Gene expression is typically understood to be controlled by their promoters. What controls promoter function is less understood, thus limiting the ability to modify gene expression in plants to obtain desired changes in trait performance. The discovery of cis-regulatory DNA elements is starting to change our approach to this challenge. These are DNA sequences elsewhere in the genome that affect the ability of promoters to control genes and their expression. We are using newly developed single-cell genomic methods to systematically and precisely identify, analyze, and validate cis-regulatory DNA elements important for soybean seed development, including the synthesis and accumulation of oil and protein and their precursors as well as for understanding responses to soybean cyst nematode infection. We have prepared single-cell genomic libraries from 11 major tissues including seed, which will allow us to identify the location of cis-regulatory elements. We have released these data publicly to all soybean researchers via https://soybean-atlas.com/ and soon via Soybase. We have also been innovating methods to isolate soybean cells after infection with soybean cyst nematodes so we can study how cells respond to this devastating disease. To understand how the cis-regulatory elements control gene expression, we are measuring gene expression at single cell type resolution from the same eleven tissues. We are also implementing the use of novel technologies to spatially identify which cells genes are active within, which serves to validate our previous research and serves as a resource to the entire soybean community. We have successfully developed spatial transcriptomics to map soybean gene expression at the highest resolution ever, thus far. In parallel, we have identified cis-regulatory DNA elements associated with key genes important for protein and oil production in the seed based on previous USB supported research. These cis-regulatory elements are being targeted by genome editing methods to produce plants with more or less gene expression and potentially expand the tissues and organs in which the genes are expressed. We cannot at this time predict which of these events will occur, but what we can do is select plants with desired alterations to protein or oil production.

This project has successfully produced the first genome-wide atlas of soybean gene regulatory activity at single-cell resolution. This project led to the first atlas of cis-regulatory sequence activity, gene expression at spatial resolution for any crop plant. This is a milestone for the soybean research community analogous to producing the first reference genome sequence, as it will facilitate soybean trait improvement by both academic and industry. We have created a roadmap that will help researchers use genome-editing to improve soybean traits with pinpoint accuracy. This single-cell atlas has also successfully guided our genome-editing plans to improve soybean tolerate soybean cyst nematode infection and to alter protein/oil content in developing seeds. In summary, this project provided unprecedented resolution of cellular activity that will be useful to soybean labs all over the world.