Updated April 30, 2020:
Project Report: October 1, 2018-September 30, 2019 (Year 1 Final Report)

Objective 1: Diversify the genetic base of SCN resistance in soybean.
Although a large number of sources of resistance to SCN are available to soybean breeders, PI 88788 is the resistance source for over 90% of the varieties available to growers in the North Central US. The effectiveness of PI 88788 resistance has decreased over time as the nematodes have adapted to this resistance source. Research over the past 20 years has resulted in the identification of resistance genes from several other sources of resistance. However, the resistance genes from novel sources largely have not been transferred into elite soybean varieties. Research is needed to overcome the bottlenecks that have slowed the incorporation of these new genes into commercial varieties.

Brief Summary of Accomplishments:
1.1 Develop and evaluate germplasm with new combinations of resistance genes in high-yielding backgrounds (Diers, Scaboo).
Progress has been made in developing varieties with alternative sources of resistance. In the 2018 SCN MG III prelim regional test, the second highest yielding line had resistance from PI 88788 combined with the chromosome 15 and 18 genes from G. soja. In the MG IV test, the second highest yielding line also had the same gene combination. The test of the population segregating for resistances genes Rhg1, Rhg4, the two G. soja genes, and the gene on chromosome 10 was completed in two locations in both Illinois and Missouri. When the effects of the resistance genes were analyzed across the field locations, we found that only Rhg4 was associated with significantly greater yield, whereas the other genes had no detectable impact on yield or a significant negative impact on yield. One issue with these tests is that the nematode populations were low in the experimental locations, which made it difficult to detect a positive impact for having SCN resistance. Early generation populations segregating for Rhg1 and Rhg4 and the chromosome 11 QTL derived from cultivars in the mid-south (i.e. S11-20124C released by Shannon et al., 2019) which have sources of each of the targeted genes and QTL from PI 90763, PI 437654, and/or PI 88788 have also been developed.

Objective 2: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Past research efforts to determine the inheritance of SCN virulence (i.e., the ability of the nematode to reproduce on resistant varieties) led to the identification of the reproduction on resistant varieties or ror genes, which were shown to be inherited in both dominant and recessive manners. However, since the initial discovery of these genes there has been no further information published concerning their sequence identity or mechanism in conferring SCN virulence. Genome and transcriptome comparisons of SCN populations that differ in virulence on resistant soybean have the potential to identify genes underlying virulence, determine the mechanism/s of virulence, and lead to the development of molecular diagnostic tools to assess for virulence in field populations.

Brief Summary of Accomplishments:
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: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 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).

The project investigators met in St. Louis in November 2018 to establish the details of the nematode sequencing experiments to be conducted in Illinois, Missouri, and Iowa in 2019 in order to address this objective. Additional discussions occurred via teleconference and email in early 2019.

We have successfully assembled and published the first version of the SCN genome (BMC Genomics 2019; 20:119). We have also completed and published detailed analysis of “spliced leader preference in SCN” (Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1. Scientific Reports 2019, 9; 1356). Currently we are working on scaffolding a second SCN assembly with Chicago and Hi-C to get closer to a pseudomolecule assembly.

We have sequenced the genomes of five additional virulent SCN inbred populations that differ in their ability to reproduce on resistant varieties (i.e., different HG types). All primary raw sequencing data from all strains and technologies has been deposited under embargo into NCBI SRA repository with required BioProject and BioSample accessions. Draft assemblies have been generated using both Oxford Nanopore and Illumina technologies and these assemblies meet or exceed the quality metrics of many published draft genomes for nematodes. However, we wish to publish higher quality genomes given the complex nature of the SCN genome and our desire to locate the genes important for virulence, which are likely repetitive in nature and may be hard to resolve from draft genomes. We therefore determined strategies and negotiated contracts with Dovetail Genomics for improving current SCN assemblies using their Chicago and Hi-C library services and HiRise assembly software. For this, we reared nematode material for the five aforementioned virulent SCN inbred populations, an avirulent population, and the SCN inbred field population used in rotation studies. This material was shipped to Dovetail and subjected to Chicago and Hi-C library generation and assembly. We have now received the Dovetail assemblies for four of the five HG types. For the assemblies that we have received, quality metrics are excellent, with all of them as good or better than the published TN10 genome sequence. We are now sequencing two additional HG types and annotating the genomes to identify effector sequences.

The adapted populations under Obj 2.2 have also been further selected, typed, and are currently being reared for collection of nematode material for sequencing. We have evaluated and rejected wtdbg2 as an improved assembler compared to SMARTdenovo for SCN. We tested the newest Oxford Nanopore basecaller, guppy 2.3.5 and compared basecalling accuracy to previous versions. We have settled on a set of Oxford assembly software that produces optimal results. We have also developed a methodology for Pacific Biosciences sequencing and have submitted the Polar Genomics DNA for Pacific Biosciences sequencing, where our own DNA was previously not successful. We are waiting for the data to confirm the effectiveness of our method in this organism.

We also negotiated a contract with Polar Genomics for DNA extraction services for long-read technologies. We found that although we can extract DNA in our laboratory that works well for Illumina or Sanger sequencing, our own protocols yield inconsistent data from Oxford sequencing and are insufficient for Pacific Biosciences. We have found the Polar Genomics DNA works well where our own DNA gave insufficiently high quality data. We have thus contracted with Polar Genomics for DNA for the remaining unsequenced nematode lines to be sequenced using Pacific Biosciences technology.

Simultaneously, we continue to work on improving our SCNBase website. 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. We have added tutorials to SCNBase describing how to upload different data types to the web repository. We have also generated a Github repository for all the scripts needed to create files for SCNBase. A total of 43 annotation tracks have been added to SCNBase. We have developed a method for annotating genes with orthologous species that significantly increases the proportion of annotated genes with higher confidence. We have also added a “content-by-category” link to the SCNBase homepage that details many of these new analyses/annotations and the methods necessary to derive such results. The SCNBase paper has been written and is currently under review by collaborators before submission to the “Database” journal. Meanwhile, we continue to add newly generated SCN genome/transcriptome related data to this repository to make it available to our collaborators as well as the SCN community worldwide. 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.

In support of Objective 2, we developed transcriptomic resources from an avirulent and a highly virulent population of SCN. We isolated esophageal gland cells from each of these populations and constructed RNA-seq libraries from pools of 100 gland cells from each population. Samples representing three independent biological replications of each population were generated. Sequencing of some of these libraries confirmed their utility in allowing us to validate the candidacy of recently discovered novel effector candidates from our prior work (Scientific Reports 2018; 8:2505). Several novel candidate effector candidates were cloned utilizing the SCN genome sequence. Additional sequence analysis and confirmation of gland expression was initiated. Once sequencing is complete in year 2, in-depth studies of transcript differences between these populations and how that may relate to virulence will be conducted.

Successful SCN parasitism relies on syncytium establishment and its continued maintenance via long-term host defense suppression. These complex molecular tasks are accomplished by SCN injecting a suite of effectors into the host tissue. As a part of this project, while we have started to identify novel and population specific effectors or effector variants, we also conducted in-depth molecular characterization of previously identified effectors. 32E03 is one such effector that we studied extensively. This nuclear localized effector specifically targets and modifies the activity of the genomic DNA associated proteins. Due to these epigenetic modifications, we discovered that the expression levels of specific host genes, rDNA, were altered, which helps the cyst nematodes establish infections. We published this study in a high impact, peer-reviewed journal (The Plant Cell 2018, 30 (11) 2795-2812). Simultaneously, we conducted detailed functional characterization studies of the 28B03 and 2D01/16B09 classes of effectors. Our data show that the 28B03 effector is involved in host defense suppression and the 2D01 effector may be modulating cell wall reprogramming for feeding site formation. We determined that the 28B03 effector modulates phosphorylation status of a defense-related membrane bound protein by interfering with a kinase cascade and the 2D01 effector protein interacts with the kinase domain of a receptor required to regulate cell wall modification while protecting plants from pathogen attack.

Objective 3. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
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. The study was moved to phase II field trials in year 1 of this project.

Brief Summary of Accomplishments:
3.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum).
The project investigators met in St. Louis in November 2018 to establish the details of the field experiments conducted in Illinois, Missouri, and Iowa in 2019 in order to address this objective. Additional discussions occurred via teleconference and email in early 2019. The investigators discussed various experimental details such as the structure and size of the microplots to be used, the experimental design and layout of the experiments, the infestation methods to be used to introduce SCN into the soil within the microplots, and collection of soil samples. Fields for this study were identified in all three states. Seed was increased by the University of Illinois and University of Missouri and was packaged for shipment and exchange among Universities. Also, investigators in each state cultured populations of SCN in pots in the greenhouse in the fall of 2018 and maintained cultures through the winter to increase the nematode numbers for infestation of the microplots that comprise the field experiments. In Fall 2018 and January 2019, two new graduate students were hired at University of Missouri and Iowa State University and have begun working on experiments in this objective of the project. The microplots in each state were infested and planted on schedule, soil samples were taken after infestation at planting to determine initial egg population densities in each microplot and HG type tests were conducted on soil samples taken from susceptible plots at 60 days post-planting to determine the baseline HG type of the SCN populations at each location. Subsequent analysis and field sampling was continued during the fall and winter of 2019.

Objective 4. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
We will communicate the results of our studies to SCN Coalition members, which includes extension plant pathologists and nematologists at the land-grant universities in the 12 NCSRP states and researchers at partnering private companies, for the development of education/outreach materials on how to best manage SCN using strategic rotation of resistance varieties for sustainable soybean production. We will ensure that messages are relevant and pertinent to the local conditions with SCN in each state.

Brief Summary of Accomplishments:
4.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum).
The SCN Coalition is a national educational campaign funded by NCSRP, USB, and private industry to educate farmers and agribusiness personnel who advise farmers about the current situation with SCN resistance as well as about new research developments, including new and genetically diverse sources of SCN resistance. In year 1 of the project (FY2019), project Co-PI Tylka gave 25 radio interviews, including one on WHO radio’s “The Big Show” that reached listeners in IL, IA, MN, MO, NE, and WI. Another notable interview given was with Tyne Morgan; the video interview played on US Farm Report (500,000 viewers) at Commodity Classic in February 2019. During year 1, Co-PI Tylka gave 14 presentations and a webinar, including a presentation to 400 crop advisers at the Indiana Certified Crop Advisors annual meeting in Indianapolis in December 2018. Tylka also had one-on-one conversations with approximately 100 farmers at the Farm Progress Show in Decatur, IL, in August 2019. In these interviews, presentations and conversations, Tylka described the loss of effectiveness of PI 88788 resistance and the need for novel resistance (including mention of this project). A news conference was organized by the SCN Coalition at Commodity Classic in Orlando on February 28, 2019. At the press conference, PI Mitchum presented the six goals of the recently released “National Soybean Nematode Strategic Plan”. Goal 2 of the plan is to “Discover, leverage and enhance native nematode resistance in soybean.” Our current NCSRP-funded research project was mentioned at the press conference as an example of work under Goal 2 of the strategic plan. A press release was distributed on February 28 in conjunction with the press conference, and it also mentioned the current NCSRP-funded research project. Numerous news articles highlighting the research being conducted under this project have been released since. Several project PIs also informed plant pathologists, nematologists, plant breeders, industry partners, and growers on the results of our work at the 2019 Soybean Breeder’s Workshop held in St. Louis, February 11-13, 2019.

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

Brief Summary of Accomplishments:
5.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario.
During the reporting year, results from the 2018 trials were analyzed, summarized into a report and provided to cooperators and other interested parties. Because of the need for breeders to make quick decisions on advancing experimental lines, we made the preliminary analysis available as soon as possible after harvest. The first preliminary draft was made available on December 13 and a final version was made available on January 11. This 2018 report includes results from the testing of 182 experimental lines and checks were grown in 39 locations in 11 states and one Canadian province. In addition, soil samples from the test locations were analyzed for SCN egg number and HG type and the lines in the test were evaluated for resistance to two SCN isolates. All soil samples had an HG type of 2.5.7 except one that had an HG type 1.2.5.7. This means that at all test locations the nematodes can overcome resistance from PI 88788. The egg counts from locations ranged from 40 to 8160 eggs / 100 cc of soil. Progress has been made in developing varieties with resistance other than from PI 88788. In the MG II test, the highest and second highest yielding lines have high levels of resistance from PI 437654 to an HG 2.5.7 population. As mentioned previously, the second highest yielding line in the MG III prelim test and the MG IV test had resistance from PI 88788 combined with the chromosome 15 and 18 genes from G. soja.

The 2019 test was planned and seed of the test entries was distributed to cooperators. This test included 190 experimental lines and checks grown in 29 locations in 10 states and one Canadian province. At the end of this reporting period, the results from these tests had not been provided to test organizers.

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 Phase II of an integrated, collaborative, and multi-state project among plant breeders, molecular biologists, bioinformaticians, and nematologists was initiated. Our proposed objectives specifically addressed 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.

We achieved the following during the year 1 funding cycle:
Soybean yield results of 182 publicly developed SCN-resistant experimental lines and checks grown in 39 locations in 11 states and one Canadian province were summarized into a report and provided to cooperators and other interested parties. Soil samples from the test locations were analyzed for SCN egg number and HG type and the lines in the test were evaluated for resistance to two SCN isolates.

Significant progress was made in developing soybean varieties with SCN resistance other than from PI 88788. In the MG II test, the highest and second highest yielding lines have high levels of resistance from PI 437654 to an SCN HG 2.5.7 population. The second highest yielding line in the MG III prelim test and the MG IV test had resistance from PI 88788 combined with the chromosome 15 and 18 genes from G. soja.

Three-year field experiments were initiated in Illinois, Missouri, and Iowa to determine the most effective SCN resistance rotation practices to combat SCN. Microplots in each state were infested and planted on schedule, soil samples were taken to determine initial egg population densities in each microplot and HG type tests were conducted on soil samples taken from susceptible plots to determine the baseline HG type of the SCN populations at each location.

The first version of the SCN genome was successfully assembled and published. A second SCN assembly is underway to get closer to a pseudomolecule (higher resolution) assembly.

The genome sequences of five virulent SCN HG types were generated. Draft assemblies were completed and are being improved further to help surmount the complex nature of the SCN genome in order to locate the genes important for virulence, which are likely repetitive in nature and may be hard to resolve from draft genomes.

A field population of SCN adapted to reproduce on the different types of resistance (PI 88788, Peking, G. soja) was further selected to generate SCN virulent populations typed and reared for collection of material needed for long-read genome sequencing and population genetic studies.

The SCNBase website was improved. A significant increase in “web traffic” to this web portal suggests that it is generating considerable interest in the SCN community from all over the world. SCNBase provides prime visibility and impact of the collective data procured through farmer investments in research, which will enable others in a coordinated fight against this pathogen.

Whole nematode and gland-enriched transcriptomic resources from avirulent and highly virulent populations of SCN were developed. Sequencing confirmed utility for identifying SCN effector candidates and gene cloning was facilitated by access to the SCN genome sequence. Genome-assisted studies of transcript differences between SCN populations and how that may relate to virulence are being initiated.

SCN parasitism relies on feeding site establishment in soybean roots and its continued maintenance via long-term host defense suppression by injecting a suite of effectors into the host tissue. Identification of novel and population specific effectors or effector variants was initiated, and in-depth molecular characterization identified effectors that modify host plant DNA and suppress plant defenses to cause disease.

The SCN Coalition educated farmers and agribusiness personnel who advise farmers about the current situation with SCN resistance as well as about new research developments, including new and genetically diverse sources of SCN resistance. Twenty-five radio interviews that reached listeners in IL, IA, MN, MO, NE, and WI, a video interview on US Farm Report (500,000 viewers), 14 presentations and a webinar, including a presentation to 400 crop advisers at the Indiana Certified Crop Advisors annual meeting, one-on-one conversations with approximately 100 farmers at the Farm Progress Show, a news conference at Commodity Classic highlighting the launch of the “National Soybean Nematode Strategic Plan,” and numerous news articles highlighting the research being conducted under this project.