Project Details:

Title:
Utilizing Unique Genetic Diversity to Combine Elevated Protein Concentration with High Yield in New Varieties and Experimental Lines (1820-152-0106-B)

Parent Project: This is the first year of this project.
Checkoff Organization:United Soybean Board
Categories:Seed composition, Breeding & genetics, Sustainability
Organization Project Code:1820-152-0106-B
Project Year:2018
Lead Principal Investigator:George Graef (University of Nebraska)
Co-Principal Investigators:
Asheesh Singh (Iowa State University)
William Schapaugh (Kansas State University)
Brian Diers (University of Illinois at Urbana-Champaign)
Aaron Lorenz (University of Minnesota)
Hari Krishnan (University of Missouri)
David Hyten (University of Nebraska at Lincoln)
Steven Clough (USDA/ARS-University of Illinois)
Rusty Smith (USDA/ARS-University of Illinois)
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Keywords: high protein, diversity, breeding, high protein, diversity

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

To increase soybean value for the entire value chain, selection of soybean germplasm and identification of soybean genes controlling protein synthesis and storage along with the development of markers for those genes, provide the opportunity to create soybeans with higher protein levels and will aid in the development of commercial soybean varieties with an improved nutritional bundle.
The proposed work combines unique and highly productive genetic resources with evaluation and selection for improved seed compositional balance to create new soybean lines with the physiological capacity to simultaneously improve protein concentration, increase yield, and retain oil concentration. Our established cooperative testing with all major industry partners facilitates their identification, adoption, and use of superior soybean lines to develop and deliver new products with improved nutritional bundle to US soybean producers. This project strengthens our partnership with industry programs and improves profitability for US soybean farmers.

Project Objectives

1) Develop and release improved soybean lines that are higher in seed protein concentration and have improved nutritional bundle and superior yield compared with current high-yield cultivars.
2) Characterize high protein sources for the presence of the major protein gene on chromosomes 15 and 20.
3) Map new genes for high protein concentration from Glycine tomentella.
4) Characterize protein composition and amino acid profiles of G. tomentella-derived lines and selected experimental soybean lines.
5) Improve seed quality in early planting soybean production system (ESPS).

Project Deliverables

1. Distribute final report of all field and seed composition data for experimental lines tested jointly with commercial companies to all participants and other interested soybean breeders by January 31
2. Continue and strengthen industry cooperative evaluations of new, diverse, high-yield lines with improved seed composition and nutritional bundle, facilitating exchange and adoption of germplasm by industry programs
3. Develop a common, testing-only MTA acceptable to all commercial, university, and USDA participants to facilitate exchange of seeds for the cooperative tests by January 31, 2018
4. Make germplasm releases of experimental lines with improved yield and enhanced protein concentration and nutritional bundle to be used by both public and private sector breeders to develop new varieties
5. Develop a list of high-protein germplasm accessions indicating the presence or absence of the high-protein gene on chromosome 20 that can help select new sources of high protein that are genetically different from what is currently being used
6. Provide information on genetic markers to identify the specific genes on chromosomes 15 and 20 that increase protein concentration
7. Obtain preliminary data indicating if the genes controlling high protein concentration in G. tomentella-derived lines are different from the major genes known in soybean
8. Identify specific protein components that are responsible for the increased protein concentration in experimental lines derived from G. tomentella
9. Provide data on protein quality for both high protein soybean lines and high protein G. tomentella-derived lines that will help select lines with higher concentration of sulfur-containing amino acids
10. Identify genes in elite and exotic germplasm pools that influence seed protein concentration
11. Identify unique loci for yield and seed composition traits in exotic sources that are not present in the commercial gene pool
12. Develop at least one new soybean line with superior yield and seed composition and quality for the ESPS in the southern US

Progress of Work

Update:
The 2017 Diversity Coop Tests had 90 entries in MG II, III, and IV. The MG I tests were arranged separately between UNM and companies for the earlier MG locations. The MGII-IV tests were grown by University of Nebraska, Kansas State University, Iowa State University, USDA-ARS in Illinois, and company locations in IL, IA, IN, and MN. Companies growing the Diversity Coop Tests this year included Dow, Pioneer, Syngenta, KG AriProducts. Bayer and Monsanto have grown the tests in past years, and are interested in growing the tests again, but with the mergers this year they did not participate in 2017. We completed all the data collection and are analyzing the regional data from all cooperators. We will test seed composition on the top 50% yield entries from at least 5 environments. Final data summary reports will be shared with all cooperators by the end of January, as indicated in our Timeline and the deliverable above.

In addition, the companies that did not grow lines this year are interested in receiving the data and in transferring lines from the 2017 tests for their evaluation and use. We will keep in contact with all companies to share information and facilitate their involvement and use of the material.

Population development - ISU, KS, NE, MN, IL and USDA developed new crosses, F2-F4 generations, progeny rows, and preliminary multi-location yield tests. These efforts involve more than 100 new crosses, over 100 advanced generation populations, and more than 6,000 new lines in progeny rows and first stage yield tests.

More than 25 experimental were shared with industry partners through MTAs for their further evaluation and use in breeding programs.

Multiple lines with diverse pedigree are entered in the USDA Uniform Soybean Tests Northern States in MG I, MGII, MG III, and MG IV tests.

Based on preliminary analysis of the regional data received to date, the #1 yielding line in the Uniform Prelim II Test is U15-927115, which has genetic diversity in the pedigree. Several other diversity lines yield as well or better than the checks in the test. The line U14-103015 is the #2 yielding line in the UT I test, which comes from a cross of two LG lines, selected from one of Randy Nelson’s diverse populations.

ISU and other cooperators plan to enter new lines derived from their diversity crosses into the 2018 USDA Uniform tests as well.

At the Stoneville, MS location, yield plots experienced Dicamba damage early in their reproductive cycle via the local producer next to our fields. Therefore, only some plots were harvested and their yield and maturity estimates should be considered tentative. Because of the limited number of harvested plots, lines in MG III and IV early tests were combined for analysis and lines in MG IV late through early VI tests were combined for analysis.

For the MG III/IVE tests, ten breeding lines exceeded the top-yielding commercial checks (Progeny 4211 and 94Y23), which had yields of 61.7 and 60.2 bu/ac, respectively. Four of these lines were 25% exotic germplasm (PI 587982A), three of the lines were 50% exotic germplasm (PIs 587982A and 603756), and one line was 12.5% exotically-derived (PI 587982A).

For the MG IVL/VIE tests, the seed yield of one breeding line (10049-142-31) exceeded (77.2 bu/ac) that of the best checks (UA5612 and AG4835RR2), which yielded 75.2 and 65.3 bu/ac, respectively. However, ten breeding lines had yields that exceeded that of AG4835RR2. Five of those breeding lines had 25% exotic pedigrees (PIs 587982A and 603756), two had 50% exotic pedigrees (PIs 587982A, 603756, and 588016), and two had 19% exotic predigrees (PIs 427099, 438151, and 445830).

Seed from 33 populations of plants that each have recombination in unique positions near where the chromosome 15 gene is located were harvested in the field and each plant from the populations were threshed individually. The seed were cleaned and seed from plants from the populations are being tested for protein and oil concentration using NIR. These plants from about ½ of the populations have been tested with genetic markers. As soon as more protein and oil analyses are completed, the results from these tests will be analyzed.

Two populations were grown in the field during 2017 to confirm the candidate gene we identified for the chr 20 protein gene. The plants from the populations were harvested in the field and individually threshed. They will soon be tested for protein and oil concentration.

Multi-location yield tests to evaluate G. tomentella-derived lines for yield, agronomic traits, and seed composition were grown in Illinois, Iowa, Nebraska, and Kansas. The field data is being analyzed and we will be getting all the post-harvest data, including seed composition, during the next quarter.

Information on protein subunit composition was provided in the previous quarter. Seeds are now submitted for amino acid analyses, and those results should be available for the next report.

All major companies participated in the 2017 Diversity Coop Tests or requested lines and data from the tests.

All industry partners have transferred lines from the Diversity program to their breeding programs.

Other public breeding programs are using lines from the Diversity program in their breeding and development efforts

Yield and field data are being analyzed, and composition data are being collected. A report on regional yield, composition, and stability will be included with the March quarterly report.

Updated December 4, 2018:

View uploaded report Word file

Updated December 4, 2018:
Program Objectives:
• Develop and release improved soybean lines that are higher in seed protein concentration and have improved nutritional bundle and superior yield compared with current high-yield cultivars.
• Characterize high protein sources for the presence of the major protein gene on chromosomes 15 and 20.
• Map new genes for high protein concentration from Glycine tomentella
• Characterize protein composition and amino acid profiles of G. tomentella-derived lines and selected experimental soybean lines.
• Improve seed quality in early planting soybean production system (ESPS).

General brief summary of current status of plots:
All cooperators report good planting and initial growing conditions for all of the Diversity Protein Tests. At least one location had significant rain in early June with some standing water, but plots will be monitored for potential damage and subsequent disease issues.

Progress for the current period is reported below for each Deliverable and KPI.

Expected Outputs/Deliverables:
• Distribute final report of all field and seed composition data for experimental lines tested jointly with commercial companies to all participants and other interested soybean breeders by January 31
• Continue and strengthen industry cooperative evaluations of new, diverse, high-yield lines with improved seed composition and nutritional bundle, facilitating exchange and adoption of germplasm by industry programs
o We completed transfer of over 135 lines in the 2018 USB Diversity Protein Tests to project cooperators and our industry partners. Due to ongoing mergers, Pioneer and Monsanto are not able to participate this year. But we have participation from Syngenta, Dow, Schillinger Genetics, and KAPI on the industry side. These tests are MG2, MG3, and MG4, with a preliminary and an advanced test in each MG at 9-12 locations each. I spoke with people from the other companies, and they are interested in rejoining our program next year.
o UMN has maintained communication with Corteva and Bayer Crop Sciences regarding germplasm sharing. They are particularly interested in germplasm from our program with diverse genetic backgrounds. We discussed the possibility of shared testing with them during the summer of 2018, but the major mergers that have happened during the last year and are ongoing prevented us from doing this. We hope to resume the cooperative testing next summer.
• Develop a common, testing-only MTA acceptable to all commercial, university, and USDA participants to facilitate exchange of seeds for the cooperative tests by January 31, 2018
o We will pursue this during the summer 2018. I have been talking with people from Monsanto and other companies regarding key items and protocols that will facilitate this. One important consideration is that some companies require testing for adventitious presence (AP) in all seed lots coming from outside their system. That adds expense and time. In addition, at least one company requires that MTAs for the current season be completed before January 15 of that year. That will be challenging. One approach we talked about is developing a preliminary test for entries that are in their first year of testing, and an advanced test for entries that are retained for a second or third year of testing. That will allow those industry partners who require more time for AP testing and MTA processing to participate in the advanced tests the second year. Another option we discussed is outsourcing of that company testing. That was just a suggestion in a discussion; we don’t know if it is possible or will be allowed by the company. But these may present options for 100% participation by our industry partners in the USB Diversity Protein Tests.
o Minnesota is doing this independently since we are the only University testing with companies in the earlier MG zone.
• Make germplasm releases of experimental lines with improved yield and enhanced protein concentration and nutritional bundle to be used by both public and private sector breeders to develop new varieties
o Breeding for yield and quality using diverse sources is ongoing at UMN. The following activities are related to the overall breeding effort in 2018:
? We made breeder’s seed for one line (MSC09-774089) which is being tested for the third year in regional tests in 2018. This line has PI 567516C as a parent. If this line again yields 95% of the check in 2018, we will release this line as a germplasm release.
• Develop a list of high-protein germplasm accessions indicating the presence or absence of the high-protein gene on chromosome 20 that can help select new sources of high protein that are genetically different from what is currently being used
• Provide information on genetic markers to identify the specific genes on chromosomes 15 and 20 that increase protein concentration
• Obtain preliminary data indicating if the genes controlling high protein concentration in G. tomentella-derived lines are different from the major genes known in soybean
• Identify specific protein components that are responsible for the increased protein concentration in experimental lines derived from G. tomentella
• Provide data on protein quality for both high protein soybean lines and high protein G. tomentella-derived lines that will help select lines with higher concentration of sulfur-containing amino acids
• Identify genes in elite and exotic germplasm pools that influence seed protein concentration
o We used two long-term recurrent selection populations to investigate how selection for increasing seed protein concentration might affect seed protein composition and quality. The UP2 population was developed from seven 100% exotic, unadapted, high-protein soybean accessions. The UP3 population was developed from 100% elite, high-yielding lines with average or above-average seed protein concentration. High-resolution two-dimensional (2-D) analysis of seed proteins of 7 exotic parent lines of UP2 and the 10 highest protein lines after five cycles of recurrent selection has been completed. This analysis demonstrated several of the UP2 high protein lines (UPPC7(S3)-0115, UPPC7(S3)-0230, UPPC7(S3)-0064, UPPC7(S3)-0033) failed to accumulate Gy4 protein. Interestingly, all these high protein lines accumulated a unique high-molecular weight protein. The identity of this protein is currently being investigated by mass spectrometry. The relationship, if any, between the high-protein trait and the presence of this unique high-molecular weight protein needs further investigation.
o Amino acid profile of 7 exotic parent lines of UP2 and the 10 highest protein lines after five cycles of recurrent selection and 7 original elite parental lines of UP3 and the 10 highest protein lines after five cycles of recurrent selection was determined by high-performance liquid chromatography. Based on this analysis the following conclusions can be derived.
? Methionine content of UP3 high protein lines (1.2%) is slightly lower than their parents (1.4%).
? Cysteine content of UP3 high protein lines are very similar to that of parents.
? Alanine, Isoleucine, Threonine and Valine content of UP3 high protein lines are slightly lower than their parents.
? UP3 high protein lines have higher content of Arginine and Serine than their parents.
? Methionine and cysteine content of UP2 high protein lines is very similar to that of parents.
? Threonine and valine content of UP2 high protein lines are slightly lower than their parents.
• Identify unique loci for yield and seed composition traits in exotic sources that are not present in the commercial gene pool
• Develop at least one new soybean line with superior yield and seed composition and quality for the ESPS in the southern US
o Yield trials in the early production system of the Mid-south at Stoneville, MS were planted 18 April 2018, with the breeding nursery planted 20 April 2018. Field conditions were cool and wet in early April, but progressed to hot and dry in May. We initiated furrow irrigation the 8th and 9th of June. The plots look very good and rows will soon be lapping (covering the inter-row spaces). So far, there has been no Dicamba drift damage from our neighbors.

Key Performance Indicators:
• Most or all major US commercial soybean programs participate in the cooperative wide-area evaluation of our diverse, high-yield soybean lines with improved seed protein concentration and nutritional bundle
o Yes, we have participation from Syngenta, Dow, Schillinger Genetics, and KAPI on the industry side. Pioneer and Monsanto are interested in rejoining our program next year.
• Companies use selected lines from our diversity tests in their breeding programs
o UMN is also still working through licensing agreement on four diverse lines being transferred to Monsanto. Once again, the buyout of Monsanto by Bayer has significantly delayed this. We have reviewed another list of lines with them, which they are interested in transferring for crossing in their program once a boilerplate license agreement can be established.
o University of Nebraska is finalizing a MTA for transfer of 13 diverse, high-yield lines to Monsanto during 2018.
• Other public researchers use diverse, high-yield, high-protein lines in their breeding programs
o Many high-yield lines with diverse pedigree and improved seed composition have been entered into the USDA Uniform Soybean Yield Tests Northern States, and are used by other state and USDA soybean breeding programs in their breeding efforts.
• Genetic markers for protein QTL on Chr 15 and Chr 20 are available for use by breeding programs
• Candidate genes for the protein QTL on Chr 20 and Chr 15 are identified
o Brian Diers, University of Illinois - During the past quarter, progress has been made in cloning the high protein genes on chromosomes 15 and 20. For the chromosome 15 gene, we are fine mapping it by identifying recombinants close to the gene and testing the progeny of these recombinants for protein and oil and with markers. Using data from several populations that were field tested in 2017, we have tentatively placed the gene into a 267 kb interval. To further narrow the interval, we have planted in the field this spring approximately 40 plant rows derived from each of 9 selected plants that have recombination in the 267 kb interval. These plant rows will be harvested this fall and tested for protein and oil concentration.
o To confirm the gene candidate we have identified for the chromosome 20 high protein gene, Tom Clemente at the University of Nebraska made transgenic plants that knock out the low protein allele. At this point, we don’t know whether we need to knock out the low or high protein allele of the candidate gene to show its function. This summer we will grow plants that have a functional low protein allele or have this allele knocked out to determine the effect of knocking out this allele. To determine the impact of knocking out the high protein allele, we will make crosses between the transgenic plants and a source of the high protein allele to develop germplasm for future tests.

• Soybean lines developed in this program show stable performance for improved yield and seed composition over locations and years
o Several lines from the USDA, ARS breeding program at Illinois and the University of Nebraska program have shown superior yield and seed compositional quality over years and locations. Those were highlighted in the March report in the attached 2017 Diversity Program Yield Test report.
o MSC09-774089 has showed stable yield over the past three years. It will be release as a germplasm release in 2019 if yield is again good in 2018 regional trials. Breeder’s seed of this line has been made.
• At least one new commercial soybean variety from an industry partner results from crosses made with lines from this program!

Updated December 4, 2018:

Program Objectives:
• Develop and release improved soybean lines that are higher in seed protein concentration and have improved nutritional bundle and superior yield compared with current high-yield cultivars.
• Characterize high protein sources for the presence of the major protein gene on chromosomes 15 and 20.
• Map new genes for high protein concentration from Glycine tomentella
• Characterize protein composition and amino acid profiles of G. tomentella-derived lines and selected experimental soybean lines.
• Improve seed quality in early planting soybean production system (ESPS).

General brief summary of current status of plots:
For this period from mid-June to mid-September, the Diversity Cooperative Tests were growing in the field at locations throughout the Midwest with USDA, university, and industry partners. Some initial maturity dates on early plots were begun.

Progress for the current period is reported below for each Deliverable and KPI.

Expected Outputs/Deliverables:
• Distribute final report of all field and seed composition data for experimental lines tested jointly with commercial companies to all participants and other interested soybean breeders by January 31
• Continue and strengthen industry cooperative evaluations of new, diverse, high-yield lines with improved seed composition and nutritional bundle, facilitating exchange and adoption of germplasm by industry programs
• Develop a common, testing-only MTA acceptable to all commercial, university, and USDA participants to facilitate exchange of seeds for the cooperative tests by January 31, 2018
• Make germplasm releases of experimental lines with improved yield and enhanced protein concentration and nutritional bundle to be used by both public and private sector breeders to develop new varieties
o For plots in Mississippi, all plots received damage from Dicamba drift during the spring/summer. The damage could have come multiple times from multiple points. Differences for damage among breeding lines were noted. Harvest of late III/early IV yield trials has been completed. Harvest of mid-to-late IVs is temporarily on hold due to rain bands associated with Tropical Depression Gordan and associated storms from the gulf. We expect seed damage from this harvest delay, which will hopefully allow for the separation of genotypes for seed damage. The paper work for a germplasm release has been submitted to the National level and is awaiting approval.
o New crosses were made to develop new populations with diverse germplasm and improved seed protein/oil/carbohydrate composition.
• Develop a list of high-protein germplasm accessions indicating the presence or absence of the high-protein gene on chromosome 20 that can help select new sources of high protein that are genetically different from what is currently being used
o High protein accessions from the germplasm collection were identified that do not have the chromosome 20 high protein allele. Nine of these accessions were planted in the crossing block (IL) and were crossed with germplasm with normal protein levels to develop germplasm that can be used to map alternative alleles that can increase protein levels and potentially not have a negative impact on yield.
• Provide information on genetic markers to identify the specific genes on chromosomes 15 and 20 that increase protein concentration
• Obtain preliminary data indicating if the genes controlling high protein concentration in G. tomentella-derived lines are different from the major genes known in soybean
• Identify specific protein components that are responsible for the increased protein concentration in experimental lines derived from G. tomentella
• Provide data on protein quality for both high protein soybean lines and high protein G. tomentella-derived lines that will help select lines with higher concentration of sulfur-containing amino acids
• Identify genes in elite and exotic germplasm pools that influence seed protein concentration
o High-resolution two-dimensional (2-D) analysis of the UP2 high protein lines (UPPC7(S3)-0115, UPPC7(S3)-0230, UPPC7(S3)-0064, and UPPC7(S3)-0033) revealed the accumulation of a unique high-molecular weight protein in these lines. We have partially purified this unique protein and identified this protein by mass spectrometry. MALDI-TOF-MS analysis of this protein followed by peptide mass searches revealed significant peptide matches with soybean 11S glycinin protein. Antibodies raised against soybean glycinin protein also reacted against this unique protein indicating that this protein is most likely an unprocessed glycinin precursor. This possibility is currently being verified by cloning the glycinin gene from these UP2 high protein lines.
• Identify unique loci for yield and seed composition traits in exotic sources that are not present in the commercial gene pool
• Develop at least one new soybean line with superior yield and seed composition and quality for the ESPS in the southern US

Key Performance Indicators:
• Most or all major US commercial soybean programs participate in the cooperative wide-area evaluation of our diverse, high-yield soybean lines with improved seed protein concentration and nutritional bundle
o Yes, we have participation from Syngenta, Dow, Schillinger Genetics, and KAPI on the industry side. Pioneer and Monsanto are interested in rejoining our program next year.
• Companies use selected lines from our diversity tests in their breeding programs
o UMN is also still working through licensing agreement on four diverse lines being transferred to Monsanto. Once again, the buyout of Monsanto by Bayer has significantly delayed this. We have reviewed another list of lines with them, which they are interested in transferring for crossing in their program once a boilerplate license agreement can be established.
o University of Nebraska is finalizing a MTA for transfer of 13 diverse, high-yield lines to Monsanto during 2018.
• Other public researchers use diverse, high-yield, high-protein lines in their breeding programs
o Many high-yield lines with diverse pedigree and improved seed composition have been entered into the USDA Uniform Soybean Yield Tests Northern States, and are used by other state and USDA soybean breeding programs in their breeding efforts.
• Genetic markers for protein QTL on Chr 15 and Chr 20 are available for use by breeding programs
• Candidate genes for the protein QTL on Chr 20 and Chr 15 are identified
o Brian Diers, University of Illinois - During the past quarter, progress has been made in cloning the high protein genes on chromosomes 15 and 20. For the chromosome 15 gene, we are fine mapping it by identifying recombinants close to the gene and testing the progeny of these recombinants for protein and oil and with markers. Using data from several populations that were field tested in 2017, we have tentatively placed the gene into a 267 kb interval. To further narrow the interval, we have planted in the field this spring approximately 40 plant rows derived from each of 9 selected plants that have recombination in the 267 kb interval. These plant rows will be harvested this fall and tested for protein and oil concentration.
o To confirm the gene candidate we have identified for the chromosome 20 high protein gene, Tom Clemente at the University of Nebraska made transgenic plants that knock out the low protein allele. At this point, we don’t know whether we need to knock out the low or high protein allele of the candidate gene to show its function. Populations of plants that are segregating for the knock out of the low protein allele are growing in the greenhouse. The plants are filling pods and should be ready to harvest in about a month.

• Soybean lines developed in this program show stable performance for improved yield and seed composition over locations and years
o Several lines from the USDA, ARS breeding program at Illinois and the University of Nebraska program have shown superior yield and seed compositional quality over years and locations. Those were highlighted in the March report in the attached 2017 Diversity Program Yield Test report.
o MSC09-774089 has showed stable yield over the past three years. It will be release as a germplasm release in 2019 if yield is again good in 2018 regional trials. Breeder’s seed of this line has been made.
• At least one new commercial soybean variety from an industry partner results from crosses made with lines from this program!

Final Project Results

Updated November 2, 2018:

View uploaded report Word file

Benefit to Soybean Farmers

In time, soybean varieties with more protein will be available to growers. This soybean will produce a superior meal that will be in demand by animal producers for inclusion in poultry and swine feed.

Performance Metrics

1. Most or all major US commercial soybean programs participate in the cooperative wide-area evaluation of our diverse, high-yield soybean lines with improved seed protein concentration and nutritional bundle
2. Companies use selected lines from our diversity tests in their breeding programs
3. Other public researchers use diverse, high-yield, high-protein lines in their breeding programs
4. Genetic markers for protein QTL on Chr 15 and Chr 20 are available for use by breeding programs
5. Candidate genes for the protein QTL on Chr 20 and Chr 15 are identified
6. Soybean lines developed in this program show stable performance for improved yield and seed composition over locations and years
7. At least one new commercial soybean variety from an industry partner results from crosses made with lines from this program!

Project Years