The vision for this project is to resequence the genomes and gene space (the DNA that represents the gene-encoding regions) for a sub-collection of chemically mutagenized soybean lines. From this, we will perform bioinformatic analysis of nearly all of the soybean genes to identify DNA base changes created by the mutagenesis. Work from the first 20 months of this project has been very promising. To date, we have developed and grown over 1,000 unique mutant families. Approximately half of this population was grown in the field in 2018 as M2 plants. Mutant phenotypes were observed and cataloged in the E/E population, indicating successful mutagenesis. A subset of 50 M2 individuals were resequenced to 20x genome coverage to identify nearly all mutations that occurred in these plants. Preliminary analysis of these data indicate that several thousand new mutations were generated in each plant. Furthermore, we are currently performing Molecular Inversion Probe (MIP)-based sequencing (Turner et al. 2009) on 371 unique M2 plants to identify mutations specifically in the gene-encoding regions. We will use these data to build a public database of the mutant variants, such that any soybean researcher can search for mutant alleles in any gene of interest. In the 2019 field, we expanded this population by growing more M2 plants. Furthermore, we performed more detailed phenotypic assessments of M3 families, identifying both stable and segregating mutant phenotypes.

Objective 1: Increase the number of unique mutant lines.
In Year 1, we generated pure seed stocks and performed initial E/E mutagenesis. Due to seed sensitivity to the mutagenesis treatment, the M1 plants need to be grown in greenhouse conditions to germinate and develop into mature plants. The M2 generation was grown in the field in 2018, and M3 seed was harvested. However, the initial M1 generation was only ~400 plants due to greenhouse limitations. To increase the size of the population, we planted and harvested a second batch of mutagenized M1 materials in the greenhouse in the summer of 2018. The M2 seeds were harvested and will be planted in the field in 2019. Our goal is to have M3 seed harvested for over 1,000 independent mutant families by the Fall of 2019.

Objective 2: Perform genome and gene space resequencing on a collection of M2-derived soybean E/E mutants.
For the new batch of M2 plants grown in 2019, DNA will be collected from each plant and a minimum of 20 M2 individuals will be resequenced to 20x genome coverage to increase the number of lines that can be used for reverse genetics by the public. Furthermore, we will perform MIP-based sequencing on ~400 M2 plants to identify mutations in the gene space for approximately 20,000 genes. We will also use the MIP technology to resequence the full gene space for approximately 300 high-priority genes that are known (or hypothesized) to control major agricultural traits. The MIP method will provide mutation information from a much greater portion of the population than could be accomplished on a similar budget using other methods.

Objective 3: Map the location of genes underlying specific mutant phenotypes.
A subset of M3 lines will be grown as M2:3 families in the 2019 field. These rows will be phenotyped to identify evidence for clear genetic segregation for morphological traits (e.g., differences in architecture, coloration, trichome morphology, etc.). A minimum of three families will be subjected to bulk segregant sequencing (Campbell et al. 2016) to map the chromosomal location that carries the causative mutation. Resequencing of the parent lines may be used to validate the mapped location, and identify candidate mutations and genes that cause the mutant trait. This work will be performed as a proof-of-concept and template for how other soybean researchers can use this population to identify the functions of soybean genes.

Objective 4: Create a searchable public database that hosts all of the sequence polymorphism information for the resequenced mutant plants.
For this objective, we will work with Soybase (USDA-ARS, Ames, IA) to develop a database that publicly shares all of the mutation information for each of the resequenced plants from Objective 2. This database will be designed as an internet web-tool that enables true “reverse genetics,” whereby anyone can search their specific gene(s) of interest by simply typing in their gene model number, and the web-tool will return a list of mutant lines that carry a mutation for that gene. The web-tool will also include a seed request space. This database will be designed to be expandable so that it can continuously incorporate new mutation information as more mutant lines are resequenced in the future. Ideally, the database will be maintained in perpetuity.

Objective 5: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.
As described in the objectives above, the combination of resequencing and database development make it possible to create a user-friendly platform for seed requests and delivery. To make this possible, M3 seeds from the 2018 field harvest are now in cold storage. In these conditions, soybean seeds can be maintained for many years with only a gradual loss in germination. This will allow maintenance of the collection for many years and meet the demands of researchers who want to use this resource in future years. At the time when seed germination is jeopardized by the duration of the storage, the M3 plants can be grown, harvesting the M4 seed for long-term storage in the same manner. M3 seed harvested in 2019 will also be introduced into cold storage for this purpose.

None to date. Future deliverable goals include:

1) Thousands of new soybean mutant lines adapted to the North Central region that exhibit a wide range of traits, including yield, seed composition, disease resistance/susceptibility, nematode resistance/susceptibility, insect pest resistance/susceptibility, response to water, nutrients, climate, soil and environmental conditions. These lines will be available for researchers interested in identifying genes underlying these traits and breeders interested in using the novel alleles derived from these populations.

2) An internet-searchable database wherein researchers can identify lines that are carrying mutations for any given gene of interest, including an interface to order specific mutant seed stocks. This will be an unprecedented resource for the soybean research community.

Updated May 1, 2020:
Objective 1: Increase the number of unique mutant lines.
To increase the size of the population, we planted and harvested a second batch of mutagenized M1 materials in the greenhouse in the summer of 2018. The M2 seeds were harvested and then planted in the field in 2019. We have harvested our goal of M3 seed, over 1,000 independent mutant families for this project.

Objective 2: Perform genome and gene space resequencing on a collection of M2-derived soybean E/E mutants.
We have been working hard to refine our sequence analysis pipeline to accurately call mutations on the initial 50 M2 lines that have been sequenced at the whole genome level. We are in the process of defining these calls so that these data can be used by the research community. The MIP work has shown promising early results, however the data collection is currently disrupted by the COVID19 lab hibernations.

Objective 3: Map the location of genes underlying specific mutant phenotypes.
A subset of M3 lines were grown as M2:3 families in the 2019 field. These rows were phenotyped to identify evidence for clear genetic segregation for morphological traits (e.g., differences in architecture, coloration, trichome morphology, etc.). Tissue was collected from individual plants with varying phenotypes from 10 M2:3 families grown in the field or greenhouse for bulk segregant mapping.

Objective 4: Create a searchable public database that hosts all of the sequence polymorphism information for the resequenced mutant plants.
This will be performed in the future, when we have accurately called the mutations in our population and we are confident that they are ready to share with the community.

Objective 5: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.
M2:3 seeds have been harvested for all of our lines from the 2018 and 2019 field seasons. A small number of lines did not have adequate seed for long-term storage. We grew and harvested M2:4 seeds for these populations. These materials will be transferred to long-term cold storage.

Updated October 30, 2020:
Progress overview: 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: A third batch of seeds were mutagenized and grown in the greenhouse during the winter of 2019-20, this time using MN1312CN as the genetic background. MN1312CN is a modern cultivar well adapted to Minnesota and less prone to lodging than MN1806CN. Approximately 400 seeds were harvested and planted in the field during the summer of 2020, of which about 220 germinated and were harvested this October. These lines have been phenotypically evaluated for various traits (such as leaf color, plant architecture, height, fertility) and tissue was collected from each plant for future genotyping applications. A subset of these lines will be grown as M3 rows 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 previously, in 2018 a set of fifty mutant plants, some with interesting characteristics important to agronomic traits, were selected for whole genome resequencing to discover novel mutations. In this reporting period we developed a pipeline to analyze this dataset which minimizes the false mutation discovery rate, resulting in a set of high confidence SNPs for these lines. Among these fifty lines approximately 605,000 mutagenesis derived SNPs were discovered, with a range of about 4000-24000 SNPs per M2 plant. These SNPs occur on all twenty chromosomes of the soybean genome, and we are now investigating which occur in known genes. Considering over 1200 independent M1 families have been generated thus far, and average mutation rate of 12000 mutations per line would imply that this collection contains 14.4 million novel SNPs throughout the soybean genome.

We have also developed several sets of M3 families segregating for unique phenotypes to highlight the potential of this mutant collection for gene discovery. Whole-genome sequencing will be performed on these families in conjunction with bulked segregant analysis to identify the causal SNPs.

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 3000 M2 plants grown during the 2019 field season. Seed from an additional 220 M2 plants from the MN1312CN background were harvested in the 2020 field season.

Deliverables: None to date.

Performance Metrics:

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. This project provides a new mutant genetic resource for public use, including breeding and gene discovery efforts. This population will have the advantage of having already identified mutations in specific genes, such that researchers can do targeted breeding and gene discovery work on gene with previously predicted or known functions. Here are some measurables so far generated by this project, mostly within the realm of resource development:

4300 M2-generation mutant plants were phenotyped in the field in 2018 or 2019 for morphological traits; M3 seed has been harvested and stored for almost all of these plants

50 plants have been sequenced at the whole-genome scale to identify all new mutations

220 new M2 plants belonging to 30 families with the 'MN1312CN' background were grown in the field to expand the size of this mutant population and provide even greater genetic diversity.

Researchers throughout the soybean community will be able to order seeds from this population in the future. Common targets of mutation breeding are yield and seed composition traits.

A poster describing this project, its current progress, and future plans was presented in 2019. Reference: Mulkey SE, Patil G, Roessler J, Stec AO, Belzile F, Hyten DL, Stupar RM. Development of an ENU and EMS Induced Mutant Resource for Functional Genomics in Soybean. Plant and Animal Genome Conference, San Diego, CA, Jan 2019.

Novel chemical mutant populations were developed in the soybean genotypes 'MN1806CN' and 'MN1312CN' using a combination of ENU and EMS mutagenesis. These populations demonstrated a range of unique traits in the M2 generation, indicating successful mutagenesis. We have sequenced the whole genomes of 50 mutant plants from this population to identify which genes and regulatory regions have mutations. Further sequencing is in progress on several sets of mutants to identify the mutations controlling interesting traits. When fully analyzed, 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.

The specific goal of this project is to develop enabling resources for gene discovery and germplasm development for traits critical to soybean growers, namely yield enhancement and seed composition improvement. The long-term benefit of these resources will be to enable breeding capacities to more effectively and efficiently improve soybean varieties for farmers.

1) A total of 70 mutagenized lines (M2 generation) will be resequenced across the entire genome and all mutations within gene-encoding regions will be cataloged. Approximately 750 M2 lines will be MIP resequenced to identify mutations specifically within portions of the gene space.

2) A database will be developed that allows users to identify the full set of mutations within their genes of interest.