Update:
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

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.