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 stacking all four genes into one line to test our hypothesis that resistance mechanisms encoded four genes, PSS25, PSS30, GmDS1 and GmSAMT2, are distinct and complementary to each other and together they will provide soybean with stable and robust SCN and SDS resistance, 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.

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.

Update:
Project Report
Iowa Soybean Association
March 31, 2021

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 November 1, 2020 to March 30, 2021

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 November 1, 2020 to March 30, 2021, mostly under the Objectives 2 and 3.

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 the generation of the segregating lines for various combinations of transgenes. To identify the segregating lines carrying expected combinations of transgenes, we conducted -polymerase chain termination reactions (PCR) using synthesized oligo-nucleotide primers, specific to the transgenes. The PCR assays of 335 plants identified 37 plants carrying four genes and 74 plants carrying combinations of two or three genes.

Stacked Lines Carrying Two Transgenes:
Earlier we reported that 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 Prom1-GmSAMT2, screened to identify the F3 families that are homozygous for both genes. The F2 plants were grown in the field during the summer of 2020 and PCR was conducted to identify the homozygous plants. We identified six putative homozygous F3 families, 84, 86, 87, and 89, carrying Prom2-PSS25 and Prom3-GmSAMT2, and 93, and 94, containing Prom2-PSS25 and Prom1-GmSAMT2 transgenes. As of today, we have harvested seeds from at least eight individual F3 plants from each of the F3 families 84, 86, 87, 89, 93, and 94. The F4 plants homozygous for both PSS25 and GmSAMT2 genes will be analyzed during this summer for SDS and SN resistance.

Stacked Lines Carrying Three Transgenes:
Earlier we reported development of two lines homozygous for Prom3-GmDS1 and Prom2-PSS30. These two lines were used to hybridize individually with transgenic lines carrying either Prom2-PSS25 or Prom3-GmSAMT2 gene. We also reported the identification of four F2 plants carrying three genes Prom2-PSS30, Prom3-GmDS1 and Prom3-GmSAMT2 and four other F2 plants carrying Prom2-PSS30, Prom3-GmDS1 and Prom2-PSS25. The seeds of these eight plants were sown in the greenhouse and seedlings were screened for the presence of three transgenes.

We also identified addition stacked lines carrying combinations of three genes from the segregating population generated by crossing two parental lines, each carrying two transgenes: (i) Prom3-GmDS1 and Prom2-PSS30, and (ii) Prom2-PSS25 and Prom3-SAMT2 (activities under Objective 3). From PCR assays of 201 progenies of the three lines carrying all four genes, we were able to identify plants carrying combinations of three genes in addition to lines carrying all four genes (Objective 3).

Among these F3 plants, 15 carry Prom2-PSS30, Prom3-GmDS1 and Prom2-PSS25 transgenes; 23 plants contain Prom2-PSS30, Prom3-GmDS1 and Prom3-GmSAMT2; 19 plants harbor Prom2-PSS25, Prom3-GmDS1 and Prom3-GmSAMT2; and 13 plants carry Prom2-PSS30, Prom2-PSS25 and Prom2-GmSAMT2.

Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2.
Earlier we reported three F1 plants that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2 developed by crossing two F1s. F2 seeds of these plants were planted in two greenhouses to raise the F2s. The F2 plants were evaluated by conducting PCR to identify plants carrying all four transgenes. Thirty-eight plants carrying the four transgenes were identified.

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 were presented in our last report and 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 an earlier semi-annual report. Enhanced SCN resistance was observed for the stacked lines as compared to either parent carrying GmDS1 or Pss30.

We will have seeds harvested for planting this summer from most of the plants that carry combinations of two, three and all four transgenes considered for this study. This summer, these lines will be evaluated for responses to F. virguliforme under field condition and for SCN resistance this summer in the greenhouse.

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 have identified plants that 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 generated 70 lines carrying combinations of three transgenes and 38 lines carrying all four genes. We will be evaluating most of these lines along with lines with two genes and parental lines with single transgenes for responses to F. virguliforme this summer under field condition and for SCN resistance in greenhouse. Overall, we are in the right track towards accomplishing the goal of this project.

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 (earlier report).
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 earlier; partially ahead of the deadline. Planned to conduct this summer since we have now gathered seeds of lines with stacked four, three and two transgenes.
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 by our deadline.
Overall self-evaluation: We are making progresses as expected.

Update:
Project Report
Iowa Soybean Association
October 31, 2021
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 April 1, 2021 to October 31, 2021
Overview: 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 the Year 3 of the 3-year project, end on September 30, 2021. ISA recently approved additional six months until March 31, 2022 to continue this project under a no-cost extension.
In our previous report submitted on April 30, 2021, we reported the progresses made from November 1, 2020 to March 31, 2021, mostly under the Objectives 2 and 3.
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 the generation of the segregating lines for various combinations of transgenes. To identify the segregating lines carrying expected combinations of transgenes, we conducted -polymerase chain termination reactions (PCR) using synthesized oligo-nucleotide primers, specific to the transgenes. In the previous reports, we mentioned the identification of 335 plants carrying various combinations of transgenes. We also reported that among these plants, 37 contained four transgenes and 74 carried combinations of two or three genes. We harvested seeds from these plants in the greenhouse between April and July 2021. Seeds of 40 transgenic lines, harvested before the first week of June, 2021, were planted along with control nontransgenic cultivars in the field this summer, 2021 to determine the responses of the transgenic soybean lines carrying combinations of transgenes to F. virguliforme.

Stacked Lines Carrying Two Transgenes:
Previously, we harvested seeds from at least eight individual F3 plants from each of the F3 homozygous families, viz., 84, 86, 87, and 89 carrying Prom2-PSS25 and Prom3-GmSAMT2, and 93 and 94 carrying Prom2-PSS25 x Prom1-GmSAMT2. The F4 plants homozygous for both PSS25 and GmSAMT2 genes were grown during this summer for SDS resistance, along with the three F4 lines carrying the two transgenes Prom3-DS1 and Prom2-Pss30 identified and analyzed in the summer of 2020.

Stacked Lines Carrying Three Transgenes:
Earlier we identified F3 plants carrying a combination of three transgenes. Eighteen of those lines including five carrying Prom3-GmDS1, Prom2-PSS30 and Prom2-PSS25 transgenes; 11 carrying Prom3-GmDS1, Prom2-PSS30 and Prom3-GmSAMT2; 19 plants harboring Prom2-PSS25, Prom3-GmDS1 and Prom3-GmSAMT2; and two carrying Prom2-PSS30, Prom2-PSS25 and Prom3-GmSAMT2 transgenes were planted in the field to evaluate their responses to F. virguliforme.

Objective 3. Identify Williams 82 lines that carry all four fusion transgenes: Prom2-PSS25, Prom2-PSS30, GmDS1 and GmSAMT2.
Earlier we reported three F1 plants that carry all four transgenes: PSS25, PSS30, GmDS1 and GmSAMT2 developed by crossing two F1s. F2 seeds of these plants were planted in two greenhouses to raise the F2s. The F2 plants were evaluated by conducting PCR to identify plants carrying all four transgenes. Thirty-eight plants carrying all four transgenes were identified. From April to July 2021, seeds from these lines were harvested. Seeds of eight lines were randomly planted in three blocks of the field. At least 30 seeds of each line were planted in individual rows of each block. For the rest five lines, the number of seeds were low and were only grown with other eight lines in the fourth block just for seed increase.
To identify the individual plants carrying all four transgenes in homozygous condition, leaf samples were collected from randomly selected over 800 single plants. The pods of these selected plants have been harvested. The seeds from these lines will be shortly threshed. DNA is being extracted from these 800 samples and will be used to conduct molecular analysis for identifying the homozygous plants containing all four fusion transgenes. In four-gene segregation with independent assortment (genes are not linked, segregate independently), screening of a large segregating population (256 plants) is required for identifying a single individual with all four genes in homozygous condition. From screening 800 F2 plants, we expect to obtain only three homozygous F2 plants. The homozygous individual plant for all four genes will be investigated this winter for responses to F. virguliforme and SCN in growth chambers and greenhouse conditions, respectively.
Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
In our original proposal, we planned to generate lines containing only three fusion genes. We expanded the scope of the project and generated lines that carry all four fusion transgenes. Lines carrying two or three fusion genes along with population segregating all four fusion genes were planted this summer in the field to evaluate their responses against F. virguliforme infection. Unfortunately, acceptable SDS symptoms were not developed this summer in our experimental plot.

This year, the soybean growing season was very dry. We continuously irrigated the plot from the 4th week of August when the soybean lines just reached the R3 stage (starting to form pods). We irrigated twice a day, ¼ inch water in the mid-day and 1/4th inch water at dusk. The soil was muddy and moist with high humidity. Despite our effort of maintaining the ideal condition for SDS foliar disease development, the disease appeared only in a limited number of plants of some of the rows. The scored data are not meaningful to report. This is the first time we failed to observe any meaningful SDS foliar disease development since 2013, the year we started to conduct field trial for SDS resistance screening.

Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDS1 and GmSAMT2 fusion genes for resistance to H. glycines

We have harvested seeds from most of the plants that carry combinations of two, three and all four transgenes considered for this study. The lines that are homozygous for the fusion gene combinations will be identified and evaluated for responses to F. virguliforme and SCN infection under growth chamber and greenhouse conditions, respectively, by March 31, 2022, the new extended last date for this project.

Key Performance Indicators/Performance Metrics: We expect to accomplish in Year 3 of the project the following.
1. The F1 and F2 populations will be evaluated and lines with either two, three or four transgenes will be generated.
Self-evaluation: We have identified soybean genotypes carrying either two, three or four fusion gene combinations. Seeds of these plants have been harvested.

2. Levels of SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known.
Self-evaluation: Responses of lines carrying two fusion genes to F. virguliforme and SCN have been reported earlier. We failed to observe the foliar SDS symptoms among the transgenic lines this summer. We however plan to identify the homozygous lines for 2, 3 and 4 gene combinations and then evaluated them for their responses against F. virguliforme and SCN by March 31, 2022 (under the no-cost extension).

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 for Deliverables: Timelines and milestones for deliverables are:
1. We will deliver the seeds of genotypes carrying all four transgenes by May, 2021.
Self-evaluation: Seeds of line genotypes carrying all four transgenes or fusion genes were obtained and grown in the field this summer. Seeds of these lines have been harvested.
2. SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known by September, 2021.
Self-evaluation: We have shown earlier that two genes further enhance SDS and SCN resistance due to complementary effects between the two fusion or transgenes. We failed to get the foliar SDS symptoms this year despite we irrigated routinely during the reproductive phase starting R3 stage. The season was very dry and we observe only sporadic foliar symptoms among the transgenic lines.
We expect to identify homozygous lines for 3 and 4 gene combinations and evaluate the lines carrying 2, 3 and 4 genes for SDS and SCN resistance by March 31, 2022.

View uploaded report

Update:

Update:
Final Report
Iowa Soybean Association
May 2, 2022

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

Overview: 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 SCN and SDS diseases. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens, SCN and F. virguliforme, 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 transgenes considered were identified in previous transgenic studies conducted in our laboratory with the support from Iowa Soybean Association, Consortium of Plant Biotechnology Research, and United States Department of Agriculture - National Institute of Food and Agriculture (USDA-NIFA). The four genes use distinct mechanisms to confer both SCN and SDS resistance, when overexpressed in transgenic soybean plants (e.g., Ngaki et al. 2021: Plant Biotechnology Journal 19: 502–516 https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13479; Kambakam et al. 2021: Plant Journal 107:1432-1446 https://onlinelibrary.wiley.com/doi/10.1111/tpj.15392). Of the four genes, two are from soybean and two are from Arabidopsis thaliana. The two soybean genes, GmDR1 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, GmDR1 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 four transgenes selected for this study govern novel mechanisms for SCN and SDS resistances. Therefore, outcome of this proposed research is expected to be highly significant because the SCN and SDS resistances governed by these genes will complement the resistance mechanisms currently available in soybean cultivars. The project will therefore lead to development of soybean lines with robust resistance against two most serious soybean pathogens, SCN and F. virguliforme, that together suppress soybean yield valued at close to $2 billions. Furthermore, GmDR1 also provides tolerances to soybean aphids and spider mites, which are serious pests of soybean (Ngaki et al. 2021: Plant Biotechnology Journal 19: 502–516 https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13479). Therefore, this project is expected to significantly improve the soybean growers’ farm economy.

In this project, our goal was to stack all four transgenes into single soybean plants and then determine if the SCN and SDS resistances of the resultant soybean lines with all four transgenes are further improved.

We proposed to conduct following five objectives to accomplish the goal of this 3-year project.

1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDR1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDR1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to H. glycines.

Here we report the progress made under each project during the entire project period starting October 1, 2019.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDR1, among the transgenic soybean lines.

In this objective, we have successfully mapped all four transgenes to be stacked into single plants in Year 1 of the project. Results confirmed that all four transgenes are unlinked and stacking them into a single plant is feasible.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2.

In 2020, we reported the generation of the segregating lines for various combinations of transgenes. To identify the segregating lines carrying expected combinations of transgenes, we conducted -polymerase chain termination reactions (PCR) using synthesized oligo-nucleotide primers, specific to the transgenes. In the previous reports, we mentioned the identification of 335 plants carrying various combinations of transgenes. We also reported that among these plants, 37 contained four transgenes and 74 carried combinations of two or three genes. We harvested seeds from these plants in the greenhouse between April and July 2021. Seeds of 40 transgenic lines, harvested before the first week of June, 2021, were planted along with control nontransgenic cultivars in the field this summer, 2021 to determine the responses of the transgenic soybean lines carrying combinations of transgenes to F. virguliforme.

Stacked Lines Carrying Two Transgenes:
We harvested seeds from at least eight individual F3 plants from each of the F3 homozygous families, viz., 84, 86, 87, and 89 carrying Prom2-PSS25 and Prom3-GmSAMT2, and 93 and 94 carrying Prom2-PSS25 x Prom1-GmSAMT2. The F4 plants homozygous for both PSS25 and GmSAMT2 genes were grown during this summer for SDS resistance, along with the three F4 lines carrying the two transgenes Prom3-DS1 and Prom2-Pss30 identified and analyzed in the summer of 2020.

Stacked Lines Carrying Three Transgenes:
In 2020, we identified F3 plants carrying a combination of three transgenes. Eighteen of those lines including five carrying Prom3-GmDR1, Prom2-PSS30 and Prom2-PSS25 transgenes; 11 carrying Prom3-GmDR1, Prom2-PSS30 and Prom3-GmSAMT2; 19 plants harboring Prom2-PSS25, Prom3-GmDR1 and Prom3-GmSAMT2; and two carrying Prom2-PSS30, Prom2-PSS25 and Prom3-GmSAMT2 transgenes were planted in the field to evaluate their responses to F. virguliforme. Thirteen F3 plants carrying a combination of three transgenes from Prom2-PSS30, Prom3-GmDR1, Prom3-SAMT2, and Prom2-PSS25 were also grown in the field for SDS infection and/or seed increase.

Objective 3. Identify Williams 82 lines that carry all four fusion transgenes: Prom2-PSS25, Prom2-PSS30, GmDR1 and GmSAMT2.

In our original proposal, we proposed to stack two combinations of four genes; viz., (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2. In Year 2 of the project, we added this objective to stack all four transgenes in single plants. To obtain all four transgenes in one transgenic plant, we generated two F1 plants carrying either (i) Prom3-DS1 and Prom2-Pss30 or (ii) Prom2-PSS25 and Prom3-SAMT2 genes.

In 2020, we reported three F1 plants that carry all four transgenes: PSS25, PSS30, GmDR1 and GmSAMT2 developed by crossing the two F1s. F2 seeds of these plants were planted in two greenhouses to raise the F2s. The F2 plants were evaluated by conducting PCR to identify plants carrying all four transgenes. Thirty-eight plants carrying all four transgenes were identified. From April to July 2021, seeds from these lines were harvested. Seeds of eight lines were randomly planted in three blocks of the experimental plot. At least 30 seeds of each line were planted in individual rows of each block. For the rest five lines, the number of seeds were low and were only grown with other eight lines in the fourth block just for seed increase.

To identify the individual plants carrying all four transgenes in homozygous condition, leaf samples were collected from randomly selected over 800 single plants. The pods of these selected plants were then harvested at maturity.
In November and December of 2021, the seeds of the harvested pods were threshed and stored in cold room in individual seed envelopes.

In January and February, DNA was prepared from 700 of the 800 leaf samples harvested earlier in the summer of 2021. We conducted molecular analysis for identifying the plants containing all four fusion transgenes. In four-gene segregation with independent assortment (genes are not linked, segregate independently), screening of a large segregating population of 256 individuals is required for identifying a single individual with all four genes in homozygous condition with a low probability. From screening 800 F2 plants, we expect to obtain only three homozygous F2 plants with an approximately 95% certainty of finding a single homozygous plant for all four transgenes. In February, we analyzed DNA of the first batch of 96 plants individually for each of the four transgenes, Prom2-PSS30, Prom3-GmDR1, Prom3-SAMT2, and Prom2-PSS25. Nearly half (40 plants) of the 96 plants showed PCR amplification for all four transgenes. Of the 40 plants positive for all four transgenes, 32 showing strong PCR amplification for all four transgenes were selected for further investigation.

Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to F. virguliforme.

In our original proposal, we planned to generate lines containing only three fusion genes. We expanded the scope of the project and generated lines that carry all four fusion transgenes. Lines carrying two or three fusion genes along with population segregating all four fusion genes were planted the summer of 2021 in the field to evaluate their responses against F. virguliforme infection. Unfortunately, acceptable SDS symptoms were not developed in the experimental plot.

This year, the soybean growing season was very dry. We continuously irrigated the plot from the 4th week of August when the soybean lines just reached the R3 stage (starting to form pods). We irrigated twice a day, ¼ inch water in the mid-day and 1/4th inch water at dusk. The soil was muddy and moist with high humidity. Despite our effort of maintaining the ideal condition for SDS foliar disease development, the disease appeared only in a limited number of plants of some of the rows. The scored data are not meaningful to report. This is the first time we failed to observe any meaningful SDS foliar disease development since 2013, the year we started to conduct field trial for SDS resistance screening.

Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to H. glycines

We have evaluated transgenic soybean lines carrying both PSS30 and GmDR1 for responses to H. glycines infection. The lines carrying both genes were more tolerant to the pathogen than lines carrying either of the transgenes.

Of the selected 32 plants with strong PCR amplifications for all four transgenes in Objective 3, 19 were selected randomly for responses to SCN infection. For each genotype, six seedlings were individually infected with SCN. Thirty days following sowing soybean seeds in soil containing SCN cysts, the numbers of the newly developed cysts on soybean roots of individual plants were counted under a microscope by Madeline Thomson, an undergraduate student. A total of 79 plants representing the selected 19 random genotypes, containing all four transgenes along with four plants of A95-684043 (A95), an SCN resistant control line, and six plants of transgenes recipient nontransgenic Williams 82 line were investigated for responses to SCN infection.

The important take-home message from this study is that we were able to identify 17 individual segregants from nine lines carrying all four transgenes that are highly SCN resistant. We identified five plants that exhibited two or three progenies with FI below 20. Progenies of these five plants will be grown to develop the next generation for identifying lines homozygous for all four genes. These lines will also be tested for possible SDS resistance in the field this summer; and in 2023 through 2025, if we can secure a grant.

Key Performance Indicators/Performance Metrics: We expect to accomplish in Year 3 of the project the following.

1. The F1 and F2 populations will be evaluated and lines with either two, three or four transgenes will be generated.
Self-evaluation: We have identified soybean genotypes carrying either two, three or four fusion gene combinations. Seeds of these plants were harvested.

2. Levels of SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known.
Self-evaluation: Responses of lines carrying two fusion genes to F. virguliforme and SCN have been reported earlier. We failed to observe the foliar SDS symptoms among the transgenic lines in the summer of 2021. We however plan to identify the homozygous lines for 2, 3 and 4 gene combinations and then evaluated them for their responses against F. virguliforme and SCN under the support of a possible support of renewal 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.



Timelines and Milestone for Deliverables: Timelines and milestones for deliverables are:

1. We will deliver the seeds of genotypes carrying all four transgenes by May, 2021.
Self-evaluation: Seeds of thirteen genotypes carrying all four transgenes or fusion genes were generated in greenhouse and planted in the field in the summer of 2021.

2. SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known by September, 2021.
Self-evaluation: We have shown earlier that two genes further enhance SDS and SCN resistance due to complementary effects between the two transgenes. We failed to get the foliar SDS symptoms in 2021 despite we irrigated routinely during the reproductive phase starting R3 stage. The season was very dry and we observed only sporadic foliar symptoms among the transgenic lines. We have shown that 17 progenies of nine lines carrying all four transgenes exhibited enhanced SCN resistance to level, which is comparable to that of an SCN resistant soybean cultivar. Progenies of three of the nine lines showed very few SCN cysts.

View uploaded report

Final Project Results
Iowa Soybean Association
May 2, 2022

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

Final Report of a project that was started in 2019.

Overview: 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 SCN and SDS diseases. Our long-term goal is to alleviate soybean yield suppression from these two most serious pathogens 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 transgenes considered were identified in previous transgenic studies conducted in our laboratory with the support from Iowa Soybean Association, Consortium of Plant Biotechnology Research, and United States Department of Agriculture - National Institute of Food and Agriculture (USDA-NIFA). The four genes use distinct mechanisms to confer both SCN and SDS resistance, when overexpressed in transgenic soybean plants (e.g., Ngaki et al. 2021: Plant Biotechnology Journal 19: 502–516 https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13479; Kambakam et al. 2021: Plant Journal 107:1432-1446 https://onlinelibrary.wiley.com/doi/10.1111/tpj.15392). Of the four genes, two are from soybean and two are from Arabidopsis thaliana. The two soybean genes, GmDR1 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, GmDR1 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. virguliforme.

The four transgenes selected for this study govern novel mechanisms for SCN and SDS resistances. Therefore, outcome of this proposed research is expected to be highly significant because the SCN and SDS resistances governed by these genes will complement the resistance mechanisms currently available in soybean cultivars. The project will therefore lead to development of soybean lines with robust resistance against two most serious soybean pathogens, SCN and F. virguliforme, that together suppress soybean yield valued at close to $2 billions. Furthermore, GmDR1 also provides tolerances to soybean aphids and spider mites, which are serious pests of soybean (Ngaki et al. 2021: Plant Biotechnology Journal 19: 502–516 https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13479). Therefore, this project is expected to significantly improve the soybean growers’ farm economy.

In this project, our goal was to stack all four transgenes into single soybean plants and then determine if the SCN and SDS resistances of the resultant soybean lines with all four transgenes are improved further.

We proposed to conduct following five objectives to accomplish the goal of this 3-year project.

1. Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDR1, among the transgenic soybean lines.
2. Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2.
3. Objective 3. Identify Williams 82 lines that carry all four transgenes: PSS25, PSS30, GmDR1 and GmSAMT2.
4. Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to F. virguliforme.
5. Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to H. glycines.

The accomplishments in this project are described under each of the five objectives.

Objective 1. Map the four fusion genes, PSS25, PSS30, GmSAMT2 and GmDR1, among the transgenic soybean lines.

In this objective, we have successfully mapped all four transgenes to be stacked into single plants in Year 1 of the project. Results confirmed that all four transgenes are unlinked and stacking them into a single plant is feasible.

Objective 2. Identify Williams 82 lines that carry combinations of three fusion genes: (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2.

We proposed to stack two combinations of four genes; viz., (i) PSS25, PSS30 and GmDR1 and (ii) PSS30, GmDR1 and GmSAMT2. First, we generated a line carrying both GmDR1 and PSS30. We then crossed this line individually to two transgenic lines carrying either PSS25 or GmSAMT2 transgene. We generated five lines carrying the PSS25, PSS30 and GmDR1 gene combination and 11 lines carrying the combination of PSS30, GmDR1 and GmSAMT2 genes.

Objective 3. Identify Williams 82 lines that carry all four fusion transgenes: Prom2-PSS25, Prom2-PSS30, Prom3-GmDR1 and Prom3-GmSAMT2.

In Year 2 of the project, we added this objective to stack all four transgenes in single plants. To obtain all four transgenes in one transgenic plant, we generated two F1 plants carrying either (i) Prom3-DR1 and Prom2-Pss30 or (ii) Prom2-PSS25 and Prom3-SAMT2 genes. By crossing these two F1 plants, we were able to get a segregating population that segregated for all four transgenes. We conducted polymerase chain termination reaction (PCR) to identify the lines that carry all four transgenes. Thirty-eight plants carrying all four transgenes were identified in greenhouse. Seeds of eight of these lines were randomly planted in three blocks in the field during the summer of 2021. At least 30 seeds of each line were planted along with F. virguliforme inoculum in individual rows of each of the three blocks during the summer of 2021. The number of seeds of five additional lines were low and were grown with seeds of the eight lines in the fourth block just for seed increase.

To identify the individual plants carrying all four transgenes, leaf samples were collected from randomly selected over 800 single plants from the field grown 2021 crop. The pods of these plants were harvested and threshed individually in 2021. DNA of the harvested 700 of 800 leaf samples were prepared. We conducted PCR of individual plants for all four transgenes. In four-gene segregation with independent assortment (as the four genes are not linked and they segregate independently), screening of a large segregating population (256 plants) is required to identify a single individual plant with all four genes in homozygous condition. From screening 800 F2 plants, we expect to obtain on average three homozygous F2 plants. This means that from screening all 800 plants, one homozygous plant may be identified with a certainty of 95%.

We conducted PCR of the first group DNA samples of 96 plants for all four transgenes. We observed that only 40 plants contain all four transgenes, PSS25, PSS30, GmDR1 and GmSAMT2. Of the 40 plants, 32 exhibited strong amplification for all four transgenes and were considered for further studies.

Objective 4. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to F. virguliforme.

We have evaluated transgenic soybean lines carrying both PSS30 and GmDR1 for responses to F. virguliforme infection. The lines carrying both genes were more tolerant to the pathogen than lines carrying either of the transgenes. This experiment was conducted in growth chamber.

In our original proposal, we planned to generate lines containing only three fusion genes. We expanded the scope of the project and generated lines that carry all four fusion transgenes. Lines carrying two or three fusion genes along with population segregating all four fusion genes were planted this summer in the field to evaluate their responses against F. virguliforme infection. Unfortunately, acceptable SDS symptoms were not developed this summer in our experimental plot. In the summer of 2021, the soybean growing season was very dry. We continuously irrigated the plot from the 4th week of August when the soybean lines just reached the R3 stage (starting to form pods). We irrigated twice a day, ¼ inch water in the mid-day and 1/4th inch water at dusk. The soil was muddy and moist with high humidity. Despite our effort of maintaining the ideal condition for SDS foliar disease development, the disease appeared only in a limited number of plants of some of the rows. The scored data are not meaningful to report. This is the first time we failed to observe any meaningful SDS foliar disease development since 2013, the year we started to conduct field trial for SDS resistance screening.

Objective 5. Evaluate Williams 82 lines carrying PSS25, PSS30, GmDR1 and GmSAMT2 fusion genes for resistance to H. glycines.

We have evaluated transgenic soybean lines carrying both PSS30 and GmDR1 for responses to H. glycines infection. The lines carrying both genes were more tolerant to the pathogen than lines carrying either of the transgenes.

Of the selected 32 plants with strong PCR amplifications for all four transgenes in Objective 3, 19 were selected for responses to SCN infection. For each genotype, six seedlings were individually infected with SCN. Thirty days following sowing soybean seeds in soil containing SCN cysts, the numbers of the newly developed cysts on soybean roots of individual plants were counted under a microscope by Ms. Madeline Thomson, an undergraduate student. A total of 79 plants representing the selected 19 random genotypes, four plants of A95-684043 (A95), an SCN resistant control line, and six plants of transgenes recipient nontransgenic Williams 82 line were investigated for responses to SCN infection. The responses of the 79 progenies of the 19 plants containing all four transgenes to SCN was determined 30 days following sowing of soybean seeds in SCN HG Type 2.5.7 infested soil, and female indices (FI) (FI defined as the percentages of cyst numbers of a plant over the average cyst number of six Williams 82 nontransgenic plants) were calculated for each plant. We observed that 17 progenies from nine of the 19 selected plants exhibited female indexes = 20%, which are comparable to that in the SCN-resistant A95-684043 control line and could be suitable for protecting soybean from SCN. The plants were heterozygous and segregating for all four transgenes. This experiment is being repeated to confirm the impact of the four genes in soybean’s immunity against SCN. The progenies of these nine plants will be grown in the field this summer to identify the homozygous lines for all four transgenes and also for responses to F. virguliforme.

The important take-home message from this study is that we were able to identify 17 individual segregants from nine lines carrying all four transgenes that are highly SCN resistant. We identified five plants (Plant # 29, 63, 66, 80, and 94) that exhibited two or three progenies with FI < 20%. Progenies of these five plants will be grown to develop the next generation for identifying lines homozygous for all four genes. These lines will also be tested for possible SDS resistance in the field this summer; and will be further investigated for SCN and SDS resistance in 2023 through 2025, if we can secure a grant. The top 17 transgenic plants exhibited female indices of = 20%, which are comparable to the SCN-resistant A95-684043 line. Plants 63-5, 80-3, and 80-4 have the smallest female indices of one or less than one percent. These data indicate that the four transgenes together could enhance SCN resistance in soybean.

Key Performance Indicators/Performance Metrics: We expect to accomplish in Year 3 of the project the following.

1. The F1 and F2 populations will be evaluated and lines with either two, three or four transgenes will be generated.
Self-evaluation: We have identified soybean genotypes carrying either two, three or four fusion gene combinations. Seeds of these plants have been harvested.

2. Levels of SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known.
Self-evaluation: Responses of lines carrying two fusion genes to F. virguliforme and SCN have been reported earlier. We failed to observe the foliar SDS symptoms among the transgenic lines this summer. We however plan to identify the homozygous lines for 2, 3 and 4 gene combinations and then evaluated them for their responses against F. virguliforme and SCN under the support of a possible support of renewal 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.

Timelines and Milestone for Deliverables: Timelines and milestones for deliverables are:

1. We will deliver the seeds of genotypes carrying all four transgenes by May, 2021.
Self-evaluation: Seeds of thirteen genotypes carrying all four transgenes or fusion genes were generated in greenhouse and planted in the field in the summer of 2021.

2. SCN and SDS resistances of lines carrying 2, 3 or 4 fusion gene combinations will be known by September, 2021.
Self-evaluation: We have shown earlier that two genes further enhance SDS and SCN resistance due to complementary effects between the two transgenes. We failed to get the foliar SDS symptoms in 2021 despite we irrigated routinely during the reproductive phase starting R3 stage. The season was very dry and we observed only sporadic foliar symptoms among the transgenic lines. We have shown that 17 progenies of nine lines carrying all four transgenes exhibited enhanced SCN resistance to level, which is comparable to that of an SCN resistant soybean cultivar. Progenies of three of the nine lines showed very few SCN cysts.

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.