The US soybean commercial germplasm pool is narrow, with just 17 accessions contributing ~90% of the genes to the current US commercial germplasm. This severely limits potential for future progress to improve yield and composition. Programs within individual companies are even narrower as a result of intense selection and limited germplasm exchange during the past 20 years. In addition to the narrow genetic base, the general negative association between yield and seed protein concentration has resulted in a protein decrease of about 2 percentage points during the last 85 years of breeding. Unless breeders refocus efforts on protein concentration and overall balance of soybean seed composition, this decrease in seed protein concentration will likely continue. Soybean processors expect about 11 pounds of oil per bushel and need to produce a soybean meal with 48% protein. There is a range in soybean seed protein and oil concentrations that will result in the desired products (Figure 1). This comprehensive, coordinated program includes applied development efforts to transfer genetically diverse, high-yielding lines with improved nutrition bundle to our commercial partners for incorporation into their programs, and basic research to identify genes and gene functions related to seed composition, particularly high protein. The genomics component provides DNA sequence information as well as identified genomic regions that are transferred to elite germplasm from exotic sources. The genomics information will aid in modeling and prediction of breeding value, and enable implementation of genomic mating schemes both in this coordinated project and in commercial breeding programs.

Updated January 24, 2021:
March 15, 2020 Report
This project has the following four objectives. A brief summary of the project status for the current quarter is given under each objective. The objectives form a coordinated, comprehensive program with both basic and applied research objectives to address these challenges and achieve our goals.

1) Use PI accessions from the USDA Soybean Germplasm Collection that have never before been used in US soybean breeding programs for yield improvement to develop and release soybean lines with significantly increased genetic diversity, greater productivity, higher seed protein concentration, and improved nutrition bundle compared with current high-yield cultivars. This activity is tightly linked with our industry partners for wide-area cooperative evaluation throughout the major soybean production region in the US in Maturity Groups 0, I, II, III, IV, and V.

We conducted the 2019 Diversity Cooperative Tests with USDA, university and industry partners evaluating MG 0, I, II, III, IV and V soybean lines with improved yield and seed protein. The 2019 Diversity Coop Tests have more than 340 entries in 12 tests being grown in 20 locations in 7 states. The 2019 Diversity Coop Test report and summary was shared with cooperators in January 2020 and results were presented at the Soybean Breeders Workshop and USB Composition Workshop in St. Louis in March.

We expect results from the soluble carbohydrate and amino acid composition tests at the end of March. Those results will be added to the summary report and redistributed to all cooperators so they can use the information for selection of parent lines for 2020 crossing.


2) Identify and characterize candidate genes for the large-effect QTLs on Chr 15 and Chr 20, including the development and evaluation of transgenic lines to evaluate effects of gene candidates in different genetic backgrounds in greenhouse and field environments. This objective also includes the analysis of the genetically diverse germplasm developed by all cooperators for allele status at the Chr 15 and Chr 20 QTLs.

- The most likely gene candidate for the QTL is Glyma.20G085100 based on consistency of the allelic state in populations that the QTL was mapped.

- A hair-pin element was transformed into soybean to knock down the expression the candidate gene.

- Preliminary results show slightly higher protein and lower oil concentration for two T2 plants, no difference for two others.

- The plants and lines of these and additional transformation events will be grown in the field in 2020 in Nebraska for evaluation of seed composition and other traits.

3) Characterize protein composition, amino acid profiles, and soluble carbohydrate composition of seeds from our developed high-protein, high-yield lines from diverse G. max accessions and of selected experimental extreme high-protein lines from long-term selection programs.

Awaiting analysis results for amino acids and soluble carbohydrate composition for the high-yield experimental lines and the unltra-high protein lines. We expect results for both at the end of March.

4) Increase genetic diversity, seed composition, and seed quality in the early-planting soybean production system (ESPS) in southern US production areas.

The improved lines DS31-243 and DS1260-260 that we reported on in the last quarter, have improved seed quality scores that result in lower dockage for producers at the elevator. Both lines also produce a soybean meal with 48% or more protein and virtually zero dollar loss (discount) due to seed damage, compared to a high of more than $3,000 per 100 acres loss for one of the commercial checks.

Updated January 24, 2021:
September 15, 2020 Report
This project has four main objectives that form a coordinated, comprehensive program with both basic and applied research objectives to address these challenges and achieve our goals. For the previous three quarterly reports, we have itemized accomplishments as deliverables and KPIs by objective. For this fourth quarter report, we provide a general summary of the current status of the field plots including the Diversity Cooperative Test with our industry partners, and general status. There is not too much to specifically report regarding new results until after harvest and analysis of the 2020 data. We will plan to provide some of those preliminary results for the December 2020 quarterly report.

Programs initiated more than 200 new populations for genetically diverse, high-yielding lines with improved balance of seed protein and oil.

Diversity Coop Tests are going well, with the exception of some Dicamba damage at some locations, and loss of plots due to that damage plus severe hail or other environmental conditions. But overall, the mult-location cooperative tests with university, USDA, and industry programs is doing well and we look forward to good data after harvest for our yield, agronomic, and compositional quality analyses.

Initiated transformation for mutation of genes that underlie the QTL regions on Chr 10 and Chr 20. Field plots planted with the soybean events that carry the RNAi element targeting down-regulation of the gene call that underlies the protein QTL on chromosome 20 are maturing. Harvest is expected by the second week of October. Data will be ascertained on various agronomics, 100 seed weight, plot weight along with total protein and oil, and amino acid profile.

Research focused on fine mapping a gene on chromosome 15 that is important for increasing seed protein concentration progressed during the past quarter. For this work, we used results from previous field testing of 415 experimental lines from nine populations. Each population was derived from a plant that had a cross over event in a different position across the 450,000 base pair (450 kb) interval the protein gene was previously mapped. Attempts to use this field data previously to fine map the gene failed because we had poor quality marker data. To fix this problem, DNA was re-isolated from the lines and marker tests were completed using the new DNA. The new marker data were excellent and was used to position the protein gene relative to the cross over event in each population. These tests allowed us to position the gene to a 72.7 kb interval with a high degree of certainty and likely to a 50.9 kb interval. Within the 72.7 kb interval, there are 11 gene models in the soybean genome assembly 2. Among these genes, three have putative functions suggesting that they could be the protein gene. One of the three is listed as a late embryogenesis abundant protein and two as nodulin MtN21/EamA-like transporter family proteins. Further research is needed to identify which of these genes is the primary gene candidate for this important protein gene.

A draft of our research publication entitled “Biochemical characterization of UP2Y lines generated from a long-term breeding program using exotic soybean germplasm reveal that it is possible to increase the concentration of protein without lowering the quality” is currently being reviewed by coauthors.

We have conducted transcriptome analysis of two high protein lines (UP2PC7(S3)-0115 and UP2PC7(S3)-0230), two founder lines (PI404177 and MD83-2048) along with a standard soybean cultivar (Maverick). The RNAseq data will be analyzed during the next quarter.

Evaluation of seed quality for several 2020 Diversity Coop Test entries in MG 2 and 3 will begin at the MS location in the next week or two after exposure of mature soybean plants to hot humid conditions in Mississippi.

Updated December 7, 2021:
This project will significantly increase the genetic diversity in US soybean germplasm in both the public and private sectors, expanding the genetic potential for improved seed composition, productivity in diverse and changing environments, and profitability for US soybean farmers. The ultimate goal is to have at least one commercial seed company commercialize a new soybean variety that is derived from lines and information from this project in the next 5-10 years. The lasting impact of this project will be the transformation of the US commercial soybean germplasm pool to include 3x or more of the PI accessions than in the current gene pool, setting the stage for continued, long-term success for US soybean farmers.

Annual reports summarizing the multi-environment, multi-year tests that include yield and other important agronomic traits, seed protein and oil concentration, calculation of seed oil yield (lb/bu), meal protein percentage, meal protein yield (lb/bu), amino acid composition, composition for major soluble carbohydrates (sucrose, raffinose, stachyose), seed weight, and other information were developed each year. These Diversity Cooperative Tests involved 11 or more university, USDA and industry partners each year with 18 or more locations evaluating 220+ diverse, high-yielding soybean lines each year in Maturity Groups I, II, III and IV. Copies of the annual reports were shared each February with the USB/Smith Bucklin representatives working with this project and should be on file and available to USB members who want to review them. All cooperators received the annual report for their use in decision making for line advancements, crossing, and other research.

This was an interdisciplinary project that involved breeding, genetics, genomics, and biotechnology, working on different aspects of soybean genetic diversity to improve soybean yield, meal protein, and profitability for US soybean farmers. The diverse lines developed and tested in this program have 3x to 4x the contributions from genetically diverse soybean accessions compared to the foundation of the current US commercial germplasm pool.

All 400+ genetically diverse experimental lines were evaluated by our industry partners as well. Several lines were transferred to industry breeding programs through MTAs and/or license agreements for use in their development of commercial cultivars.

Results from our analysis of amino acid composition in the elite, high-yielding lines in the Diversity Cooperative Tests across the north central USA in Maturity Groups I, II, III and IV, indicate that there is no difference in amino acid composition in the high-yield lines with increased seed protein concentration or better oil/protein balance. Similar results were seen from our evaluation of amino acid composition of the ultra-high protein lines from long-term selection in either exotic or elite genetic backgrounds. Seed protein concentrations exceeded 55% on a dry matter basis, with no decline in protein quality as protein concentration increased. In fact, in some lines, we actually saw an increase in the percentage of sulfur-containing amino acids in the higher-protein lines. The observation that there are no negative relationships between sulfur containing amino acids and yield is good news for soybean farmers and for breeding. We can develop and high-yielding lines that have high-quality amino acid composition in the high-protein meal.
Fine mapping of the QTL regions on Chromosomes 15 and 20 led to identification of candidate genes in each of those regions. Modulation of gene expression through transformation, and evaluation of the transformed progenies in greenhouse and field environments showed slight increase in seed protein concentration in some transgenic lines, potentially confirming the targeted candidate gene as the causal gene for the high-protein phenotype.
Significant progress was made with development of lines with improved seed quality for use in the early soybean production system in the southern US. In addition to improved resistance to heat and fungal damage, improved seed protein concentration also was achieved.

Our DNA sequencing of all of the entries in the Diversity Cooperative Tests allowed prediction of cross performance and more informed selection of parents for population development. We also are developing tools for better visualization of genetically diverse regions on each chromosome for every line, so specific regions may be targeted for enhancing favorable genetic diversity in commercial germplasm.

These accomplishments lay the foundation for sustained and enhanced growth in productivity, quality, and profitability for soybean farmers.