1a. Objectives (from AD-416)
1. Strategically expand the USDA Soybean Germplasm Collection, conserve and distribute available genetic diversity in genus Glycine, and evaluate genetic resources in the collection. 2. Develop experimental lines derived from exotic germplasm with high yield and/or modified seed composition and map the loci associated with these traits. 3. Elucidate genetic mechanisms of resistance to sudden death syndrome, white mold, and soybean rust in diverse soybean germplasm.
1b. Approach (from AD-416)
Identify genes associated with defense to various pathogens such as Fusarium solani, Sclerotinia sclerotiorum, and Phakopsora pachyrhizi by comparing genomic mRNA levels between resistant and susceptible lines. Candidate genes related to defense will be characterized by functional molecular studies and will be located on the physical map to determine if gene is from a region of the genome associated with any known QTLs for resistance to the specific disease. Analyze soybean interactions with Sclerotinia by analyzing effects of oxalic acid on soybean. Examine physiological conditions that might enhance soybean susceptibility to rust disease caused by Phakopsora pachyrhizi. Strategically expand the USDA Soybean Germplasm Collection to better represent the diversity of the genus Glycine. Conserve, evaluate and distribute available genetic diversity in genus Glycine. Develop experimental lines derived from exotic germplasm with high yield, high protein concentration and/or high oil concentration. Map and confirm quantitative trait loci for yield, and protein and oil concentration with the positive allele coming from exotic germplasm.
3. Progress Report
Over 3200 plots were planted for seed replacement for the USDA Soybean Germplasm Collection. During the first 6 months of 2009 we distributed over 40,000 seed samples in response to 558 requests. Over 3700 four-row yield plots are being grown to identify yield genes from exotic soybean germplasm or to confirm genes tentatively identified in previous research. The objective is to find yield enhancing genes from exotic germplasm that can improve the yield of U.S. commercial varieties. Over 100 accessions from the USDA Soybean Germplasm Collection are being planted at 6 locations to confirm resistance to soybean rust identified in previous research. Nearly 2400 four-row yield plots and 3800 one-row yield plots were planted to identify high yielding experimental lines derived from exotic germplasm. These lines have pedigrees that range from 13 to 100% exotic germplasm. Two hundred fifty one advanced experimental lines are being cooperatively tested with soybean breeders in private industry. Over 1800 experimental lines were analyzed to select for high oil concentration, and 1100 lines were analyzed for high protein concentration, and 100 lines were analyzed to select for high or low isoflavone concentration. Genome expression in roots responding to Fusarium virguliforme was analyzed for 3 biological replicates for both resistant and susceptible genotypes. Determined that 2500 of the 38,000 genes analyzed were significantly changing in expression level during these interactions at 5 and 7 days post inoculation. Finished analysis of experiments designed to determine soybean responses to specific type-three-secretion-system effectors released by plant-invasive bacteria. Identified over 6630 significantly changing genes out of 38,000 in the soybean leaf response to Psuedomonas syringae and 3050 changing in response to Bradyrhizobium japonicum. qRT-PCR has been performed on 20 genes to verify the B. japonicum data. RNA has been purified from an additional time point to be used for verification of the P. syringae study. Completed and published our Sclerotinia stem infection study. The final analysis was the mapping of genes that were differentially expressed during the resistance response and determining their location relative to known Sclerotinia resistance QTL. Six of these genes were within 500 kb of a known resistance QTL. We conducted third microarray replication of soybean leaves infiltrated with the Sclerotinia virulence factor, oxalic acid. We obtained RNA of 3 biological replicates for qRT-PCR verification of our microarray study involving the oxalate oxidase transgenic and have begun the qRT-PCR analysis of 17 genes. Conducted analysis of D1 subunit of photosystem II during pathogen attack. Conducted qRT-PCR on this and 14 other chloroplast genes from leaves inoculated with P. syringae and conducted western blots using a D1-specific antibody on proteins extracted from similarly infected leaves. Implementing functional analysis tools for our lab to allow RNAi studies in soybean roots and leaves. We successfully made RNAi constructs to 6 defense-candidate genes. These constructs were transformed in soybean roots and restricted nodulation.
1. New high yielding experimental line developed from exotic germplasm. The soybean germplasm line LG04-6863 was released. It has shown high yield potential in tests across the southern US with 38% of its pedigree from plant introductions and unique diversity not known in soybeans adapted to southern US environments. LG04-6863 (relative maturity 4.4) has yielded 97, 100 and 101% of yields of Asgrow AG4403 (3833, 4047, 3193 kg/ha-1), a widely grown Roundup Ready cultivar of similar maturity, across 15 southeastern Missouri tests; eight locations in the USB Southern Early Group IV Diversity Trials and eight locations in the USDA Uniform Preliminary Early Group IV Test – Southern States, respectively. Maturity, height and lodging in these tests for LG04-6863 were similar to Asgrow AG4403. This high yielding line with new genetic diversity will provide both public and commercial soybean breeders with an important resource for developing future soybean varieties.
2. Mapped 6 genes associated with defense to white mold disease to be very close to known resistance markers to white mold. White mold is a disease caused by the fungal pathogen Sclerotinia sclerotiorum and is found to occur throughout the world affecting many valuable crop plants if the environment is favorable. In soybeans, there is no immunity and the disease can be serious if the climate is very cool and wet during the flowering period. Using gene expression analysis that looked at expression of as many genes as possible from the soybean genome (we looked about 38,000 genes) we identified 6 genes that were significantly changing in response to the pathogen and also were located very closely to areas of the genome that breeders had determined to be associated with resistance using classical genetic mapping methods. These 6 genes are good candidates for being the defense-associated gene within these regions. Geneticist can now use the sequences of these genes to design genetic markers to verify whether these are exact markers for white mold defense genes.
3. Verified six genes as having a role in plant-microbe interactions. We analyzed the expression of genes across multiple studies involving soybean response to microbes and selected a small set of genes for gene suppression study. We selected genes based on their apparent importance in both disease resistance and nodulation. We made genetic constructs to six of these genes and sent then to our collaborators at the Danforth Plant Center in St. Louis where they were used to greatly reduce the expression of each of these specific genes. Reduced expression of each resulted in poor nodulation, indicating a positive role of each in establishment of proper nodulation.
5. Significant Activities that Support Special Target Populations
We participated in the University of Illinois Research Apprentice Program II (RAPII) and hosted a high school student from June 14 to August 1, 2009. The RAP program is designed to expose high-school students from under-represented communities to Agriculture-related research. An African-American high-school student from Chicago, participated in a project involving the genetic analysis of a soybean mutant. She did some work in the field (collect leaf discs and phenotypic data from a segregating population) as well as work in the lab (RNA extractions, small gene expression study, and a chemical assay). At the end of the program, she presented a research paper, a poster and gave a 12 minute PowerPoint presentation. She was one of several students to win awards for ‘Outstanding Scholarship’ and ‘Outstanding Presentation’.
Bilgin, D.D., Delucia, E.H., Clough, S.J. 2009. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR. Nature Protocols. 4:333-340.