Project Details:

Title:
Discovering and finally understanding the functions of genes that underlie major agricultural traits in soybean

Parent Project: This is the first year of this project.
Checkoff Organization:North Central Soybean Research Program
Categories:Breeding & genetics
Organization Project Code:NCSRP
Project Year:2018
Lead Principal Investigator:Robert Stupar (University of Minnesota)
Co-Principal Investigators:
Gary Muehlbauer (University of Minnesota)
Amy Skog (University of Minnesota)
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

Click a section heading to display its contents.

Project Summary

The RFP for this funding program states a specific goal of “gene discovery and germplasm development” for traits critical to soybean growers, namely yield enhancement and seed composition improvement. Furthermore, the program includes basic and applied research aims across a wide range of additional traits, including soybean disease, nematode, insect pest, response to water, nutrients, climate, soil and environmental conditions. When done right, mutant population resources have the potential to increase our understanding of the genes that underlie all of these traits (and beyond). This proposal aims to build a better community soybean mutant resource of the North Central Region, thereby enabling both public- and private-sector researchers to directly investigate the function(s) for any gene of their choosing. While most projects have the capacity to add knowledge to a specific trait or process, this project has the capacity to accelerate numerous other projects and enable researchers working on virtually any trait or process controlled by genetics.

The co-PIs of this project have spent much of the past decade developing and using soybean mutant materials. This includes spontaneous mutants (e.g., Campbell et al., 2014), as well as those derived from irradiation (fast neutron; e.g. Bolon et al. 2011; Dobbels et al. 2017) and gene editing (e.g., CRISPR/Cas; Michno et al., 2015) mutagenesis. Furthermore, the genetic materials (i.e., mutants) from these projects can be used by other researchers across the county to study their genes and traits of interest. For example, mutant lines from the fast neutron population developed at the University of Minnesota have been sent to over 30 research groups over the past seven years. However, the majority of seed requests we receive are from colleagues looking for mutant alleles for a specific soybean gene. Unfortunately, the fast neutron population is not adequate for meeting these types of requests, as genotyping these lines is slow and expensive because gene deletions, which are difficult to identify with affordable assays, are the main type of mutation found in these lines. In fact, there are no easy populations or platforms from which to quickly order mutant lines for any specific gene in the entire soybean community. Even in the case of TILLING populations, alleles much be searched for on a gene-by-gene basis, requiring both wet-lab work and computational work. At best, mutations for a small number of genes can be identified, but it is very time-consuming.

Given this experience, the co-PIs have learned many lessons, and observed how other crop communities have developed and used their mutant collections. Recently, the PI and his former graduate student published a comprehensive list of recommendations for developing mutant resources in soybean that will make them more useful to the research community (Campbell and Stupar, 2016). This list includes considerations for seed source, seed purity (minimizing background genetic heterogeneity), mutagenesis source, genotyping, phenotyping, seed storage, and other details. Based on these criteria, the co-PIs have recently initiated a new project that they hope will provide a unique “reverse genetics” platform for the soybean research community. This platform 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).

Project Objectives

Objective 1: Perform genome and exome resequencing on a collection of M2-derived soybean ENU mutants.
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.
Objective 3: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.

Project Deliverables

Key performance indicators:
1) In year one, a minimum of fifty ENU mutagenized lines will be resequenced and all mutations within gene-encoding regions will be cataloged.

2) In year one, a database will be developed that allows users to identify the full set of mutations identified within their genes of interest.

Deliverables:
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. This would be an unprecedented resource for the soybean research community. This will include an interface to order specific mutant seed stocks.

Timelines and Milestones:
October 2017: M2 seed harvested from M1 plants (one seed per plant)
Winter 2017-2018: A small grow out of M2 individuals in the greenhouse. The first set of plants will be resequenced.
Summer 2018: A large grow out of M2 individuals and continued resequencing of select individuals. Database development.
Fall 2018: Harvest of M3 seeds and initiation of storage.
Subsequent years will include sequencing of greater numbers of lines and database updates, improvement and management.

Progress of Work

Updated January 25, 2019:
This report covers progress from 10/1/2017 to 3/31/2018. 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: 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 chemically-treated seed was directly planted in the field in Saint Paul in May of 2017, and the resulting M1 mutant generation germinated well. However, the chemically-treated M1 seedlings were particularly sensitive to abiotic stresses. Following several hard rains and a hail storm, the mutant seedlings were essentially destroyed and the field population was abandoned. We repeated the mutagenesis treatment a second time in the fall and germinated the seeds in a greenhouse. Under greenhouse conditions, a larger proportion of the seedlings survived. They plants were grown in small flats with short daylengths to accelerate maturity; we will harvest the seed by the end of March, 2018. The M2 seeds will then be planted in the 2018 field (these should be field-hearty, as they will not be chemically treated). We expect to extract DNA from ~400 independent plants, and fifty will be resequenced measure the rate of mutagenesis in the population.

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: None to date, as the resequencing data will need to be accomplished before we can meet this objective.

Objective 3: Store M3 seeds for the collection. Distribute seed to users in the research community based on their specific needs and requests.
Progress: The current plan is to harvest the first round of M3 seeds following a field grow-out of the population in 2018.

Deliverables: None to date.

Updated January 25, 2019:
This report covers progress from 4/1/2018 to 9/30/2018. 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: 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 under greenhouse conditions in small flats with short daylengths to accelerate maturity. We harvested the M2 seed and planted it in the 2018 field. We took preliminary field notes on the individuals and identified some mutated phenotypes. We collected leaf tissue from ~400 independent plants and are in the process of isolating DNA for each individual. We plan on resequencing approximately 50 plants to measure the rate of mutagenesis in the population. Furthermore, to increase the size of the population, we planted another batch of mutagenized M1 materials in the greenhouse this summer. We are hoping to harvest M2 seeds this fall from nearly 1,000 individuals and grow the M2 materials in the field in 2019.

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: None to date, as the resequencing data will need to be accomplished before we can meet this objective.

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 will soon harvest the first round of M3 seeds from the 2018 field. Harvest will be done on a plant-by-plant basis to ensure purity of each mutant line.

Deliverables: None to date.

Final Project Results

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.

Benefit to Soybean Farmers

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:

1368 M2-generation mutant plants were phenotyped in the 2018 field for morphological traits; M3 seed has been harvested and stored for almost all of these plants

50 plants are being sequenced at the whole-genome scale to identify all new mutations

371 plants will be sequenced for gene-only regions to identify mutations in specific regions of genes

Approximately 900 new M2 families will be grown in the filed in 2019 to expand the size of this mutant population, allowing for the discovery of a wider range of mutations in more genes in the future

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 recently presented. 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.

Project Years