Project Details:

Title:
High Oleic/Low Lin (HOLL) Soybean Breeding (Year 1 of 1720-162-0109)

Parent Project: This is the first year of this project.
Checkoff Organization:United Soybean Board
Categories:Seed composition, Breeding & genetics
Organization Project Code:1720-162-0109
Project Year:2017
Lead Principal Investigator:Kristin Bilyeu (USDA-ARS)
Co-Principal Investigators:
Zenglu Li (University of Georgia)
Brian Diers (University of Illinois at Urbana-Champaign)
Aaron Lorenz (University of Minnesota)
Pengyin Chen (University of Missouri)
Henry Nguyen (University of Missouri)
Andrew Scaboo (University of Missouri)
Grover Shannon (University of Missouri)
Vince Pantalone (University of Tennessee-Institute of Agriculture)
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Keywords: high oleic, low linolenic, breeding, FAD2 gene

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

The US soybean producer needs high yielding high oleic/low linolenic soybean varieties to provide the market with high value, highly functional soybean oil with zero trans fats. This project will conduct soybean variety development and research targeted to all US maturity groups (00-VIII), including non-GMO sources with and without herbicide traits.

Project Objectives

Objective 1: Develop and release competitive yielding non-GMO and herbicide resistant soybean varieties containing the high oleic/low linolenic (HOLL) acid trait in all US maturity groups.
Objective 2: Determine the optimum allele combinations to produce environmentally stable high oleic/low linolenic acid soybean varieties for US soybean production environments.
Objective 3: Determine yield potential and seed composition interactions of the high oleic acid trait along with other oil and meal traits.

Project Deliverables

1. Non-GMO and herbicide resistant soybean varieties with >70% oleic acid/<3% linolenic acid and competitive yields in MG 00-VIII.
2. Strategy for backcrossing and forward crossing four genes to achieve target fatty acid profiles in different production environments. Extension recommendations for producers using HOLL soybean varieties.
3. Research report on the status of the high oleic trait interactions with other oil and meal traits. Soybean germplasm with HOLL trait plus low saturated fatty acids. Soybean germplasm with HOLL trait plus enhanced nutritional energy meal/ENEM trait.

Progress of Work

Update:
There was a delay in getting the agreement between USB and USDA finalized in a timely manner. None of the sub-awards have been signed yet, and therefore none of the six university collaborators have had access to any funds for this project since August 30, 2016.

Hurricane Otto that caused flooding and destruction in Costa Rica has led to loss of some materials for at least the southeast Missouri program and my USDA research.

Progress continues otherwise as specified in the proposal.

During this reporting period all project participants sent their location samples for the 2016 environmental stability study, and we were able to complete fatty acid analyses. The average oleic acid across locations and dates was 84.0% for the indel version and 81.7% for the S117N version. Both values are higher than in 2015 (83.1% and 79.9%). I did not request weather data for either year for all of the locations, but the weather station observations for our Illinois and southeast Missouri locations indicate increases in August mean air temperatures from 2015 to 2016 of 4.1 oF and 2.9 oF, respectively. The average linolenic acid for all gene combinations in southeast Missouri, Tennessee, and Arkansas was below the 3% threshold in 2016 with one exception for the two gene combination in Arkansas. A table is attached below with the 2016 environmental stability study results.

Because the University of Arkansas has a vacancy in the soybean breeder position, I have worked with Moldir Orazaly to further their HOLL program. They grew F2 plants segregating for all four HOLL genes of their first two converted high yielding parents that emerged from the backcrossing program. We helped them identify lines to harvest and advance in the winter nursery. The southeast Missouri program only recently received their F2 seeds, and they are chipping the seeds and analyzing for fatty acids before sending to their winter nursery. The University of Tennessee and northern Missouri programs are finalizing their genotyping selections for their HOLL conversions that emerged from the backcrossing program early enough to be grown in their 2016 fields.

In 2016, Andrew Scaboo conducted a one location yield test with three replications. My best yielding research line was high oleic with one low lin gene and yielded 107% of the checks average. The best S117N FAD2-1A HOLL line yielded 98% of the checks, while the best indel FAD2-1A HOLL line yielded 92% of the checks. The lines were developed by a combination of forward crossing and backcrossing to reduce genomic contributions from the original plant introduction or mutant parent sources of the variant genes. Dr. Scaboo also tested HOLL lines from his forward crossing program in 2016 in one location, two or three replication tests. He identified three HOLL lines with competitive yields (107%, 118%, and 118% of the public checks).

Update:
This project is for the most part continuing as scheduled. The paperwork to finalize funding for all project participants was completed today (March 3, 2017) even though we had an October 1, 2016 start date.

The project team had a comprehensive meeting on February 12, 2017. A public research update was presented as part of the USB Seed Composition Workshop. High yields were reported with the high oleic/low lin trait in conventional and herbicide resistant varieties under development. Fatty acid profiles were relatively stable across environments, and either three or four gene selections were required to hit the oleic and linolenic acid targets. New nominations were made of recurrent parents into the dedicated backcrossing program. Team members compared and discussed best strategies to identify HOLL lines exiting the backcrossing program. Effort is focused on rapid release of high yielding HOLL varieties from MG 00 to VIII.

Update:
This project is for the most part continuing as scheduled. All team members conducted research and activities related to planting this quarter.

The project team had a brief meeting on June 14, 2017. A research update was presented as part of the meeting that involved USB board members, Missouri Soybean Merchandising Council board members and staff, a private company breeder, and technology transfer representatives from the Universities and USDA involved in the project. Kelly Whiting and Greg Luce organized the meeting, and it was successful in developing a core of current knowledge of the HOLL project and initiating the routes for commercialization relationships for HOLL soybeans that exit the project.

Dr. Li and I also made a brief visit to the Puerto Rico field site in May to address some issues related to improving communication to maintain the workflow that the project requires. The winter nursery is a critical component of the project, and it is succeeding it meeting the objectives. We became aware of potential issues in communication and wanted to deal with all of the parties involved directly. We also assessed the status of each of the cycles that were in the field.

Project team members reported that they had requested and received backcrossed seed from Puerto Rico. Depending on each recurrent parent, some of the materials will need to be screened for the 1/256 that have the four gene combination, and other materials will be grown to generate seed for that screening step.

This field season will have most programs evaluating yield for a set of HOLL lines.

Update:
Kristin Bilyeu, USDA/ARS Columbia, Missouri

Research is progressing for this period as planned. A third year of environmental stability analyses are in field experiments in all project team locations.
From the individual reports below, I calculate there are 756 HOLL lines in multi-location field trials across maturity groups II-VII, with the majority of the lines tested in three or more environments.


Aaron Lorenz, University of Minnesota
Backcrossing
Our first set of “stacked F2s” were returned last winter. These were from the M07-297007 (MG I) recurrent parent. We screened over 2500 seeds and identified four segregants homozygous for all four genes and those are now growing in plant rows in Saint Paul. The plants look very good, being healthy and highly uniform across as well as within rows. We will bulk seed from each row and send to Chile for another seed increase so seed can be more quickly multiplied if agronomic performance is indeed superior. These lines will be entered into 2018 advanced yield trials (3 locs, 2 reps).
The next set of “stacked F2s” were returned in May from recurrent parent M07-296048 (MG 0). About ~7000 seeds were returned. We both planted seeds in the field (~3500 planted, ~2300 germinated) and chipped seeds in the lab. For the field, tissue was collected and sent to UGA for marker analysis. We expect to receive the results soon. Once the marker results are in hand, we will harvest plants homozygous for all four genes and send them to Chile for a seed increase so they can be included in yield trials in 2018. Plants are heterozygous at the FAD3 loci but homozygous at the FAD2 loci will be sent to Chile for plant pulls to obtain more homozygotes. These will then be planted in 2018 plant rows.
The remaining seeds from this population are being genotyped at UMN after selection using single-seed NIR and seed chipping. All seeds have been scanned with the NIR and those with readings higher than 50% oleic were selected for chipping. We have chipped and genotyped ~1000 seeds so far. We have found four individuals homozygous for all four genes. These will be planted in the growth chamber for seed increase over the winter. The heterozygotes at FAD3 will be planted in the greenhouse to harvest pods and obtain more homozygotes (two generations) for plant rows in summer of 2018. The remaining 500 seeds selected by NIR will be chipped soon and genotyped. We are using these populations to assess the success of our single seed NIR based on a previous calibration. If this is unsuccessful, we will begin the development of a new calibration this winter.
The most recent sets of “stacked F2s” were sent in July. The recurrent parents were M09-160019 (MG 0) and M09-288155 (MG I). A total of 3000 seeds were sent. These have been scanned with NIR, chipped, and extracted. Markers are currently being run on these and heterozygous/homozygous individuals will be planted in the GC/GH for generational advancement and seed multiplication over the winter time.
In summary, no HOLL lines are in yield trials in 2017 (although we have many HO) but we anticipate entering many into both PYTs and AYTs in 2018 based on all the stacked F2s sent and screened.
We nominated eight new recurrent parents, including both RR1 and RR2 parents and ranging from 0.08 to 1.5 in maturity.
Forward crossing
In 2016, 10 crosses were designed that are segregating for both FAD2 and FAD3 loci. These are now in the F2 breeding nurseries in Saint Paul in 2017. All plants look good. We will advance these through our normal breeding program and genotype at the F4:5 generation just before they enter plant rows in 2019.
In 2017, we designed and successfully made four crosses segregating for high oleic and low linolenic. This number is down as we hoped to use plants from the M07-296048 stacked F2 pop but we did not get the genotype information on time.

Briand Diers, University of Illinois

During the past quarter, the University of Illinois breeding program has been developing and testing experimental lines that have all four genes required to achieve oil with both greater than 80% oleic acid and less than 3% linolenic acid. Two-row yield plots are being grown of 135 experimental lines that have this four gene combination. This includes 30 lines that have a relative maturity (RM) ranging from 2.5-3.0, 65 lines with a RM from 3.1-3.6, and 45 lines with a RM from 3.7-4.2. All lines are being tested in two locations in Illinois. The 2.5-3.0 lines are also being tested in one location in Iowa and Nebraska, the 3.1-3.6 lines in one location in Nebraska and Missouri and the 3.7-4.2 lines in one location in Missouri. Lines performing well in the 2017 tests will be evaluate in uniform tests in 2018.

Plant rows of over 1300 new experimental lines are being grown that have the four gene combination. Visual selections will be made and selected lines will be yield tested in 2018. In addition, 60 populations of F3 plants are being grown that were developed from F2 plants that were selected as being homozygous for all four gene or three genes and segregating for the fourth. Markers were used to select homozygous plants for the fourth gene and plants homozygous for all four genes will be selected and harvested this fall from the populations.

F1 plants from the Puerto Rico backcrossing of the four genes into two Illinois adapted backgrounds (one is Roundup Ready and the other is conventional) are being grown in the field. These were tested with markers and plants heterozygous for all four genes were identified. F2 progeny from these F1 plants will be grown in the greenhouse this fall to identify plants that are homozygous for all four genes.

Crosses were made for 40 combinations between plants selected as being homozygous for all four genes or homozygous for three and segregating for the fourth. F1s plant from these crosses will be grown in a greenhouse this fall and F1 plants that are homozygous for all four genes will be selected which will give us large populations that we will be able to use to select for yield and other agronomic traits. An additional 26 cross combinations were made between new, high yielding cultivars and experimental lines and plants homozygous for the four genes. These crosses are being done to bring in new genes for high yield into the oleic breeding effort.


Andrew Scaboo, University of Missouri

Advanced Yield Trails:
We have approximately 130 HOLL lines (Conv./RR1/RR2) in yield trials at 6 locations in MO during 2017. The conventional lines are also tested in IL, NE, and IA at one location in each state. In MO, these tests have been maintained well during the growing season and we expect good yields and data. These lines will have two years of yield testing after this year, and the best lines will advance to the USDA regional trials during 2018 and 2019, with potential for release during 2019.

We have 12 RR2 and 2 conventional HOLL lines from the USB backcrossing program in yield trails at two locations in MO during 2017, although these tests were planted in early July of 2017. These lines will be fast-tracked to USDA regional trials during 2018 and 2019, with potential for release during 2019.

Preliminary Yield Trials:
We have over 300 HOLL lines in preliminary yield trials at 4 locations in MO. These tests have been maintained well during the growing season and we expect good yields and data. Material in this stage of testing needs three more years of field data collection before release.

Progeny Row Trials:
We have selected approximately 500 progeny rows via MAS, which carry all four HOLL mutant genes, for advancing to preliminary yield trials during 2018. These tests have been maintained well during the growing season and we expect good yields and data.

Crossing and Generational Advancement Trials:
We have continued to make crosses for both our forward and backcrossing HOLL breeding program. We cross during the summer and winter breeding cycles, and we were successful during the spring and summer of 2017. These tests have been maintained well during the growing season and we expect good F1 – F3 and BC1or2F1 seed production.

Pengyin Chen, University of Missouri Delta Center
1) Promising high oleic lines in seed increase – Two promising high oleic lines were increased in Costa Rica with a nearly 45X production. Seed returned from Costa Rica were planted to a 3-4 acre breeder seed production in preparation for release.

Vince Pantalone, University of Tennessee

In 2017, we have one quad mutant HOLL line in yield testing for which our elite cultivar Ellis is the recurrent parent. We also have ~90 quad mutant HOLL progeny rows for which Ellis is the recurrent parent. Additionally, we have ~10 quad mutant HOLL progeny rows for which our high yield line with resistance to glyphosate, TN13-4710R2, is the recurrent parent. These rows will serve as sources for the next year of field testing, when they will be compared with other promising breeding lines and elite check cultivars in replicated, multi-environment yield trials.

We have ~40 lines with HO and ~60 lines with LL in yield trials. These lines are represented in a variety of tests in MGs IV and V. Most of the lines are in conventional backgrounds, but there are four HO and ten LL lines with glyphosate resistance traits. Approximately 2/3 of the lines tested are in the first year of testing, while the remaining lines are split, with ~1/6 in repeated field testing after having excelled in field trials in previous years, and the remaining ~1/6 in graduate student tests. Among the graduate student projects are field tests examining gene combinations and trait stability for HO, LL, and HOLL.

Further, we currently have ~50 rows planted of (BC3_HO × BC3_LL) TN13-5538R1, which are derived from F1 plants confirmed to be quad het for HOLL. We are currently screening this population with SNPs, and expect to find quad mutants at a rate of 1/256. Once identified, we will conduct a winter nursery single plant production and perform progeny row field testing next summer. This is an exciting possibility, as TN13-5538R1 is a newly released cultivar by our program with high yield, resistance to races 2 and 5 of SCN and resistance to SDS, as well as resistance to glyphosate herbicide.

Additionally, we have breeding populations containing HO and LL traits at every generation of development, from F2 to F6 and beyond. We are currently screening these populations with SNP markers to confirm successful transmission of these important traits. Plants that are phenotypically selected for advancement for be further confirmed for HOLL traits using gas chromatography. We have also been continually adding to our future pool of HOLL soybeans in elite genetic backgrounds through both forward and backcrosses. Seventeen of our new cross combinations last month included at least one parent containing HO, LL, or HOLL traits, which we will seek to incorporate into high yielding soybean cultivars in the coming years.

Moldir Orazaly (Leandro Mozzoni), University of Arkansas

HOLL Advanced test from backcrossing program:
In 2017, seven backcrossed lines with confirmed HO and LL genes were planted in two AR locations (Stuttgart and Keiser) with three replications each. These seven lines are derived from two different recurrent parents: R10-197RY and UA 5715GT, which are a high yielding roundup ready 2 line and roundup ready 1 variety, respectively. Both recurrent parents were included in the yield test so we can compare HOLL lines’ performance against recurrent parents without HOLL.

Backcrossed stacked seeds:
We received HOLL stacked seeds in 2017 summer and planted them in Fayetteville, AR. These seeds are derived from one conventional high yielding variety, UA 5615C, and one roundup ready-2 line, R11-89RY. A total of 27 seeds derived from UA 5615C and 33 seeds derived from R11-89RY were germinated. DNA samples of these plants were submitted to Dr. Li’s lab. Once the plants with HOLL genes are identified, they will be harvested and sent to winter nursery to increase seed for 2018 yield trial.

HOLL Advanced test from forward crossing program:
A total of ten advanced HOLL lines with 70 – 86.9 oleic and 0.6-2.7 linolenic lines are being tested in three AR locations (Keiser, Pinetree, and Stuttgart) with 3 replications in each location.

HO advanced lines from forward crossing program:
A total of 13 lines with high oleic trait only are being advanced in three AR locations (Stuttgart, Keiser, Pinetree) with three replications in each location.

LL advanced lines from forward crossing program:
A total of 23 advanced lines with 0.8-2.6% linolenic fatty acid are being evaluated in three AR locations with three replications in each location.

Zenglu Li, University of Georgia

Objective 1: Develop and release competitive yielding non-GMO and herbicide resistant soybean varieties containing the high oleic/low linolenic (HOLL) acid trait in all US maturity groups.

2017 USDA Uniform Test

In 2016 UGA advanced yield trials at four locations, two conventional lines G15PR-342 and G15PR-302 yielded above best check in the test. These lines also have oleic content >75% and linolenic content <3%. In 2018, we entered these two lines into USDA Preliminary Uniform Test MGVII.

2017 UGA Advanced Yield Test

Based on the 2016 UGA yield trials at 2 locations, we have advanced 12 high oleic conventional lines into 2017 UGA advanced yield trials at four locations. All these lines yielded at least 103% of the best check in the test and have oleic content >75% and linolenic content <3%.

2017 UGA 1st Year Yield Test

During the 2016-2017 winter, we grew plant rows from 5 pedigrees in Puerto Rico. One of pedigrees is RR1, two are RR2Y and two are conventional. A total of 136 lines from these 5 pedigrees were included in
2017 UGA 1st Year Yield Tests by using commercial RR2Y cultivars as checks in each test. All these lines possess four genes (2 high oleic and 2 low linolenic). Recurrent parents of these lines are currently released soybean cultivars. Planting at two locations were completed although there were some delay due to excess rain in May and June.

Pipeline of HOLL lines at UGA
Stacked F2 seed from three pedigrees were planted in our crossing block for genotyping and seed increase. These stacked F2 plants are segregating for 4 genes (HOLL). Over 2500 samples were collected from these plants for genotyping and genotyping is in progress. All three recurrent parents are RR2Y lines. They are in the USDA Uniform Tests and in the pipeline for release. The selected stacked F2 plants will be planted as plant rows in Puerto Rico this winter for seed increase and selected rows will be entered into yield trials in 2018.

High-oleic, low-linolenic (HOLL) Nursery and Genotyping Update
(January – August 2017)

Lab Genotyping
Since January 2017, we have received 21,697 samples from the Puerto Rico HOLL backcrossing nursery for seven programs MN, IL, MO (2), AR, TN, and GA (Table 1). We have also received F1 samples from IL, AR, and TN programs (213 samples) as well as F2 samples from MN program (2,276 samples) in addition to the above-mentioned HOLL F1 and F2 samples received for the GA program. All of these samples were genotyped on time and selections made for their respective crosses.
In the spring of 2017, 36 new elite lines representing MGs 0.5 – 8 and RR1, RR2Y and conventional were sent to Puerto Rico from the 7 programs for parallel backcrossing of HO and LL traits using various donors. Genotyping and backcrossing for 2017 Cycle 1 was completed in April 2017 and 2017 Cycle 2 was completed in early August 2017. The third cycle of this program is planned to be planted in the fall of 2017.

Nursery updates
The HOLL nursery in Puerto Rico has since received two convection ovens for the purpose of drying leaf samples prior to shipment to reduce expenses on shipping materials and methods of shipment.
Although hurricane Irma inflicted significant damage to numerous islands in the Caribbean, 2016 cycle 4 and 2017 cycle 2 plants were unaffected due to preparatory measures taken.

Final Project Results

Updated December 2, 2018:
See Year 3 for Final Report (FY19)

See Year 3 for Final Report (FY19)

Benefit to Soybean Farmers

Provides greater choice, availability, and seed costs for high oleic varieties. Premiums for high oleic.

Performance Metrics

High yielding high oleic + low linolenic varieties in both non-GMO and herbicide trait versions will be developed and available for release to producers – either directly from the university or licensed to private seed companies. Specific KPIs are listed below by objective:

1. The outcomes of this project are high oleic/low linolenic acid soybean varieties with the first releases, up to 8 varieties, available in 2018, with up to 157 backcrossed high oleic/low linolenic acid soybean lines available in the pipeline through 2021 for yield test and release. Utilization of HOLL varieties by producers will contribute to the strategic goal of USB to grow high oleic varieties on 18 million soybean acres by 2023, and thereby recapture lost market share in the oilseed market.

1. The USDA lab will complete high oleic/low linolenic stability tests with soybean germplasm containing two, three, and four gene combinations in MG 0-VIII and confirm the need for a four gene combination for maximum breeding efficiency by February 2017. The target audiences of seed companies, processors, oil end users, and farmers will benefit from a stable and reliable source of high oleic/low linolenic acid soybeans across US production environments and will begin to adopt varieties, or advance material toward commercialization.
2. Yield tests will be analyzed for potential effects of trait combination for high oleic and low saturates as well as high oleic and enhanced nutritional energy meal traits from field trials in 2016 and 2017. Soybean producers and seed companies will benefit from research directed at developing soybean varieties with value added traits by beginning to adopt and use these results to advance toward commercialization.

Project Years