Updated October 1, 2019:
Progress overview: This report covers progress from 10/1/2018 to 9/30/2019. Overall, the project addresses the RFP’s stated goal of “gene discovery and germplasm development” for traits critical to soybean growers, namely yield enhancement and seed composition improvement. The heart of the project focuses on developing soybean mutant genetic resources that will increase our understanding of the genes that underlie traits of agronomic importance. The development of these resources has been designed to overcome factors that have limited similar projects in the past; such factors include issues with seed source, seed purity (minimizing background genetic heterogeneity), mutagenesis source, genotyping, phenotyping, and seed storage. The new mutant resources will allow researchers to identify mutant stocks for their gene(s) of interest by simply searching a genomic database that will be developed by this project. This will provide an unparalleled public resource in which researchers can quickly identify the mutations, order mutant seeds, and test the agricultural function and importance of the gene(s). Our research objectives and current progress are summarized below.

Objective 1: Generate greater numbers of unique mutant lines.
Progress: As described in previous reports, we increased the size of the population by planting and harvesting a second batch of mutagenized M1 materials (using the ENU/EMS combination, as described below in Obj 2) in the greenhouse in the summer of 2018. The M2 seeds were harvested and were planted in the field in 2019. These lines have been phenotypically evaluated this past summer (including traits such as lodging resistance, increased pod number, and plant architecture) and tissue was collected from each plant for future genotyping applications. We plan to harvest and store seed this fall. A subset of these lines will be grown as M3 row in the field next year.

Objective 2: Perform genome and gene space resequencing on a collection of M2-derived soybean ENU/EMS mutants.
Progress: As described in previous reports, the first batch of seeds were mutagenized using a combination of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) chemicals. The plants were grown under greenhouse conditions in small flats with short daylengths to accelerate maturity. We harvested the M2 seeds and planted them in the 2018 field. We took preliminary field notes on the individuals and identified some mutated phenotypes. A set of fifty-two mutant plants, some with interesting characteristics important to agronomic traits, were selected for whole genome resequencing to discover novel mutations. Two parent plants of the MN1806CN mutant population were also sequenced to verify that mutations discovered are in fact novel and not present in the parent line. In the reporting period, we have continued to analyze this dataset. We have tested several different analysis pipelines to optimize true mutation detections and minimize the false mutation discovery errors. This work is still ongoing.

We are also in the process of performing cost-efficient, low read-depth sequencing on thousands of genes for 371 unique M2 plants. This work is based on the Molecular Inversion Probe (MIP) protocol (Turner et al. 2009) modified to work in soybean by co-PI David Hyten. Each of the 371 individuals in the mutant population will be resequenced in 200 bp targeted portions of the gene space for approximately 20,000 genes. A subset of “high priority genes” (those most likely to have desirable outcomes) will be sequenced in their entirety. In this reporting period, we have designed the MIPs assays and initiated the sequence work.

Objective 3: Create a searchable public database that hosts all of the sequence polymorphism information for the resequenced mutant plants.
Progress: None to date, as the resequencing data will need to be fully analyzed before we can meet this objective.

Objective 4: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.
Progress: M3 seed was successfully harvested and stored from approximately 1150 M2 plants grown during the 2018 field season. Some mutants from 2018 displayed phenotypes with delayed maturity or reduced fertility, which in turn reduced the total number of seeds harvested. To ensure that these genotypes will be available to the scientific community, M3 plants were grown in rows this summer and will be bulk harvested to produce M4 seed. Roughly 400 M3 lines belonging to 135 M2 families are currently growing in the field for additional phenotyping. A subset of lines/families that displayed novel segregation for important traits (plant height, fertility, canopy architecture, etc.) have been identified, as they may be useful for cloning genes of interest. Lastly, to increase the overall size of the population, five plants each of an additional 900 M2 families are currently grown in the field. M3 seed will be harvested this fall and added to our storage collection.

Deliverables: None to date.

A novel chemical mutant population was developed in the soybean genotype ‘MN1806CN’ using a combination of ENU and EMS mutagenesis. This population demonstrated a range of unique traits in the M2 generation, indicating successful mutagenesis. We are currently sequencing the whole genomes of 50 mutant plants from this population to identify which genes and regulatory regions have mutations. Furthermore, we are also using MIPs sequencing to gather mutation information specifically on smaller gene regions for a larger set of mutants (371 plants). This sequencing data will be shared on a public database, allowing researchers to identify mutations in genes of interest and order these lines from our population. This will allow for more efficient breeding with novel traits and discovery of gene functions.