Project Details:

Title:
Manipulating a major gene governing seed reserves as a means to maintain yield and oil while increasing protein

Parent Project: This is the first year of this project.
Checkoff Organization:North Central Soybean Research Program
Categories:Seed composition, Breeding & genetics, Industrial use – Meal
Organization Project Code:097817-00
Project Year:2020
Lead Principal Investigator:Matthew Hudson (University of Illinois at Urbana-Champaign)
Co-Principal Investigators:
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Our groups have fine mapped the high protein QTL cqSeedProtein-03 and recently identified a gene that is the basis of this QTL. This gene is Glyma.20G085100 and a survey of soybean germplasm shows that there is a 321bp deletion in the high protein allele. We generated transgenic plants carrying an RNAi construct targeting this gene. Data from a
greenhouse-grown population segregating for the transgene shows that those plants with the transgene have on average 2% higher protein. In this project, we will further characterize these transgenic events and additional events, perform field trials, introgress the transgene into different varieties, attempt to recreate the high protein phenotype using CRISPR/Cas9, and further our knowledge of an additional high protein locus on Chromosome15.

Project Objectives

1) Yield trials. We will perform yield trials of the transgenic plant material already developed, after ensuring that we have homozygous transgenic events in the Thorne background. We will also trial other material developed in the remaining objectives below. The yield trials will be conducted to test the impact of the transgenes on yield and protein and oil concentration.

2) Introgression. We will introgress the RNAi transgene from Thorne into high-yielding elite lines developed by the Diers group, to determine its effects on protein content and yield in a commercially relevant current variety.

3) CRSIPR/Cas9 editing. We will develop knockout lines in Glyma.20G085100 using the CRISPR/Cas9 system, in order to generate specific edits that we anticipate will maintain the high yielding, high-seed-protein phenotype of the transgenic lines. The transgenic RNAi lines are tightly regulated and have several regulatory hurdles before they can be commercialized, but CRISPR-derived edited lines, once cleaned by back-crossing to elite material, lack the transgenic DNA present in transgenic varieties.

4) Identification of the causative locus of the protein content gene located on Chromosome 15. We are close to identifying the molecular basis of a second protein content gene located on Chromosome 15 using fine mapping. We will extend the fine mapping data and use whole-genome sequencing of the line to identify the causative gene at this locus for future editing experiments.

Project Deliverables

We are applying to NCSRP for one year of funding to initiate this project. At the end of the first year we will have reached the following milestones:

1) Field trial completed for one growing season using RNAi transgenics

2) The high protein allele for Glyma.20G085100 downregulated and the effect on protein and oil tested in both a greenhouse and field and for yield in the field.

3) CRISPR guide construct designed, created and tested in transgenic soybean roots

Progress of Work

Updated April 9, 2020:
Report on “"Manipulating a major gene governing seed reserves as a means to maintain yield and oil while increasing protein, March 2020.

At the beginning of the project in October 2019 we stated the following milestones and KPIs. A brief progress report is given for each milestone and KPI. In addition, we have found several interesting research leads that were not anticipated when the initial proposal was written. Two key insights are
1) A finding that the transgenic RNAi construct is also affecting protein, but has pleiotropic effects on maturity and seed mineral content, where the original allele does not.
2) A discovery that the low protein gene found in high-yielding conventional soybean varieties may revert (at a very low frequency) back to the high protein gene. Thus, we may be able to identify revertant alleles within existing elite lines to facilitate the creation of high protein varieties.
In addition to these novel findings, which both require further research before they can be exploited in breeding and agronomy, our project to engineer protein levels using RNAi transgenics has proceeded according to plan and all milestones and KPIs are on or ahead of schedule.

Milestones:

1) Field trial completed for one growing season using RNAi transgenics

This field trial was completed at the transgenic field facility in Nebraska and we are still analyzing the data. Preliminary indications are that at least one transgenic event shows increased protein and free amino acid levels, and that oil content does not appear to be affected. However the results are preliminary and may not be statistically significant. The interpretation of yield data has been complicated by the fact that the RNAi transgenic constructs seem to affect maturity date, with the transgenic plants maturing later than the control, untransformed line. This is unexpected as the original pro/oil allele derived from PI468916 does not seem to affect maturity.

2) The high protein allele for Glyma.20G085100 downregulated and the effect on protein and oil tested in both a greenhouse and field and for yield in the field.

We have shown downregulation of the Glyma.20G085100 gene and have good data from the greenhouse and preliminary data from the field. The effect on protein appears to vary between different transgenic events, as expected. So far we are seeing a 1-2% increase in protein content in the best lines, which is also seen under field conditions, but is not yet statistically significant with the current year’s data. Interestingly we are also seeing relatively large differences in leaf mineral content in some transgenic events. Further work and additional years of field data will be needed to verify the significance of these results.

3) CRISPR guide construct designed, created and tested in transgenic soybean roots

The CRISPR guide has been designed and has been synthesized and placed in a plasmid vector. The construct has not yet been tested. Restrictions on laboratory work may slow the testing of this vector in the coming months.

We aim to complete the following Key Performance Indicators (KPIs) by the end of Year 1:

1) Data available for multiple RNAi transgenic events for yield trials in the field

We have extensive data for two RNAi events that is currently being analysed.

2) The impact of down regulating the high protein allele tested

We have demonstrated down-regulation of the allele in transgenic plants that are in the trials.

3) At least one plant transformation plasmid completed containing a guide RNA sequence targeted to the Glyma.20G085100 gene.

Although we have the guide RNA sequence it is not yet ready for plant transformation. Laboratory facilities at the University of Illinois are currently largely closed. We anticipate this will be completed by the end of the project, assuming that University laboratories are allowed to reopen.
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View uploaded report Word file

Final Project Results

Benefit to Soybean Farmers

Soybean is a major source of protein and oil globally. The protein component is currently the most valuable reserve of the seed, which is complemented by the oil co-product. Soybean protein is used primarily in feed applications and its demand is expected to increase. Unfortunately, there is an inherent negative correlation between protein and yield in soybean. An evaluation of soybean varieties released from the 1920’s to 2010 showed that during this time frame, seed protein content was reduced by approximately 2% (20 g / kg seed). This reduction in protein makes it difficult for crushers to produce a soybean meal with 48% protein, the industry standard, resulting in loss of value to growers.

Soybean is a global commodity and sold based on volume with minimal regard to quality, which has de-incentivize breeders to select for protein as they breed for higher yields. While the changes in protein concentration are small in percentage terms, the world soybean crop is projected to be around 370 million metric tons, thus a single percentage point in protein concentration represents 3.7 million tons of protein. There is now a great deal of commercial demand for higher protein soybeans that are high yielding, but conventional breeding processes are slow to incorporate introgressed genes for high protein into elite lines. We are proposing a genetic strategy to control protein concentration in tandem with preserving high yields, without compromising oil levels.

Performance Metrics

We aim to complete the following Key Performance Indicators (KPIs) by the end of Year 1:

1) Data available for multiple RNAi transgenic events for yield trials in the field

2) The impact of down regulating the high protein allele tested

3) At least one plant transformation plasmid completed containing a guide RNA sequence targeted to the Glyma.20G085100 gene.

Project Years