Update:
Project Report
Iowa Soybean Association
October 30, 2020
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 May 1, 2020 to October 30, 2020
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; and this is the Year 3 of the project. We report here the progresses made from May 1, 2020 to October 30, 2020 under each of the five objectives.
Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
This objective was completed earlier. 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. Seven transgenes are sufficient to test the hypothesis of this project and accomplish the goal.
Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDS1 and (ii) PSS30, GmDS1 and GmSAMT2.
Earlier we reported that we have identified two lines homozygous for Prom3-GmDR1 and Prom2-PSS30 which have been used in crossing with transgenic lines carrying PSS25 and GmSAMT2 genes.

We previously reported that we planted 16 seeds of each of the 24 F2 plants (12 from the cross Prom2-PSS25 X Prom3-GmSAMT2 and 12 from the cross Prom2-PSS25 x Prom2-GmSAMT2) and screened to identify the F3 families that are homozygous for both genes, means both genes are fixed.

We have identified six putative homozygous F3 lines, four lines (84, 86, 87, 89) carrying Prom2-PSS25 and Prom3-GmSAMT2 and two lines (93, 94) with Prom2-PSS25 and Prom2-GmSAMT2. These lines are being harvested from the field and will used in phenotyping experiments.

We reported in the previous quarter that we identified 17 putative F1 plants harboring a combination of the three transgenes (GmDS1 and PSS30 transgenes with either GmSAMT2 or PSS25 transgene) and that we identified four F2 plants carrying PSS30, GmDS1 and GmSAMT2 and four F2 plants segregating. PSS30, GmDS1 and PSS25. We have harvested F3 seeds from these F2 plants containing two combinations of three transgenes. The F3 seeds will be planted in the greenhouse and homozygous plants for combination of three genes, (i) PSS30, GmDS1 and GmSAMT2, and (ii) PSS30, GmDS1 and PSS25 will be identified. F4 seeds will be harvested from the selected plants and grown for phenotyping.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
Previously, we reported hybridizing 45 F1 plants carrying both PSS25 and GmSAMT2 transgenes with two transgenic soybean plants that are homozygous for both PSS30 and GmDS1. PCR screening on the F1 plants identified three F1 plants that carry all four transgenes. We have harvested the selected plants and F2 seeds of these plants will be planted in greenhouse to raise the F3 families. The F2 plants will be screened to identify the homozygous plants for most if not all four genes. Seeds of the homozygous plants for most of the four genes will be grown in summer of 2021 in the field for phenotyping.

Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
It was proposed to conduct this objective in the summer of 2021, once we have generated stacked lines with all four genes. Since we have identified two stacked lines with two transgenes, we evaluated the lines this summer in the field to determine if there is any joint effect of the two transgenes in further enhancing SDS resistance. Results are presented below.

Phenotyping of plants carrying 2 transgenes (GmDS1 and PSS30) for SDS resistance
While waiting to develop the lines with three or all four genes, we evaluated the two lines homozygous for GmDS1 and Pss30 for responses to F. virguliforme under field condition. The field trial was conducted at the ISU Horticulture Research Station. Seeds of the two homozygous transgenic soybean lines carrying Prom2-Pss30 and Prom3-GmDS1 genes, two parents carrying single transgenes and nontransgenic Williams 82 line were planted along with F. virguliforme inoculum. The plants were scored on September 14 for SDS resistance. We observed a significant enhancement in SDS resistance between the two transgene-stacked lines carrying Prom2-Pss30 and Prom3-GmDS1 as compared to either parent carrying GmDS1 or Pss30.
Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines

It was proposed to conduct this objective in Year 3 of the project period, once we have generated stacked lines with all three or four genes, considered in this study. Since we have identified two stacked lines with two transgenes, we evaluated the lines earlier for possible further enhancement in SCN resistance and data were presented in the last semi-annual report. Results were similar to the ones for SDS resistance.

We have harvested three F1s that carry all four transgenes. Progenies of these three lines will segregate for four genes. We will grow the progenies in greenhouse; and by conducting PCR, we will identify the lines that are homozygous for most of the four transgenes. Seeds of those homozygous plants will be harvested and evaluated for SCN resistance in 2021.
KPIs/Performance Metrics: We expect to accomplish the followings by years:
1. By the end of Year 1, we will complete Objectives 1.
Self-review: We already have completed Objective 1.
2. We will have harvested the F1 seeds of the two single crosses by the end of Year 1.
Self-review: We have 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-review: We already have generated pods that are expected to carry all four transgenes. We have evaluated two stacked lines with two transgenes and showed that the lines are better than their either parent.
4. In Year 3 (Starting October 1):
a. we expect to have evaluated the F2 segregating population generated to segregate all four transgenes. We will have done phenotyping and genotyping of 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.
b. A manuscript describing the SCN resistance will be published in a peer reviewed journal by the end of Year 3.
c. 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.
Self-review: We have harvested seeds three F1s (double cross) segregating for all four genes. We will be growing the seeds in greenhouse in greenhouse in next month and identified lines carrying most of the four genes in homozygous condition. In 2021, we will evaluate the lines carrying all four genes for SDS and SCN resistances. We are in the right track.
Economic Impact/Significance
In the U.S., the total annual soybean yield suppression from SDS and SCN is approximately $1.8 billion. 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, we can expect to have significant increase in the annual soybean yield values close to $360 million in U.S. and approximately $50 million in Iowa.
Timelines and Milestone Deliveries: The following are our milestones and deliverables.
1. We will have mapped all four transgenes in individual transgenic plants by December 31, 2018. – Delivered (earlier report).
2. We will have harvested the seeds of single crosses by September 30, 2019. – Delivered (earlier report).
3. We will have harvested F2 seeds carrying three transgenes in greenhouse during the winter of 2019-2020. – Delivered (earlier report).
4. We will have harvested the F1 seeds carrying all four transgenes by October 31, 2020. We have harvested the seeds on October 29, 2020. Delivered.
5. We will have accomplished initial evaluation of homozygous plants carrying at least three transgenes for responses to both F. virguliforme and H. glycines by the end of Year 3. We have evaluated two lines carrying two transgenes and showed that stacking of two transgenes increases SDS and SCN resistance (last report) as compared to their parental lines carrying either of the two transgenes. Delivered partially ahead of the deadline.
6. It will be established if we observe complementary effects among the transgenes that use distinct genetic mechanisms to confer SDS and SCN resistance.
Preliminary data indicate that SDS and SCN resistances are enhanced further among the two stacked lines carrying two transgenes as compared to the two parental lines carrying single transgenes.
7. A peer reviewed journal article describing the responses of lines with different combinations of four transgenes (at most three genes in one genotype) to H. glycines will be published.
We expect to publish the results by the end 2021.
8. We will have identified homozygous lines for all four transgenes and made available to private seed industries by October of 2021.
We expect to get lines carrying all fours transgenes in homozygous condition by our deadline.
Overall self-evaluation: We are making progresses as proposed in the proposal.

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End of Project Final Report
Iowa Soybean Association
September 30, 2020

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

Final progress report for the period from October 1, 2019 to September 30, 2020; Year 2 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 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 will provide soybean with stable and robust SCN and SDS resistance. We therefore proposed to stack all four genes into one transgenic soybean line through hybridization of the four independent transgenic lines in two steps; in Step 1, single crosses were made between two lines; and in Step 2, the double cross was made between outcomes of the two single crosses in step 1. We also proposed to put three genes in single lines: (i) PSS30, GmDS1 and PSS25, and (ii) PSS30, GmDS1 and GmSAMT2 genes 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 enhanced SCN and SDS resistance.
To facilitate the above breeding approach for stacking all four genes into one line to test our hypothesis, we proposed to conduct the following five objectives in the 3-year project period.

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

We report here the progresses made in Year 2 starting from October 1, 2019 to September 30, 2020 under each of the objectives. Our expectation was to complete Objective 2, and partially Objective 3 during this period.
Details results are reported briefly under each of the five objectives.

Objective 1. Map the four genes, PSS25, PSS30, GmSAMT2 and GmDS1, among the transgenic soybean lines.
It is important to know where in the genome (chromosomes) the incorporated genes are localized among the transgenic soybean lines to facilitate their incorporation into a single line. If two genes are in the same place or region in the genome, then they cannot be incorporated into a single 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 the incorporated genes through soybean transformation among seven of the eight transgenic soybean lines. Based on the locations of the four genes in the genomes of seven transgenic soybean lines, we should be able to identify lines that with carry all four genes. This objective was completed in Year 1.

Objective 2. Identify Williams 82 lines that carry combinations of three genes: (i) PSS30, GmDS1 and PSS25, and (ii) PSS30, GmDS1 and GmSAMT2.
A transgenic line carrying PSS30 and GmDS1 genes was available from a previous study. In Year 1, we hybridized to transgenic lines carrying either PSS25 or GmSAMT2 to obtains F1s carrying (i) PSS30, GmDS1 and PSS25, or (ii) PSS30, GmDS1 and GmSAMT2 genes. Several hybrid plants harboring a combination of the three genes: i) PSS30, GmDS1 and PSS25, or (ii) PSS30, GmDS1 and GmSAMT2 genes were identified and grown in greenhouse. This summer, the F2 seeds of these hybrids were harvested and planted in the greenhouse for molecular analyses prior to planting in the field. Based on the data of molecular analysis of the lines, the F2 plants carrying all three genes for each of the two combinations of genes were identified and replanted in the field for seed increase this summer. We have harvested seeds of four F2 plants carrying (i) PSS30, GmDS1 and GmSAMT2 genes and four with (ii) PSS30, GmDS1 and PSS25 genes. We have harvested F3 seeds from these F2 plants. The F3 seeds will be planted in the greenhouse and homozygous plants carrying combinations of three genes: (i) PSS30, GmDS1 and GmSAMT2 genes, or (ii) PSS30, GmDS1 and PSS25 genes will be identified. In 2021, F4 seeds will be harvested from the selected plants and grown in the field for investigating the levels of SDS resistance and in the greenhouse for SCN resistance.

Objective 3. Identify Williams 82 lines that carry all four genes: PSS30, GmDS1, PSS25 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 experiment. We screened the putative F1 plants and identified 45 plants carrying both genes and were used to hybridize with the line carrying PSS30 and GmDS1genes in greenhouse. Molecular analysis of the F1 plants identified three F1 plants that carry all four genes. We have harvested the F2 seeds of the three F1 plants and planted in greenhouse. The F2 plants will be screened to identify the homozygous plants for most if not all four genes. Plants carrying all four genes will be grown in the field during the summer of 2021 and investigated for the levels of SDS resistance and in the green house for SCN resistance.

Objective 4. Evaluate Williams 82 lines carrying either ((i) PSS30, GmDS1 and PSS25, or (ii) PSS30, GmDS1 and GmSAMT2 genes for resistance against F. virguliforme.
We proposed to conduct this objective in the summer of 2021, once we generate transgenic soybean lines carrying all four genes as described under Objective 3. Since we have identified two lines with PSS30 and GmDS1 genes, we evaluated these two lines this summer in the field to determine if there is any joint effect of the two genes for further enhancing SDS resistance among transgenic soybean lines. Both lines showed enhanced SDS resistance as compared to their parental lines carrying either PSS30 or GmDS1 gene.

Objective 5. Evaluate Williams 82 lines carrying either ((i) PSS30, GmDS1 and PSS25, or (ii) PSS30, GmDS1 and GmSAMT2 genes for resistance against H. glycines.
We proposed to conduct this objective in Year 3 of the project period, when we will have generated lines with all three or four genes. Since we have identified two lines with PSS30 and GmDS1 genes, we evaluated these lines for possible further enhancement of SCN resistance. Both lines showed enhanced SCN resistance as compared to the parental lines carrying either PSS30 or GmDS1 gene.

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 by the end of Year 1.
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 genes.
Self-evaluation: We have identified three F2 plants that carry all four genes considered for this study. Objective 2 is completed.

Timelines and Milestone Deliveries: The following are the milestones to be delivered in Year 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.
Self-evaluation: We have several lines identified carrying combinations of three genes or all four genes and completed the Objective 2 as proposed. We actually evaluated the lines carrying two genes for SDS and SCN resistance, which was not proposed in the proposal.

Economic Impact/Significance
In the U.S., the total annual soybean yield suppression from SDS and SCN is approximately $1.8 billion. 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, we can expect to have significant increase in the annual soybean yield values close to $360 million in U.S. and approximately $50 million in Iowa.
Overall self-evaluation: We are in the right track of progress as proposed in the proposal.