Project Details:

Title:
Stacking Four Plant Genes to Generate Durable and Enhanced SCN and SDS Resistances in Soybean

Parent Project: This is the first year of this project.
Checkoff Organization:Iowa Soybean Association
Categories:Soybean diseases, Nematodes, Breeding & genetics
Organization Project Code:450-49-01
Project Year:2019
Lead Principal Investigator:Madan Bhattacharyya (Iowa State University)
Co-Principal Investigators:
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. Soybean is also a source of biodiesel. Soybean roots fix nitrogen through symbiosis with a rhizobacterium (Bradyrhizobia japonicum). In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN) and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions (http://extension.cropsciences.illinois.edu/fieldcrops/diseases/yield_reductions.php) from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa and as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.

Growing disease resistant soybean cultivars has been the main method of controlling SCN and SDS. The number of SCN resistance genes is limited and the majority of the cultivars carry the rgh1-b locus, which has been isolated (Cook et al. 2012: Science 338:1206-1209). The rgh1-b locus is complex and comprised of three distinct genes located in a 31 kb DNA fragment. Each of these three genes contributes to SCN resistance. Multiple copies of the 31 kb fragment are required to confer SCN resistance; and cultivars carrying a single copy of the 31 kb DNA fragment are SCN susceptible. In the United States, 95% of the SCN-resistant cultivars carry the complex rgh1-b locus derived from PI 88788 (Han et al. 2015: BMC Genomics 16:598). It is becoming evident that monoculture of this form of SCN resistance may cause breakdown of the SCN resistance leading to devastating crop losses. Therefore, incorporation of diverse SCN resistance mechanisms into single cultivars is becoming crucial to secure soybean production.

SDS resistance is partial and encoded by over 80 genetic loci each contributing small effects (Chang et al. 2018: Theor Appl Genet 131:757–773). This makes soybean breeding for SDS resistance as an arduous and time-consuming process. In an ongoing Agriculture and Food Research Initiative (AFRI) project funded by USDA-NIFA, we investigated several plant genes for their utility in enhancing SDS resistance in transgenic soybean. Of the nine plant genes investigated in transgenic soybean lines, PSS25, PSS30, GmSAMT2 and GmDS1 have shown to enhance both SDS and SCN resistances. GmDS1 also confers resistance to soybean aphids and spider mites. PSS25, PSS30, GmSAMT2 and GmDS1 encode a putative transcription factor, folate transporter, salicylic acid methyl transferase enzyme, and putative receptor protein, respectively. Therefore, each of the four genes uses a unique mechanism to govern SDS and SCN resistance.
We hypothesize that since the resistance mechanisms encoded by PSS25, PSS30, GmDS1 and GmSAMT2 are distinct from the one that is encoded by rhg1-b, the SCN resistances conferred by the four genes are complementary to each other and to that of the rhg1-b gene; and together all five genes are expected to provide soybean stable and robust resistances against both SCN and F. virguiforme isolates.

The outcome of this proposed research is expected to be highly significant because it will lead to development of soybean lines with robust resistances to the two most serious soybean pathogens, SCN and F. virguliforme. Furthermore, it is worth noting that GmDS1 also confers resistance to soybean aphids and spider mites (Ngaki et al. 2018; manuscript under revision and resubmission for Plant Biotechnology Journal). It is expected that utilization of these four genes will significantly reduce the soybean yield suppression caused routinely by SCN and SDS. Thus, this project will significantly improve soybean growers’ farm economy. Even 20% reduction in yield suppression caused by SCN and SDS will be translated to extra $400 millions farm income to the U.S. soybean growers.

Justification and Rationale: The proposed research will determine if PSS25, PSS30, GmDS1 and GmSAMT2 together can increase the extent of SCN and SDS resistance in soybean. The rationale of the proposed study is that once we establish that the four novel genes together can reduce the extent of SCN and SDS incidences, then stacking of the four genes along with rhg1-b and other SDS resistance genes in commercial cultivars will be feasible to generate robust SCN and SDS resistances; and thereby, improve soybean growers’ farm income by reducing the soybean yield suppression from the two most serious soybean pathogens, SCN and F. virguliforme. At the end of the three-year project period, we expect to deliver several desirable soybean genotypes carrying all four genes in homozygous condition. These genotypes with superior SCN and SDS resistance will then become the donors for backcrossing the four genes simultaneously into commercial SDS and SCN resistant cultivars that carry desirable natural SCN and SDS resistance genes. Thus, this project will significantly contribute towards providing durable and superior resistances against two most destructive soybean pathogens that together rip-off soybean yield valued over $2 billions annually, just in the United States. These two diseases are also very important for Iowa farm economy. It is therefore well justified to undertake this project to improve the farm economy of the soybean growers of Iowa and rest of the U.S.

Project Objectives

Objective 1. Map the four fusion transgenes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines. In each of these transgenic plants, the transgene integrates randomly into the soybean genome. To facilitate the crossing between two transgenic lines, it is ideal to know the genomic locations of the transgenes. Stacking of two transgenes by crossing is only successful, if they are localized into two distinct genomic regions, between which exchange of chromosomes can take place.

Objective 2. Identify Williams 82 lines that carry combinations of three transgenes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2. We will have F¬2 lines homozygous for both PSS30 and GmDS1 prior to start of this project. We will be growing four F2 populations segregating for PSS30 and GmDS1 transgenes this summer. These four populations were generated by hybridizing two independent events of PSS30 with two events of the GmDS1 transgene. We therefore expect that at least one population will have segregation for both genes to get a desirable line with both genes for making the crosses with either PSS25 or GmSAMT2. The crosses of the line carrying PSS30 and GmDS1 transgenes with lines carrying either PSS25 or GmSAMT2 will be conducted in the summer of 2019. F1 plants will be grown in greenhouse to obtain F2 seeds, which will be planted in the field during the summer of 2020 for crossing work proposed in Objective 3. We have already generated many F1s to stack PSS30 and GmDS1 and we do not anticipate any problem in generating the F1s for this objective.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2. In the summer of 2020, we will be growing the F2 generations of both crosses to be made in the summer of 2019 in Objective 2. Lines carrying either (i) PSS25, PSS30 and GmDS1 or (ii) PSS30, GmDS1 and GmSAMT2 will be crossed to stack all four transgenes into one soybean line. The F1s will be selfed to obtain F2 seeds in greenhouse during the winter of 2019-2020.

Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme. F2 plants from the cross between the genotype carrying PSS25, PSS30 and GmDS1 and plants carrying PSS30, GmDS1 and GmSAMT2 will be grown in the field during the summer of 2021. We will conduct quantitative reverse transcriptase PCR (qRT-PCR) of the transgenes in studying the association between expression of all four transgenes and enhanced SDS resistance.

Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines. A portion of the F2 seeds harvested in greenhouse during the winter of 2019-2020 will be evaluated for SCN resistance. We will conduct qRT-PCR to determine the association between expression of all four transgenes and enhanced SCN resistance.

Project Deliverables

We expect to deliver the followings by years:

1. By the end of Year 1, we will complete Objectives 1. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.

2. In Year 2, we expect to complete Objective 2; and have the Objective 3 partially completed. We will have generated F1 seeds of the double crosses to stack all four transgenes.

3. In Year 3, we expect to have evaluated the F2 segregating population generated to segregate all four transgenes. We will have done phenotyping and genotyping the F2 individuals to determine the association between the number of transgenes and levels of SDS or SCN resistance. Genotypes carrying all four transgenes will be identified.

4. A manuscript describing the SCN resistance will be published in a peer reviewed journal by the end of Year 3.

5. The manuscript describing the SDS resistance will be ready only after completion of this project since the foliar SDS data will most likely be collected by the September of Year 3 and we need to carry on field trial at least one more year under the support of a renewal proposal.

Timelines and Milestone for Deliverables: The following are our milestones and deliverables.

1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.
2. We will have harvested the seeds of single crosses by October 31, 2019.
3. We will have harvested the seeds of double crosses by October 31, 2020.
4. We will have harvested F2 seeds in greenhouse during the winter of 2019-2020.
5. We will have accomplished initial evaluation of the F2 plants for responses to both F. virguliforme and H. glycines before the end of the Year 3.
6. It will be established if we observe complementary effects among the four transgenes that govern distinct genetic mechanisms to confer SDS and SCN resistance.
7. A peer reviewed journal article describing the responses of F2 plants to H. glycines will be published.
8. We will have identified homozygous lines for all four transgenes will be identified and made available to private seed industries by October of 2021.

Progress of Work

Updated April 2, 2019:
Semi-annual Report
Iowa Soybean Association
April 1, 2019

Project Title: “Stacking four plant genes to provide durable and enhanced SCN and SDS resistance in soybean”
Investigator:
Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu

Progress report for the period from October 1, 2018 to March 31, 2019

Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN) and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.
In this project, we proposed to evaluate the joint or combined effect of four transgenes in improving the SCN and SDS resistance in a single soybean line. Of the four genes, two are from soybean and the other two from Arabidopsis thaliana. The two soybean genes, GmDS1 and GmSAMT2, encode a receptor-like protein and a salicylic acid methyl transferase, respectively. The two Arabidopsis thaliana genes, PSS30 and PSS25, encode a folate transporter and a putative transcription factor, respectively. Thus, the four genes use distinct mechanisms to confer both SCN and SDS resistance in transgenic soybean plants.

We hypothesize that since the resistance mechanisms encoded by PSS25, PSS30, GmDS1 and GmSAMT2 are distinct, the functions of the four genes are complementary to each other and together they are expected to provide soybean with stable and robust resistances against both SCN and F. virguiforme.
The outcome of this proposed research is expected to be highly significant because it will lead to development of soybean lines with robust resistances to the two most serious soybean pathogens, SCN and F. virguliforme. Therefore, this project will significantly improve soybean growers’ farm economy.

Goals and Objectives: The goal of this project is to significantly contribute towards developing durable resistance against the two serious soybean pathogens, SCN and F. virguliforme. We propose five objectives to reach our goal.

1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines.

This is a 3-year project. In the Year 1, we plan to complete Objective 1 and partially Objective 2.

We report here the progresses made to date under each of the Objectives 1 and 2.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
In this study, we selected eight transgenic soybean lines, each carrying an independent fusion gene. The eight fusion genes for these eight transgenic lines were created by fusing PSS25, PSS30, GmSAMT2 and GmDS1 individually to two different promoters.

During development of the eight transgenic lines, the eight fusion genes randomly integrated into the soybean genome. If any two fusion transgenes are intergrated into tightly linked genomic regions, stacking of those genes becomes very difficult and we need a large population to recover recombinants containing both genes. We therefore started with eight transgenic lines, two for each of the four genes selected for this project.
We proposed to map the position of integration for each of the eight fusion genes in this objective. Mapping will not only establish the feasibility of stacking all four genes into one line, but also to generate transgene-specific PCR markers to study the segregating population for identifying desirable recombinants.

We applied the Universal GenomeWalker method (Clontech Laboratories, Inc, Mountain View, CA 94043, USA) that was successfully applied in mapping the Tgm9 transposon insertion sites in soybean earlier by us (Sandhu et al. 2017: PLoS ONE 12(8): e0180732). The genome is digested with four restriction endonuclease enzymes independently and ligated to adapters provided in a GenomeWalker kit developed by Clontech Laboratories, Inc, (Mountain View, CA 94043, USA).
Earlier, the eight fusion genes were integrated into the soybean genome using T-DNA molecules generated in a binary vector. Primers were designed from both ends of the two T-DNA borders for amplification of the target insertion sites for each of the eight transgenes. The PCR products amplified by using T-DNA end-specific primers and adapter-specific primers were sequenced; and sequences of the PCR products were used to identify the map position of each of the six out of eight transgenes.

The eight lines included earlier in phenotypic studies were selected for this project because each carries a single transgene. The positions of the other two transgenes will be known shortly. Only two transgenes Prom2-SAMT2 and 35S-Pss25 were found to be physically linked. However, they are 1.5 megabases apart with a genetic distance of approximately 17 centi Morgan (cM) (SoyBase resources: https://soybase.org/SequenceIntro.php). We do not see any problem in recombining these two genes. We should be able to obtain 17% desirable recombinants carrying both genes as opposed to 50%, if the two genes are mapped onto two chromosomes. As of now, our mapping data suggest that we will be able to stack all four transgenes into one Williams 82 line. For example, if we select transgenic lines 107 (GmDS1), 480 (PSS30), 125 (GmSAMT2) and 79 (PSS25) for making crosses, then we should be able to stack all four transgenes into one plant by analyzing small segregating populations because all four genes are mapped to four independent chromosomes; therefore, they will segregate independently.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.

For this objective, we already have generated F2:3 populations segregating for PSS30 and GmDS1 lines in a project funded by United Soybean Board. We have initiated molecular analyses to identify segregating plants that carry both Pss30 and GmDS1 transgenes. From molecular analysis of eight F2:3 population, we have identified four populations that contain both transgenes. We will now develop PCR primers, specific to each of the transgenes, Prom3-DS1 and Prom2-Pss30 mapped to Chromosomes 16 and 9, respectively. Such primers will allow us to rapidly identify the F3 plants that are homozygous for both transgenes. The homozygous plants will then be used to make crosses with each of the Lines 125 (Prom3-SAMT2) and 79 (35S-PSS25). The crosses will be started in early May in two independent greenhouses of the Agronomy and Horticulture Departments.

Project Metrics and Performance Measures, Milestones, Deliverables and Outcomes – KPIs/Performance Metrics, Economic Impact/Significance, Timelines and Milestone Deliveries to extend state-of-the-art:

KPIs/Performance Metrics: We expect to accomplish the followings by years: The responses are presented below for Year 1 of the 3-year project period. Our progress is on track as proposed in the proposal.

1. By the end of Year 1, we will complete Objectives 1. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.

Response: We have completed mapping of the transgenes. Identified four transgenic plants that are being considered for crossing in two greenhouses. We plan to start crossing middle of May and expect to get two classes of F¬1 seeds by the end of September 30, which is the end of Year 1.

2. In Year 2, we expect to complete Objective 2; and have the Objective 3 partially completed. We will have generated F1 seeds of the double crosses to stack all four transgenes.
3. In Year 3, we expect to have evaluated the F2 segregating population generated to segregate all four transgenes. We will have done phenotyping and genotyping the F2 individuals to determine the association between the number of transgenes and levels of SDS or SCN resistance. Genotypes carrying all four transgenes will be identified.
4. A manuscript describing the SCN resistance will be published in a peer reviewed journal by the end of Year 3.
5. The manuscript describing the SDS resistance will be ready only after completion of this project since the foliar SDS data will most likely be collected by the September of Year 3 and we need to carry on field trial at least one more year under the support of a renewal proposal.

Timelines and Milestone Deliveries: The following are our milestones and deliverables for Year 1 of the 3-year project period.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.

Response: We have completed mapping the transgenes and identified four minimum transgenic lines for crossing to accomplish the goal of this project.

2. We will have harvested the seeds of single crosses by October 31, 2019.

Response: Crossing work will be started in greenhouses starting early May.

3. We will have harvested the seeds of double crosses by October 31, 2020.
4. We will have harvested F2 seeds in greenhouse during the winter of 2019-2020.
5. We will have accomplished initial evaluation of the F2 plants for responses to both F. virguliforme and H. glycines before the end of the Year 3.
6. It will be established if we observe complementary effects among the four transgenes that govern distinct genetic mechanisms to confer SDS and SCN resistance.
7. A peer reviewed journal article describing the responses of F2 plants to H. glycines will be published.
8. We will have identified homozygous lines for all four transgenes will be identified and made available to private seed industries by October of 2021.

View uploaded report PDF file

Updated October 3, 2019:
Semi-annual Report
Iowa Soybean Association
October 1, 2019

Project Title: “Stacking four plant genes to provide durable and enhanced SCN and SDS resistance in soybean”

Investigator: Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu
Iowa State University

Progress report for the period from April 1, 2019 to September 30, 2019

Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN), and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa and as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.

In this project, we proposed to evaluate the joint or combined effect of four transgenes in improving the SCN and SDS resistance of a soybean line. The four genes use distinct mechanisms to confer both SCN and SDS resistance, when overexpressed in transgenic soybean plants. Of the four genes, two are from soybean and two are from Arabidopsis thaliana. The two soybean genes, GmDS1 and GmSAMT2, encode a receptor-like protein and a salicylic acid methyl transferase, respectively. The two Arabidopsis thaliana genes, PSS30 and PSS25, encode a folate transporter and a putative transcription factor, respectively.

We hypothesize that since the resistance mechanisms encoded by PSS25, PSS30, GmDS1 and GmSAMT2 are distinct, the functions of the four genes are therefore complementary to each other and together they are expected to provide soybean with stable and robust resistances against both SCN and F. virguiforme isolates.

The outcome of this proposed research is expected to be highly significant because it will lead to development of soybean lines with robust resistance to the two most serious soybean pathogens, SCN and F. virguliforme. Therefore, this project will significantly improve soybean growers’ farm economy.

Goals and Objectives: The goal of this project is to significantly contribute towards developing durable resistance against both SCN and F. virguliforme isolates that together cause soybean yield suppression valued close to $2 billion.
We propose five objectives to reach our goal in a 3-year period.
1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines.

This is a 3-year project. In Year 1, we planned to complete Objective 1, partially Objective 2 and initiate Objective 3.

We report here the progresses made to date under each of the three objectives.
Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
In the previous semiannual report of April 1, 2019, we have reported mapping six of the eight transgenes generated from four plant genes. Later, we have identified the map position of an additional transgene, Prom2-PSS25. We repeated the genome walking experiment for the transgenic line 112; but failed. It is unknown if the 35S-PSS30 transgene formed a complex locus to interfere the polymerase chain-termination reaction (PCR) amplification of DNA during genome walking.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
We have conducted hybridization experiments to raise segregating populations. We have selected three greenhouses: (i) Agronomy Greenhouse, (ii) Horticulture Greenhouse and (iii) Plant Pathology Greenhouse to grow our materials and making the crosses
We already have developed segregating populations for the GmDS1 and PSS30 transgenes from the support of an earlier one-year ISA grant. Molecular analyses conducted through PCR revealed an F2:3 family of a cross between transgenic plants 107 and 480 carries progenies that do not segregate for either Prom3-GmDS1 or Prom2-PSS30 transgene. Therefore, this family is fixed for both transgenes and used in making the three-way crosses with each of the four transgenic plants 33, 79, 125 and 327 carrying the PSS25 and GmSAMT2 transgenes. An additional line carrying the Prom2-DS1 and 35S-Pss30 transgenes was used to make crosses with transgenic plants 33, 125 and 327. Several pods carrying putative F¬1 seeds have been set and will be harvested this October.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
We have harvested the seeds of the putative hybrids between two transgenic plants carrying GmSAMT2 and PSS25. The outcomes of these crosses will assist us in accomplishing the task of the Objective 3, which is to identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2. Once true F1s of these four crosses are identified in the greenhouse this fall, we will make crosses to the F2:3 family of a cross between transgenic plants 107 and 480 that carries both Prom3-GmDS1 and Prom2-PSS30 transgenes in homozygous condition. The F2 populations to be generated from these crosses will be investigated to identify progenies with all four transgenes in homozygous conditions.

Self-evaluation
KPIs/Performance Metrics: Self-evaluations of the progress made in Year 1 are presented below.
1. By the end of Year 1, we will complete Objectives 1.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, which is enough for reaching the goal of this proposal.

2. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.
Self-evaluation: Seeds of the two single crosses are harvested as proposed. Seeds of the three-way crosses will be harvested in October.

Timelines and Milestone Deliveries: The following are the milestones to be delivered in Year 1.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, which is enough for reaching the goal of this proposal.
2. We will have harvested the seeds of single crosses by September 30, 2019.
Self-evaluation: Seeds of the two single crosses are harvested.

Overall self-evaluation: We are in the right track of progress as proposed in the proposal.

View uploaded report PDF file

Updated March 20, 2020:
End of Project Final Report
Iowa Soybean Association
December 31, 2019

Project Title: “Stacking four plant genes to provide durable and enhanced SCN and SDS resistance in soybean”
Investigator: Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu
Iowa State University

Progress report for the period from October 1, 2018 to December 31, 2019

Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN), and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa and as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.
In this project, we proposed to evaluate the joint or combined effect of four transgenes in improving the SCN and SDS resistance of a soybean line. The four genes use distinct mechanisms to confer both SCN and SDS resistance, when overexpressed in transgenic soybean plants. Of the four genes, two are from soybean and two are from Arabidopsis thaliana. The two soybean genes, GmDS1 and GmSAMT2, encode a receptor-like protein and a salicylic acid methyl transferase, respectively. The two Arabidopsis thaliana genes, PSS30 and PSS25, encode a folate transporter and a putative transcription factor, respectively.

We hypothesize that since the resistance mechanisms encoded by PSS25, PSS30, GmDS1 and GmSAMT2 are distinct, the functions of the four genes are therefore complementary to each other and together they are expected to provide soybean with stable and robust resistances against both SCN and F. virguiforme isolates.
The outcome of this proposed research is expected to be highly significant because it will lead to development of soybean lines with robust resistance to the two most serious soybean pathogens, SCN and F. virguliforme. Therefore, this project will significantly improve soybean growers’ farm economy.

Goals and Objectives: The goal of this project is to significantly contribute towards developing durable resistance against both SCN and F. virguliforme isolates that together cause soybean yield suppression valued close to $2 billion. We propose five objectives to reach our goal in a 3-year period.

1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines.

This is a 3-year project. In Year 1, we planned to complete Objective 1, partially Objective 2 and initiate Objective 3.
We report here the progresses made from October 1, 2018 to December 31, 2019 under each of the three objectives. Objectives 4 and 5 will be conducted in Year 3.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
We have mapped seven of the eight transgenes generated from four plant genes. We however failed to map the 35S-PSS30 transgene. We repeated the genome walking experiment to map this gene; but we failed again. It is unknown if the 35S-PSS30 transgene formed a complex locus to interfere with the polymerase chain-termination reaction (PCR) in two independent genome walking experiments.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
We have conducted hybridization experiments to raise segregating populations. We have selected three greenhouses: (i) Agronomy Greenhouse, (ii) Horticulture Greenhouse and (iii) Plant Pathology Greenhouse to grow our materials and conduct hybridization experiments.

We developed segregating populations for the GmDS1 and PSS30 transgenes from the support of an earlier one-year ISA grant. Molecular analyses conducted through PCR revealed an F2:3 family of a cross between transgenic plants 107 and 480 carries progenies that do not segregate for either Prom3-GmDS1 or Prom2-PSS30 transgene. Therefore, this family is fixed for both transgenes and used in making the three-way crosses with each of the four transgenic plants 33, 79, 125 and 327 carrying either PSS25 or GmSAMT2 transgene. An additional line carrying the Prom2-DS1 and 35S-Pss30 transgenes was used to make crosses with transgenic plants 33, 125 and 327. Several pods carrying putative F1 seeds were formed. Seeds were harvested from forty-six pods obtained from the three-way crosses. We used PCR method to screen the plants grown from these seeds. We have identified 17 F1 plants, each of which harbors a combination of the three transgenes: GmDS1 and PSS30 transgenes with either GmSAMT2 or PSS25 transgene.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
We have crossed transgenic soybean plants to obtain F1s carrying both GmSAMT2 and PSS25. We have harvested the seeds of the putative hybrids between two transgenic plants, each carrying either of the two transgenes. We screened the putative F1 plants using a PCR method and identified 45 plants carrying both genes. We have started to cross the identified F1 plants carrying both PSS25 and GmSAMT2 transgenes with the plants that are homozygous for both PSS30 and GmDS1. These crosses are being made in the Agronomy greenhouse (2 rooms) and Plant Pathology greenhouse (1 room).


KPIs/Performance Metrics: Self-evaluations of the progress made in Year 1 are presented below.
1. By the end of Year 1, we will complete Objectives 1.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, enough to reach the goal of this proposal.
2. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.
Self-evaluation: Seeds of the two single crosses are harvested as proposed.
3. In Year 2, we expect to complete Objective 2; and have the Objective 3 partially completed. We will have generated F1 seeds of the double crosses to stack all four transgenes.
Self-evaluation: We have completed first three months of the Year 2 and identified the F1 seeds carrying two combinations of three transgenes as proposed.

Timelines and Milestone Deliveries: The following are the milestones to be delivered in Year 1.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, which is enough for reaching the goal of this proposal.
2. We will have harvested the seeds of single crosses by September 30, 2019.
Self-evaluation: Seeds of the two single crosses were harvested.

Overall self-evaluation: We are in the right track of progress as proposed in the proposal.

View uploaded report PDF file

Updated May 1, 2020:

Final Project Results

Updated April 8, 2020:
End of Project Final Report
Iowa Soybean Association
December 31, 2019

Project Title: “Stacking four plant genes to provide durable and enhanced SCN and SDS resistance in soybean”
Investigator: Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu
Iowa State University

Progress report for the period from October 1, 2018 to December 31, 2019

Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN), and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa and as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.
In this project, we proposed to evaluate the joint or combined effect of four transgenes in improving the SCN and SDS resistance of a soybean line. The four genes use distinct mechanisms to confer both SCN and SDS resistance, when overexpressed in transgenic soybean plants. Of the four genes, two are from soybean and two are from Arabidopsis thaliana. The two soybean genes, GmDS1 and GmSAMT2, encode a receptor-like protein and a salicylic acid methyl transferase, respectively. The two Arabidopsis thaliana genes, PSS30 and PSS25, encode a folate transporter and a putative transcription factor, respectively.

We hypothesize that since the resistance mechanisms encoded by PSS25, PSS30, GmDS1 and GmSAMT2 are distinct, the functions of the four genes are therefore complementary to each other and together they are expected to provide soybean with stable and robust resistances against both SCN and F. virguiforme isolates.
The outcome of this proposed research is expected to be highly significant because it will lead to development of soybean lines with robust resistance to the two most serious soybean pathogens, SCN and F. virguliforme. Therefore, this project will significantly improve soybean growers’ farm economy.

Goals and Objectives: The goal of this project is to significantly contribute towards developing durable resistance against both SCN and F. virguliforme isolates that together cause soybean yield suppression valued close to $2 billion. We propose five objectives to reach our goal in a 3-year period.

1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines.

This is a 3-year project. In Year 1, we planned to complete Objective 1, partially Objective 2 and initiate Objective 3.
We report here the progresses made from October 1, 2018 to December 31, 2019 under each of the three objectives. Objectives 4 and 5 will be conducted in Year 3.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
We have mapped seven of the eight transgenes generated from four plant genes. We however failed to map the 35S-PSS30 transgene. We repeated the genome walking experiment to map this gene; but we failed again. It is unknown if the 35S-PSS30 transgene formed a complex locus to interfere with the polymerase chain-termination reaction (PCR) in two independent genome walking experiments.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
We have conducted hybridization experiments to raise segregating populations. We have selected three greenhouses: (i) Agronomy Greenhouse, (ii) Horticulture Greenhouse and (iii) Plant Pathology Greenhouse to grow our materials and conduct hybridization experiments.

We developed segregating populations for the GmDS1 and PSS30 transgenes from the support of an earlier one-year ISA grant. Molecular analyses conducted through PCR revealed an F2:3 family of a cross between transgenic plants 107 and 480 carries progenies that do not segregate for either Prom3-GmDS1 or Prom2-PSS30 transgene. Therefore, this family is fixed for both transgenes and used in making the three-way crosses with each of the four transgenic plants 33, 79, 125 and 327 carrying either PSS25 or GmSAMT2 transgene. An additional line carrying the Prom2-DS1 and 35S-Pss30 transgenes was used to make crosses with transgenic plants 33, 125 and 327. Several pods carrying putative F1 seeds were formed. Seeds were harvested from forty-six pods obtained from the three-way crosses. We used PCR method to screen the plants grown from these seeds. We have identified 17 F1 plants, each of which harbors a combination of the three transgenes: GmDS1 and PSS30 transgenes with either GmSAMT2 or PSS25 transgene.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
We have crossed transgenic soybean plants to obtain F1s carrying both GmSAMT2 and PSS25. We have harvested the seeds of the putative hybrids between two transgenic plants, each carrying either of the two transgenes. We screened the putative F1 plants using a PCR method and identified 45 plants carrying both genes. We have started to cross the identified F1 plants carrying both PSS25 and GmSAMT2 transgenes with the plants that are homozygous for both PSS30 and GmDS1. These crosses are being made in the Agronomy greenhouse (2 rooms) and Plant Pathology greenhouse (1 room).


KPIs/Performance Metrics: Self-evaluations of the progress made in Year 1 are presented below.
1. By the end of Year 1, we will complete Objectives 1.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, enough to reach the goal of this proposal.
2. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.
Self-evaluation: Seeds of the two single crosses are harvested as proposed.
3. In Year 2, we expect to complete Objective 2; and have the Objective 3 partially completed. We will have generated F1 seeds of the double crosses to stack all four transgenes.
Self-evaluation: We have completed first three months of the Year 2 and identified the F1 seeds carrying two combinations of three transgenes as proposed.

Timelines and Milestone Deliveries: The following are the milestones to be delivered in Year 1.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.
Self-evaluation: We already have completed Objective 1 for all eight transgenes except one. We believe that the 35S-PSS30 transgene might have integrated in multiple copes leading to a complex transgene locus, which prevented PCR amplification in our genome walking experiment. We have however necessary physical locations of the other seven transgenes, which is enough for reaching the goal of this proposal.
2. We will have harvested the seeds of single crosses by September 30, 2019.
Self-evaluation: Seeds of the two single crosses were harvested.

Overall self-evaluation: We are in the right track of progress as proposed in the proposal.

View uploaded report PDF file

End of Project Final Report
Iowa Soybean Association
December 31, 2019

Project Title: “Stacking four plant genes to provide durable and enhanced SCN and SDS resistance in soybean”
Investigator: Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu
Iowa State University

Progress report for the period from October 1, 2018 to December 31, 2019; Year 1 of a 3-year project
Soybean is the most important legume crop that provides both protein and oil. Soybean seeds contain approximately 40% protein and 20% oil. It is an important source of animal and fish feed in addition to its major role in human nutrition. In the United States, the average annual soybean yield is valued at around $40 billion. Unfortunately, 12-15% of its yield potential is suppressed annually by pathogen attacks. Among the soybean pathogens, Heterodera glycines, commonly known as soybean cyst nematode (SCN), and Fusarium virguliforme are two of the most serious soybean pathogens. F. virguliforme causes sudden death syndrome (SDS). Soybean suffers average annual yield suppression valued close to $2 billions from the attacks of SCN and SDS. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens in Iowa and as well as in the U.S. by breeding novel SCN and SDS resistant soybean cultivars.
In our earlier transgenic research, funded by Iowa Soybean Association and USDA-NIFA-AFRI, we have identified four genes that can enhance individually against both SCN and SDS among transgenic soybean lines as compared to the nontransgenic line. Each of the four genes uses distinct genetic mechanism to confer SCN and SDS resistance. We hypothesized that since the resistance mechanisms encoded by these four genes, PSS25, PSS30, GmDS1 and GmSAMT2, are distinct, the functions of the four genes are therefore complementary to each other and together they are expected to provide soybean with stable and robust SCN and SDS resistance. We therefore propose to stack all four genes into one transgenic soybean line by hybridization or crossing of the four independent sets of transgenic lines in two steps; in Step I, single cross between two; and in Step II, the double cross is generated between outcomes of the two single crosses. We also proposed to put three genes in single lines: (i) incorporate PSS30, GmDS1 and PSS25, and (ii) PSS30, GmDS1 and GmSAMT2 into single transgenic lines. Finally, we will study the lines carrying either three (this project) or four genes (under a renewal proposal in Year 4) for possible enhancement in SCN and SDS resistances.

To facilitate the above breeding approach for stacking all four genes to test our hypothesis, we proposed to conduct following five objectives to reach our goal in the 3-year project period.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
Objective 4. Evaluate Williams 82 lines carrying either PSS25, PSS30, GmDS1 or GmSAMT2 fusion genes for resistance to F. virguliforme.
Objective 5. Evaluate Williams 82 lines carrying either PSS25, PSS30, GmDS1 or GmSAMT2 fusion genes for resistance to H. glycines.

We report here the progresses made in Year 1 spanning from October 1, 2018 to December 31, 2019 under each of the three objectives. Our expectation was to complete Objective 1, partially Objective 2 and initiate Objective 3 during this period. We have completed Objective 1, and partially completed Objective 2 and initiated Objective 3.
Details results are reported briefly under each of three objectives.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
It is important to know where the incorporated genes among the transgenic soybean genomes to facilitate their incorporation into a single line. If two genes are in the same place or locus in the genome, then they cannot be recombined into a line. We therefore investigated two independent transgenic lines for each of the four genes. We need only one for each gene to accomplish our goal. We have mapped single incorporated genes among seven of the eight transgenic soybean lines. Based on the locations of the four genes among the seven transgenic lines, we were able to identify lines that will allow incorporating all four or combinations of three genes into single lines. This objective was accomplished.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
Transgenic lines stacked with PSS30 and GmDS1 genes were available from a previous study. We have started to conducted hybridization experiments to raise two segregating populations: (i) one to combine PSS25 with PSS30 and GmDS1 and (ii) the other one GmSAMT2 with PSS30 and GmDS1. This activity was conducted in three greenhouses: (i) Agronomy Greenhouse, (ii) Horticulture Greenhouse and (iii) Plant Pathology Greenhouse to make sure that even if one greenhouse failed, we would get results.
We obtained hybrid seeds from these crosses and lines segregating for three-gene combinations are being grown in greenhouses. As proposed in the proposal, we have partially completed this objective.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
To obtain transgenic lines carrying all four genes, we hybridized transgenic lines containing GmSAMT2 and PSS25. We have harvested the seeds of the putative hybrids from this hybridization experimemt. We screened the putative F1 plants using a PCR method and identified 45 plants carrying both genes and are being used to hybridize with two lines carrying PSS30 and GmDS1 in two independent greenhouses. As proposed in the proposal, we have initiated the Objective 3.

KPIs/Performance Metrics: Self-evaluations of the progress made in Year 1 are presented below.
1. By the end of Year 1, we will complete Objectives 1.
Self-evaluation: We have completed Objective 1.
2. The Objective 2 will have partially completed. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.
Self-evaluation: Seeds of the two single crosses are harvested as proposed. Thus, the Objective 2 has been partially completed.
3. In Year 2, we expect to complete Objective 2; and have the Objective 3 partially completed. We will have generated F1 seeds of the double crosses to stack all four transgenes.
Self-evaluation: We are three months into Year 2 and made significant progress towards completing the target milestones of Year 2 and also started to make good progress for accomplishing the goal of Objective 3. We have identified the F1 seeds carrying two combinations of three transgenes as proposed and also seeds of a single cross between GmSAMT2 and PSS25.

Timelines and Milestone Deliveries: The following are the milestones to be delivered in Year 1.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018.
Self-evaluation: We have identified transgenic lines at least one for each of the four genes. suitable for hybridization and bringing all four genes into a single transgenic plant.
2. We will have harvested the seeds of single crosses by September 30, 2019.
Self-evaluation: Seeds of the two single crosses were harvested by the deadline.

Overall self-evaluation: We are in the right track of progress as proposed in the proposal.

Benefit to Soybean Farmers

In the U.S., the total soybean yield suppression from SDS and SCN together is approximate 5% of the total soybean yield. Even if we can reduce the SDS and SCN incidence by 20% through cultivation of novel SDS and SCN resistant cultivars to be generated from the outcomes of this project, one can expect to receive 1% increase in the soybean yield, which will be translated to thousands of dollars to individual soybean growers.

Performance Metrics

Project Years