments: During 2018, the testing of SCN resistant experimental lines developed by breeders in 11 north central US states and Ontario was coordinated. The tests include 182 SCN resistant experimental lines and varieties that are being grown in 104 maturity group specific trials across 39 locations. These lines are also being tested for SCN resistance and the soil samples from the environments will be evaluated for SCN population density and HG type.