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 mapping of resistance genes from many other sources of resistance. However, these genes largely have not been transferred into varieties. Research is needed to overcome the bottlenecks that have slowed the incorporation of these new genes into varieties.
Objective 1.1 Develop and evaluate germplasm with new combinations of resistance genes in high-yielding backgrounds.
Experimental lines will be developed with new combinations of resistance genes. Genes that will be combined include rhg1 from PI88788 or PI437654 (Hartwig), Rhg4 from PI437654, genes on chromosomes 15 and 18 from wild soybean G. soja, a gene from PI437654 on chromosomes 11, and genes on chromosomes 10 and 18 from PI567516C. Combinations of these genes will be developed and tested for SCN resistance. Associations with yield in SCN-infested and non-infested environments will be tested to determine whether these genes are associated with yield drag. Gene combinations that are effective in controlling SCN, and are not associated with negative yield, will be bred into elite soybean germplasm using marker-assisted selection.
Objective 1.2. Genotype experimental lines for resistance gene copy number for more effective breeding.
Experimental lines in the Northern Regional SCN Test and the Northern Uniform Test known to have SCN resistance will be evaluated for copy number at the major SCN resistance locus Rhg1. These evaluations will be done using a highly accurate molecular test. It has been shown that the number of gene copies at this locus impacts SCN resistance, with more copies conferring greater resistance. It is known that Fayette, one of the first public varieties with PI88788 resistance has 10 copies and that there is instability at this locus with some plants of the variety carrying more or less than 10 copies. Differences in the number of Rhg1 copies in experimental lines may explain why some varieties with PI88788 resistance differ in resistance level. If differences in copy number are found and these are associated with resistance levels (the resistance level of these lines are already being determined in the uniform test), this will provide breeders with a new tool on how to maximize resistance in varieties. We expect to test the copy number at Rhg1 for 200-300 experimental lines annually. In addition to testing for Rhg1 copy number, lines in the regional and uniform test that have Peking or PI437654 in their pedigree will be tested for what allele at Rhg4 they carry using another molecular test.
Objective 2: Identify SCN virulence factors and better understand how the nematode adapts to resistance
2.1. Improving SCN genome assembly and its accessibility
2.1.1. Improving genome assembly
A draft genome sequence became available for SCN in late 2013, developed by the Department of Energy - Joint Genome Institute (DOE-JGI), but this draft is insufficient to address key objectives of the SCN research community. The problems with the SCN genome are 1) SCN does not readily inbreed, so we are always sequencing a mixed population and 2) the SCN genome is very repetitive, making the genome difficult to put together. Both of these are important because the variable and repeated regions, missing from the DOE-JGI draft, are likely the home of the genes that determine virulence and HG type. To address the first issue, we will apply innovative methods such as using genetic map information being developed with USB funding for SCN, and creating cultured cell lines from SCN, using methods adapted from C. elegans and other nematode species. We will address the second problem using new technologies for repeated DNA sequencing such as the NEXTERA library system and Moleculo technology, to resolve the regions of interest. The successful method will be used on a larger scale to enhance and complete the current genome sequence.
2.1.2. SCN genome curation and annotation
Genome assembly will be useless unless stakeholders have easy access to it. In this objective we will use state of the art bioinformatics tools to annotate and curate the SCN genome. All annotations will have IDs that will provide straightforward cross-referencing based on homology between the SCN gene models and model organisms. With the collection and analysis of big data comes the responsibility to release this information in an organized and easily accessible manner. By collaborating with Dr. David Grant and his USDA-ARS group that manages SoyBase, we will create a genomic toolbox for SCN that facilitates the integration of very large sequence data sets, molecular markers, QTL data and genetic maps into an easy-to-use web interface and integrate it with the existing SoyBase resource (soybase.org). The website will include a Genome Browser for easy visualization of the SCN genome. The Genome Browsers will allow researchers and breeders to visualize data at the nucleotide level generated in this proposal and curated from previous research. Furthermore, this website will be the home of all genetic, genomic, and molecular data generated for the SCN, most of it through check-off funding. As such, this website will provide prime visibility and impact to the collective data procured through farmer investments. Other significant advantages of integrating genomic resources with the existing SoyBase infrastructure include: 1) easy cross reference of virulence genes/markers with corresponding genes in soybean, and 2) the integration can serve as a model for the integration and cross referencing of other pest genomes into SoyBase.
2.2. Conduct comparative population studies to identify genes associated with SCN virulence and evaluate their utility as novel resistance targets.
RNAseq now provides a powerful basis for comparative studies. A comparison of genome-wide gene expression in SCN populations that differ in virulence on resistant soybean has the potential to identify genes underlying virulence, determine the mechanism/s of virulence, and develop molecular diagnostic tools to assess for virulence in field populations. We have assembled a comprehensive SCN gene expression atlas from whole nematodes representing early parasitic life stages across populations of SCN that differ in their ability to reproduce on the major types of SCN-resistant soybeans. These data will be deposited to the proposed SCN-SoyBase website, used to facilitate the annotation of the SCN genome, and mine for candidate genes involved in SCN virulence. While several dozen parasitism genes encoding proteins important for SCN’s ability to cause disease have been identified to date, we expect to expand this list considerably through this global analysis. Our approach will also allow us to identify genes conserved across HG types to identify the best targets for engineering broad-based resistance. In addition, we expect that some of these genes also function in nematode adaptation to resistant soybean cultivars (i.e., virulence). Past research efforts to determine inheritance of nematode virulence led to the identification of the reproduction on resistant cultivars or ror genes shown to be inherited in both a dominant and recessive manner. However, since the initial discovery of these genes there has been no further information published concerning their sequence identity or mechanism in conferring nematode virulence. As candidate virulence genes are identified, gene function will be confirmed through biochemical and/or genetic analysis to not only better understand the mechanism/s of virulence, but to also evaluate their utility as vulnerable points of disruption to enhance resistance in soybean.
2.3 Determine what stacks of genes have unique gene mechanisms and would be beneficial in gene rotations to enhance durability of SCN resistance in soybean.
Experimental lines with resistance gene combinations developed in 1.1 will be tested in greenhouse selection experiments. SCN field populations virulent on PI 88788 (HG type 2) will be used in rotation studies with experimental lines containing various resistance gene combinations. SCN population increase will be measured after each generation for 12 generations. Following 12 generations of selection, the HG type of the population will be evaluated using a modified HG type test that includes additional resistance sources. The goal is to identify alternative resistance gene combinations that when used in rotation will reduce the selection pressure on the SCN population thereby slowing nematode adaptation to resistant cultivars.
Objective 3: Translate the results of Objectives 1 and 2 to increase the profitability of soybean for producers.
A project extension and outreach coordinator, advised by project co-PI Dr. Tylka, will be 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 will be conducted by the coordinator. Materials from land-grant universities and private seed and chemical companies will be gathered, analyzed, and compared. Starting in the initial year, after assessing the current state of SCN awareness and education, and continuing in subsequent years, the coordinator will create traditional and innovative information materials to further educate and increase awareness of several SCN-related topics, such as:
1. How SCN adapts to grow on resistant soybeans.
2. How to best manage SCN for sustainable soybean production with rotation of resistance and use of other available management tactics.
3. How advances in basic research may lead to new, innovative management strategies for SCN in the future.
The development of education/outreach materials will be done in consultation with extension plant pathologists and plant nematologists at the land-grant universities in the 12 NCSRP states to ensure that messages are relevant and pertinent to the local conditions with SCN in each state.