Project Details:

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

Parent Project: Discovering and finally understanding the functions of genes that underlie major agricultural traits in soybean
Checkoff Organization:North Central Soybean Research Program
Categories:Breeding & genetics
Organization Project Code:NCSRP
Project Year:2019
Lead Principal Investigator:Robert Stupar (University of Minnesota)
Co-Principal Investigators:
David Hyten (University of Nebraska at Lincoln)
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

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 soybean ENU/EMS lines. From this, we will perform bioinformatic analysis of nearly all of the soybean genes to identify all DNA base changes created by the mutagenesis. Furthermore, we will build a public database of these variants, such that any soybean researcher can search for mutant alleles in any gene of interest. In Year 1 of funding, we have generated pure seed stocks and performed initial ENU/EMS mutagenized seed. Many of the M1 mutants have been grown in the greenhouse and M2 generation seed has been collected. The next portion of this work will focus on generating more mutant (M1) lines, performing the genome/gene space resequencing efforts on M2 materials, and developing the mutant allele database.

Project Objectives

Objective 1: Generate greater numbers of unique mutant lines.
In our first round of mutagenesis, we harvested the seed from 400 plants in April, 2018. We would like to provide a larger population for the community and will grow a larger greenhouse-based population in the Summer of 2018 to add new lines to the current population. Our goal is to have a minimum of 1,500 independent mutant lines by the Spring of 2019.
Objective 2: Perform genome and gene space resequencing on a collection of M2-derived soybean ENU/EMS mutants.
The M2 seeds from the 400 harvested lines will be planted in the 2018 field. DNA will be collected from each plant and a minimum of thirty M2 individuals will be resequenced to measure the rate of mutagenesis in the population. Furthermore, using a MIP protocol modified to work in soybean by co-PI David Hyten, each individual in the mutant population will be resequenced in a 200 bp targeted portion of the gene space for approximately 30,000 genes. The MIP method will allow us to recover mutation information from a much greater portion of the population than could be accomplished on a similar budget using other methods.
Objective 3: Create a searchable public database that hosts all of the sequence polymorphism information for the resequenced mutant plants.
For this objective, we will 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 would return a list of mutant lines that carry a mutation for that gene. The web-tool would 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. The long-term goal would be to integrate its interface into Soybase so that it can be maintained in perpetuity.
Objective 4: 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 will make it possible to create a user-friendly platform for seed requests and delivery. To make this possible, M3 seeds from each of the M2 plants will be harvested in the Fall of 2018 and stored at -80C conditions. In these conditions, soybean seeds can be maintained for many years with only a gradual loss in germination. This will allow us to maintain the collection for many years and meet the demands of researchers who want to use this resource for many years into the future. 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.

Project 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, including an interface to order specific mutant seed stocks. This would be an unprecedented resource for the soybean research community.

Progress of Work

Updated March 30, 2019:
Progress overview: This report covers progress from 10/1/2018 to 3/31/2019. 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: To increase the size of the population, we planted and harvested 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 will be planted in the field in 2019.

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. This set of fifty-four plants were aligned to the reference genome Williams82 version 2 assembly (Wm82.a2) to verify locations of mutations. All possible sequence variants have been called on this set, and filtering of a high confidence variant dataset, which can be used by soybean researchers, has been initiated. In addition to providing a thorough examination of mutations throughout the genomes of these 50 individuals, this work will give us an understanding of the frequency and distribution of mutations throughout the entire mutant population.

We are currently in the design-phase for performing the cost-efficient, low read-depth sequencing performed 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 30,000 genes. This work will provide mutation identification for a larger number of mutant lines, but only for specific intervals within genes.

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 has been successfully harvested and stored from approximately 1150 M2 plants grown during the 2018 field season. Five plants each of an additional 900 M2 families will be grown in the field this year to increase the population size. Numerous 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 will be grown in rows and bulk harvested to produce M4 seed. Roughly 250 M3 lines belonging to 100 M2 families will also be grown in the field this year for additional phenotyping. These lines/families display novel segregation for important traits (plant height, fertility, canopy architecture, etc.) and may be useful for cloning genes of interest.

Deliverables: None to date.

Final Project Results

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.

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

371 plants are being sequenced for gene-only regions to identify mutations in specific regions of genes

Approximately 900 new M2 families are currently growing in the field 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