Update:
A description of relevant progress for principal and co-principal investigators is below for each objective and sub objective in our proposal. Our team has made tremendous progress in accomplishing our research goals, conducting field experiments, publishing refereed journal articles, and communicating our results to scientists and soybean producers. We attended a wonderful SCN conference in Savannah Georgia in December of 2022 where many project members and their respective staff and students presented research supported by this project including Dr.’s Mitchum, Tylka, Diers, and Scaboo. We are planning our next group meeting for the first week of April 2023 to discuss current research progress and goals, and we are on track to continue our cutting-edge research in soybean cyst nematode biology, management, and breeding for novel resistance.
Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Sub-objective 1.1: Combine, compare, and catalogue the genomes that compromise the SCN pan-genome. (Hudson, Baum, Mitchum)
The Baum lab finished the analysis of gene expansion in the Tylenchomorpha and have identified a number of interesting gene candidates for further analysis. Multiple genes were found to be involved in host defense suppression, host cell remodeling, manipulation of host ion concentrations, redox, and manipulating host metabolism. SCNBase has undergone some extensive updates in the past months. We clustered the predicted proteins from each of the available SCN genome assemblies (TN10 draft, TN10 pseudomolecule, and X12), so that users can more easily infer how genes are related between assemblies. In addition, we’ve aligned genes from each of the three assemblies to each other to provide better spatial interpretation of different gene predictions. We’ve also integrated BLAST databases for each of these genomes, their transcripts, and predicted proteins. There were several updates to genomic tracks in JBROWSE: transposon annotations, genes that overlap with repeats, tandem repeats, large structural variants, SNPs for all available short read data from other populations, noncoding RNAs, alignments of all H. glycines ESTs, NCBI nucleotide entries, all long read transcript sequences available, and the transcriptome from Gardner et al 2018. We combined the repetitions of all individual RNAseq samples to allow users to better evaluate expression of a gene in JBROWSE. Many of the information pages on SCNBase were updated or added. We summarized the statistics for each of the three genome assemblies and compiled these stats on a single page that is linked to SCNBase and included an explanation for better interpretation. All data that was provided with the pseudomolecule assembly of TN10 now has an improved explanation of abbreviations associated with gene conditions in the Features Database. We better defined the data types that can be found on SCNBase and have provided a list of all public SCN data. We also converted the Research page to display all papers that have been published through our NCSRP funding.
Previously, the Mitchum lab employed a dual effector prediction strategy that coupled the traditional secreted protein prediction strategy with a newly developed nematode effector prediction tool, N-Preffector, to identify novel effector candidates in a de novo transcriptome assembly of the pre-parasitic and parasitic life stages of H. glycines with potential roles in virulence. From this analysis, eight novel candidate effectors with high to moderate expression in the gland RNA-seq dataset were identified. We mined the SCN pseudomolecule genome assembly to determine the gene structures and genomic organization of these sequences and have moved forward for further analysis in sub-objective 1.3 below.
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
Previously, the Hudson group developed a successful method to extract genomic DNA from a single J2 SCN with sufficient quality and quantity for whole genome sequencing. Different DNA library construction kits were tested at Roy J. Carver Biotechnology Center on campus and the best performing one for our purposes was selected. We have now accomplished collecting DNA samples from a large number of individuals from two different SCN populations, MM1 and MM2. From the virulence perspective, MM1 and MM2 SCN populations are significantly different from each other. Although both are originally driven from a common source, they have been adapted and multiplied separately in two different ways for more than ten years to make a virulent and an avirulent population. Therefore, these data can help us to perform a comparative population genomic analysis to find SNPs and genetic regions under selection for SCN virulence. 192 samples per population were sent to the Roy J. Carver Biotechnology Center for DNA library construction and sequenced on a NovaSeq 6000. The sequence data were recently received and we are currently checking the data quality. We will then align them to our newly-generated reference-quality genome for this population and begin population genetic analysis.
The Baum group continued to build resources in the form of gland cell-specific libraries. We have continued to develop libraries for the pre-infective J2 life stage, and now have the avirulent (PA3) population library constructed. Notably, we have some encouraging preliminary data (unpublished) from a collaboration in another plant-parasitic nematode, in which we prepared an analogous library from the same pre-infective life stage. What is significant is that this data represents the first evidence that this new live single-cell library approach works very well to generate a substantial list of candidate effectors from very limited starting material. There are also other very intriguing results from this specific life stage that we look forward to confirming in SCN. Additionally, using our approach to separate gland cell types, coupled with our ability to generate single-cell libraries, we have constructed our first subventral gland-specific libraries from pre-infective J2 stages of SCN and have recently submitted this for sequencing. We are hopeful that this will yield a useful dataset from which we can perform the subtraction strategy described previously and that this strategy will yield information on which effectors are present and active in each gland type in the pre-infective J2 stage of SCN.
The Mitchum lab has completed the Pool-Seq analysis of the two pairs of SCN populations un-adapted or adapted to reproduce on resistant soybeans. Previously, we reported the identification of five genomic regions spanning four chromosomes showing strong signatures of selection, as determined by the population differentiation (FST) estimates from the entire single nucleotide polymorphism (SNP) dataset containing approximately 800 K SNPs. Although FST analyses show strongly differentiated genomic regions, they are generally known to be prone to false positives, which is why we needed to gain further confidence in these candidate regions. For this reporting period, we confirmed extra evidence of selection in these regions by conducting the Fisher’s Exact test as well as principal component analysis (PCA)- and XtX-based approaches. After filtering for the overlapping SNPs identified from multiple outlier detection methods, we discovered a total of 316 significantly overly differentiated SNPs from both pairs of SCN populations. These SNP-harboring candidate genes are now being tested for their correlation to virulence. We have also been preparing a manuscript for our Pool-Seq findings which we hope to publish this year.
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
During this most recent phase of the project, the Baum group has focused on developing tools for the scientific community to employ when conducting in planta SCN studies. Most notably, we have constructed a set of GATEWAY-compatible vectors to facilitate cloning of a gene of interest in frame with different epitopes to perform functional analysis on soybean roots. Those vectors allow the selection of transgenic roots via the mCherry fluorescence or via the expression of the novel non-invasive reporter gene RUBY (which produces a red pigmentation). To confirm functionality of those vectors, we successfully express different subcellular markers in soybean roots. Along with those vectors, we are establishing different approaches to determine and study the interacting soybean proteins for SCN effectors such as immunoprecipitation or proximity-labelling followed by mass-spectrometry.
The Mitchum group has continued characterization of the 8 novel effector candidates identified under objective 1.1 is underway. We have profiled the expression of these genes in SCN throughout the life cycle and initiated studies to confirm where they effectors potentially localize within host cells after secretion by the nematode.
Objective 2: Complete the evaluation of how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles. (Diers, Scaboo, Tylka, Mitchum)
The Scaboo group completed processing of the fall 2022 soil samples from the Missouri location. SCN egg density and HG type test results were recently distributed for data analysis. SCN from each microplot were increased and sent to the Mitchum group for archiving. We have a group meeting in April of 2023 to review the data analysis in preparation for manuscript submission.
In October 2022 the Tylka group collected two separate multi-core soil samples from all microplots in each of the two microplot experiments conducted in Iowa. One set of samples were processed at Iowa State University to determine the end-of-season SCN egg population density in each microplot. The second set of samples were sent to the University of Missouri for HG type testing to determine how soybean genotypes grown in the microplots in 2022 affected or shifted the virulence profiles of the SCN populations and how the virulence phenotypes differed from the virulence phenotype of the SCN populations used to infest the microplots initially. Overall, SCN population densities increased in all microplots over four years and year-to-year differences occurred among plots with rotated soybean genotypes, but results varied somewhat between locations. For most microplots, rotated treatments had lower SCN population densities than treatments planted with the same genotypes continuously. Gene pyramid 2 (rhg1-b+G. soja+Chr.10) rotated with PI 90763 (rhg1-a, Rhg4, rhg2) had the lowest SCN egg population density at both locations. However, this rotation caused the virulence of the SCN population to increase, as reflected by elevated female index (FI) values. In the Ames experiment, the initial SCN population used to infest the microplots had a FI of 7 on PI 90763, and the continuous PI 90763 treatment and the rotation of pyramid 2 with PI 90763 caused the FI to increase to 27 and 12.5, respectively, over three field seasons. The FI on PI 88788 remained well above 10 across all microplots, even in SCN populations not exposed to the rhg1-b gene, which PI 88788 possesses. Additional shifts in virulence were observed but were less substantial in comparison to those described above. HG Type test results of the SCN populations in samples collected from the 2022 microplots have not been received yet.
Objective 3: Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers and inform growers on effective rotation schemes designed to protect our resistant sources. (Tylka, Mitchum)
Monica Pennewitt, PhD student with Greg Tylka at Iowa State University, was selected to give a presentation at the National Soybean Nematode Conference in Savannah, Georgia in December 2022. About 200 attendees were present for Pennewitt’s presentation, which described the overall scope and organization of the experiments in objective 2 of this project. Pennewitt also presented and discussed some of the results of the microplot experiments. And between October 2022 through March 2023, Greg Tylka conducted 6 interviews with radio and newspaper/magazine journalists and gave 10 presentations to agribusiness groups (in person and virtual) about SCN. The loss of effectiveness of PI 88788 SCN resistance was discussed in every interview and presentation, and Objective 2 of our NCSRP-funded research project was mentioned and described whenever time/space permitted.
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Diers)
The Diers group sent seed to cooperators for the 2022 SCN Regional Test, received the collected data during the fall of 2022, and disseminated the complete report in February of 2023. This 2022 test includes 225 entries that range from MG 0-IV. The regional test cooperators and the Diers group have also organized this trial for 2023, and seed shipment for packaging and planting is currently underway.
Objective 5: Diversify the genetic base of SCN resistance in soybean by developing and evaluating germplasm and varieties with new combinations of resistance genes in high-yielding backgrounds. (Diers, Scaboo)
The Scaboo group has now completed successful crossing attempts (3 backcrosses) using PI 90763 as a donor parent, and LD11-2170 and SA13-1385 as recurrent parents, for three major genes associated with resistance to virulent nematode populations (rhg1-a, rhg2, and Rhg4). For each crossing attempt, we have identified desirable F1 plants using marker assisted selection, and we have sped up the process by utilizing our winter nurseries in Hawaii and Puerto Rico for the last two years. During the summer of 2022, we grew over 100 plant rows derived from selected plants, and our first yield trials of this material will be in the summer of 2023. We have also identified several lines in our breeding program with the desirable three gene stack, confirmed phenotype of resistance, and selected them for advanced testing in 2023. Additionally, we are actively identifying and introgressing new and novel QTL/genes into our breeding program for cultivar development.
The Diers group has continued to work to diversify SCN resistance away from PI 88788 based resistance in Midwestern adapted cultivars. To do this, we have continued to select for the major SCN resistance genes rhg1-a and Rhg4. During the past summer, we tested over 5000 plants to select these genes and selected over 950. These selected plants will be advanced to plant rows during the 2023 growing season. In addition, in advanced yield tests we evaluated 35 experimental lines that carried both rhg1-a and Rhg4 and 22 lines with two SCN resistance genes from G. soja.
Update:
A description of relevant progress for principal and co-principal investigators is below for each objective and sub objective in our proposal. Our team has made tremendous progress in accomplishing our research goals, conducting field experiments, publishing refereed journal articles, and communicating our results to scientists and soybean producers. We are planning our next group meeting for the winter of 2023 to discuss current research progress and goals, and we are on track to continue our cutting-edge research in soybean cyst nematode biology, management, and breeding for novel resistance.
Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Sub-objective 1.1: Combine, compare, and catalogue the genomes that compromise the SCN pan-genome. (Hudson, Baum, Mitchum)
The Baum lab has generated large amounts of RNAseq and Nanopore cDNA sequencing across seven life stages of SCN. We have used these data to improve the gene annotations of all existing SCN genomes. This annotation has resulted in a consistent 3-5% improvement in BUSCO scores across all genomes and lineages. Because each gene prediction was produced using the same pipeline, we were able run Orthofinder to cluster genes by identity to be able to assign gene names based on gene families. Thus, we now have improved annotations for all 9 SCN genomes that can be readily compared across genotypes. In addition, each gene has been annotated with functional information from NCBI NR and UniProt databases. Furthermore, we have produced a number of genome browser tracks that will be helpful to researchers, including bigwig expression tracks for RNAseq and Nanopore cDNA, two repeat tracks, known effector alignments, and all annotations used to produce the final output. Among these, we produced a robust repeats track that utilized all 9 SCN genomes to identify genome repeats, which should in theory improve transposon models, resulting in fewer spurious gene models.
In collaboration with the Mitchum Lab, the sequencing and analysis of pooled SCN populations has provided quick and promising results. In the previous report, we provided pool-seq results based on the alignment of the sequencing data to TN10 reference genome - published on SCNbase. However, alignment against one linear reference genome can lead to bias towards the alleles present in the reference haplotypes. Some procedures can be done to minimize reference bias: on one hand, pan-genomes are promising alternatives that cope with the reference bias issue, and that is one of reasons why our work on the SCN pan-genome is fundamental for the future of SCN genomics research. On the other hand, reference bias can be minimized by using multiple representative reference genomes. Therefore, we have been re-estimating the population genetic parameters using multiple representative reference genomes from our new set of references (PA3 and MM26) related to the populations addressed in pool-seq analysis. We aim to identify any possible signatures of selection that were not previously detected due to reference bias. The Hudson group have also completed the raw single-juvenile SCN whole-genome sequencing data on the individuals supplied by the Mitchum group, completed the initial analysis and started the downstream analysis. Haplotype-based statistical approaches for selection scan (XP-EHH and Rsb) and related metrics were applied to the unphased SCN genomic data. Preliminary results showed high significant (FDR corrected: p < 1E-5) candidate regions under selection for virulence in SCN genome. These preliminary results parallel with pool-seq results, but with outstanding increase in resolution. Some of these candidate regions are probably harboring the genes with the most leading role in SCN adaptation to Peking type resistant soybeans (RHG1a/RHG4). Now, our analysis focuses on phasing the genotypes and estimating the population effective size (Ne). The first will increase the performance and resolution of haplotype-based statistical methods, and the latter will be the one of first attempts to estimate this important parameter in SCN populations. Our final analysis at this step would be introducing the probable genes involved in adaptation.
The Mitchum lab has continued to focus on using the available SCN genomes to conduct genome analyses for the candidate virulence genes identified from the Pool-seq analysis described below. This involves manual annotation, mapping identified SNPs and predicting impacts on protein function, assessing candidates for signal peptides, subcellular localization, and esophageal gland expression.
Sub-objective 1.2: Resequencing of the genomes and transcriptomes of virulent SCN populations and conduct comparative analyses. (Hudson, Mitchum, Baum)
The Baum lab continues to plan for the few missing gland cell-specific library resources. Together, all gland transcriptomes will provide insights into transcriptional activity within the gland cells of the developing parasitic stages over multiple life stages. We have been making progress towards the development of this useful resource to allow comprehensive analyses. When combined with our developing genomic resources, these data will provide a comprehensive analysis of the activity of the key genes (effectors) responsible for the nematode’s development and evasion strategies in achieving its parasitic lifestyle.
In a previous report, the Mitchum lab finalized a list of 71 candidate SCN virulence genes discovered from the Pool-Seq analysis and validated some of the exon SNPs present in select candidate genes by conducting Sanger sequencing of SNP-flanking PCR products amplified from genomic DNA of individual females from Pool-Seq SCN populations. For this reporting period, we have been testing the variation of these exon SNPs for their correlation to virulence, using individual females from unrelated SCN inbred populations (i.e., SCN populations not used in Pool-Seq) with same or similar virulence profiles (HG types). If those SNPs are important for virulence, we expect that the unrelated, adapted population will also contain the same homozygous (i.e., single peak at both SNPs) form, while the unrelated, un-adapted population may have a mixture of both homozygotes and heterozygotes. Concurrently, we are piloting RNA interference studies for select candidate genes, using in vitro double-stranded RNA-soaking of pre-parasitic second-stage juveniles (ppJ2s) and host induced gene silencing of parasitic second-stage juveniles (pJ2s), in order to functionally test their role in virulence.
Sub-objective 1.3: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets. (Mitchum, Baum)
The Baum group has continued to develope tools for the scientific community to employ when conducting in planta SCN studies. In particular, we have constructed a set of GATEWAY-compatible vectors to facilitate cloning of a gene of interest in frame with different epitopes to perform functional analyses on soybean roots (epitopes of choice: eGFP / 3xHA or miniTurboID-V5 at either N or C terminus). The newly developed vector series can express a given gene of interest through the highly constitutively expressed GmUbi promoter. Those vectors allow the rapid selection of transgenic roots via the mCherry fluorescent protein or via the expression of the novel non-invasive reporter gene RUBY (which produces a red pigmentation and therefore does not require any particular microscope) located on the same T-DNA as the gene-of-interest. In addition, we have established the generation of composite soybean plants, which accelerates functional analyses of a given gene-of-interest since it does not require any sterile precautions compared to in vitro hairy root culturing and is closer to real-life conditions since those transgenic roots are generated directly from a wild-type plant. To confirm functionality of these vectors, we successfully expressed different subcellular markers in soybean roots (nuclear, actin, microtubule, plasma membrane, endoplasmic reticulum and plasmodesmata). Along with these vectors, we are establishing different approaches to determine and study the interacting soybean proteins for SCN effectors, such as immunoprecipitation or proximity-labelling followed by mass-spectrometry. Also, we have developed a second series of these same vectors expressing a gene-of-interest through a dexamethasone-inducible promoter allowing us to fine-tune expression of the gene-of-interest. Furthermore, we have been working to establish a reliable RNAi pipeline based on soaking in dsRNA. We are using a SCN gland-specific transcription factor as a proof of principle case study and plan to expand to a diverse panel of effectors.
The Mitchum group has initiated cloning and characterization studies for several virulence gene candidates identified above. These candidate virulence genes coding for putative effectors will be confirmed to be expressed in the nematode esophageal gland cells and moved forward for protein-protein interaction studies. Genome analyses will be carried out to determine copy number, gene structure, and organization in the new SCN genomes.
Objective 2: Complete the evaluation of how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles. (Monteverde, Scaboo, Tylka, Mitchum)
Between 1 September and 31 October 2023 personnel in the Tylka laboratory removed mature soybean plants from the microplots at both experimental sites. The plants were run through a plot combine to obtain the seed, which will be used to plant the microplots in 2024. Also, two separate 10-core soil samples were collected from each microplot, one to use to determine SCN egg population densities and the other to test the virulence of the SCN population in each microplot on several SCN-resistant soybean genotypes. The soil samples to determine egg population densities will be processed at Iowa State University and the samples to assess virulence will be sent to the University of Missouri for HG type testing.
Our research group had two meetings during this reporting period to finalize figures and tables associated with the analysis of this 4-year rotation project, and we hope to submit a manuscript for publication of the results in early 2024.
Objective 3: Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers and inform growers on effective rotation schemes designed to protect our resistant sources. (Tylka, Mitchum)
Tylka gave 5 radio interviews with ag media personnel between September 1 and October 31, 2023. The interviews were with the National Association of Farm Broadcasters, the American Ag Network, RFD Radio Network, AgriTalk, and the Brownfield Radio Network. In each interview the loss of effectiveness of PI 88788 SCN resistance was discussed.
Mitchum hosted Gil Gullickson, contributing editor for The Furrow magazine (a John Deere publication), for a behind the scenes look at ongoing research supported by this project and SCN Coalition efforts. Gil is writing a story to translate some of the results from this project that will highlight the importance of gaining new insights into the genes controlling virulence in the nematode and how this knowledge can inform soybean breeders as new sources of SCN-resistant soybeans are developed and deployed in strategic rotations.
Objective 4: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario. (Monteverde)
The Soybean Breeding and Genetics group at the University of Illinois is coordinating the screening of the SCN Regional Test entries, which is being done at the University of Missouri. A total of 78 lines are being tested for HG 7 screening, 44 lines are being tested for HG 2.5.7, and 18 lines for HG 1.2.5.7. Additionally, samples were collected and shipped for protein and oil content and molecular markers analyses. Our group is compiling all the phenotypic data for the SCN Regional trials entries. All maturity and lodging notes were completed at each location, and the datasheets with instructions for data submission were sent to cooperators. In late October we started harvesting the SCN plots in Urbana, and we plan to finish during the first week of November.
Objective 5: Diversify the genetic base of SCN resistance in soybean by developing and evaluating germplasm and varieties with new combinations of resistance genes in high-yielding backgrounds. (Monteverde, Scaboo)
The Scaboo group has now completed successful crossing attempts (3 backcrosses) using PI 90763 as a donor parent, and LD11-2170 and SA13-1385 as recurrent parents, for three major genes associated with resistance to virulent nematode populations (rhg1-a, rhg2, and Rhg4). For each crossing attempt, we have identified desirable F1 plants using marker assisted selection, and we have sped up the process by utilizing our winter nurseries in Hawaii and Puerto Rico for the last two years. During the summer of 2023, we grew over 10,000 F3 plants at our nursery in Hawaii, and all plants were sampled for marker assisted selection. Over 750 plants were selected carrying rhg1-a, rhg2, and Rhg4. Plant rows from these selections will be grown during the winter of 2023/2024, and preliminary yield trails will be conducted during the summer of 2024. Additionally, we are actively identifying and introgressing new and novel QTL/genes into our breeding programs’ elite cultivars for cultivar development, including the new SCN resistance gene GmSNAP02.
The Soybean Breeding and Genetics group at the University of Illinois is currently harvesting the preliminary yield trials containing the experimental lines that carry the rhg1-a/Rhg4 and the Rhg1/G. soja SCN resistance gene combinations. These PYT were grown in two different locations in the state of Illinois. Additionally, single plants from F4 populations containing these same gene combinations were previously selected with molecular markers, were harvested to be grown in plant rows next year.
View uploaded report 