Worldwide, more than half of seafood was produced by aquaculture, and soybean meal is the major protein source used in aquaculture. Since the current soybean-based aquaculture feedstocks lack EPA and DHA omega-3 fish oil fatty acids, additional costs for expensive supplementation with fish oil and astaxanthin flesh pigments were introduced. To increase the content of EPA and DHA omega-3 fish oil fatty acids in soybean germplasm, the build-test-learn model is being employed. In the build step, several transgenic alleles, some supported by the NE Soybean Board, were developed to shift carbon towards increasing the content of EPA and DHA omega-3 fish oil fatty acids. In this proposal, we address the “learn” step of the build-testlearn model on soybean events carrying transgenic alleles designed to increase quality oilaccumulation in seeds, without compromising protein along with transgenic alleles designed for the ideotype soybean-based feedstock for aquaculture. After this “learn” step, a set of key genes and their interactions in the fatty acid synthesis and regulation pathways can be identified, and these differential gene calls will help guide the build constructs to break the genetic ceiling that may be limiting the omega-3 fatty acid accumulation and the new development targets the accumulation of EPA and DHA omega-3 fatty acid more than 9%. If successful, our research can lead soybeans with high value oil traits and hence, improve the revenue of Nebraska and US soybean planters from the aquaculture feed market.
This project has been granted for the first funding cycle, Oct. 2021-Sep. 2022. With the support from NE Soybean Board, we are working for the Objective One and expect to complete it during the first funding cycle. We plan to conduct Objective Two during the second funding cycle, Oct. 2022-Sep. 2023.

Objective One: Identify the differential gene calls and altered pathways of transgenic soybeans developed for the accumulation of EPA and DHA fish oil.
Objective Two: Systematically discover key genes and their positive/negative regulations in oil synthesis by a statistical model to integrate multiple datasets of transcriptome data and reveal the linkage to phenotype.

To realize these two objectives, by investigating data to find correlated gene expression profiles, especially the negative ones, we can identify negatively regulatory genes to those genes encoding the enzymes of biosynthetic pathways as the potential candidates responsible for biological limits. As a part of multitype data integration, furthermore, enzymes for fish oil synthesis will be examined for miRNA targeting, since the protein level could be suppressed by miRNAs even their gene expression levels are high. The result could be verified by Co-PI Yu’s Lab.

In the first four months, PI Zhang and Co-PI Yu’s labs will conduct RNA-seq experiments on all RAN samples which will be collected by Co-PI Clemente’s lab. PI Zhang’s lab will spend three months to analyze the RNA-seq data and identify significantly differentially expressed genes and altered oil synthesis pathways. Next, PI Zhang’s lab will spend three months to use the statistical model to look for the significant regulatory pathways to limit oil accumulation. In the last two months, PI Zhang and Co-PI Yu’s labs will conduct experimental validation to the specific gene expression regulation. In the second year, conduct the data integration and network analysis for Objective Two. We will construct an integration model to simulate the gene network for multiple datasets and look for the statistically significant genes and pathways that regulate oil synthesis in multiple transgenic alleles. Experimental validation will be conducted.

Worldwide, more than half of seafood was produced by aquaculture, and soybean meal is the major protein source used in aquaculture. Since the current soybean-based aquaculture feedstocks lack EPA and DHA omega-3 fish oil fatty acids, additional costs for expensive supplementation with fish oil and astaxanthin flesh pigments were introduced. Some transgenic alleles were developed to have fish oil accumulation, but there are some genetic bottle necks. We will collect all data from the "build" step to learn the oil synthesis pathways and their regulation to find a solution that breaks the ceiling of oil accumulation in soybean seeds. If successful, our research can lead soybeans with high value oil traits and hence, improve the revenue of Nebraska and US soybean planters from the aquaculture feed market.