Updated January 31, 2019:
This final report covers work from 10/1/2017 to 12/31/2018. Overall, this 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: Perform genome and exome resequencing on a collection of M2-derived soybean ENU mutants.
Progress: Seeds were mutagenized using a combination of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) chemicals. The plants were grown over the winter of 2018 under greenhouse conditions in small flats with short daylengths to accelerate maturity. We harvested the M2 seed and planted it in the 2018 field. Following the conclusion of the 2018 field season, we collected tissue from 1368 M2 plants belonging to 445 M1 families. 139 M2 plants (74 families) belonged to the ‘Bert’ background, and 1229 M2 plants (371 families) belonged to the ‘MN1806CN’ background. Field notes for novel phenotypes were recorded. These included traits such as plant height, branch angle/number, leaf morphology, chlorosis, flowering time, and maturity. Out of the 1368 M2 plants, only approximately 430 appeared as ‘wild type’ (normal) for all of the traits listed above. DNA was collected from 25 plants with particularly interesting phenotypes, and 25 ‘wild type’ plants chosen at random. Whole genome sequencing of these samples is currently being performed at the University of Minnesota. Furthermore, tissue from one M2 plant of each ‘MN1806CN’ family (therefore, tissue from 371 unique plants) was sent to Dr. Hyten at the University of Nebraska for analysis using molecular inversion probes (MIPs). The MIPs method will allow us to affordably sequence of portions of thousands of gene for a greater number of plants. To increase the size of the mutant population, approximately 900 additional M1 plants were harvested from the greenhouse in December 2018. Up to five M2 plants from each of these lines will be planted in the field in 2019 for phenotypic evaluation, DNA collection, and seed harvest.
Objective 2: Create a searchable public database that hosts all of the sequence polymorphism information (SNP and small indel mutations) for the subset of sequenced mutant plants.
Progress: The resequencing data for the initial 50 mutant lines is nearing completion. When this data is ready, we will analyze it and develop the database as described.
Objective 3: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.
Progress: We have harvested the first round of M3 seeds from the 2018 field. Harvest was performed on a plant-by-plant basis to ensure purity of each mutant line. The seed is now in storage and will be available to the research community upon request. However, we do not expect requests until the searchable public database of sequence polymorphisms is up and running, and accessed by researchers.
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.