INVESTIGATING THE FREQUENCY OF SORGHUM HALEPENSE X S. BICOLOR HYBRIDIZATION AND PROGENY CHARACTERISTICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1027087
• Grant No.: 2021-33522-35357
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2021-04287
• Project Start Date: Sep 1, 2021
• Project End Date: Aug 31, 2025
• Grant Year: 2021
• Program Code: [HX]- Biotechnology Risk Assessment

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Soil & Crop Sciences

Non Technical Summary
This 'standard' research project best aligns with the program area 'characteristics, rates, and mechanisms of gene transfer to wild relatives'. The overarching goal of this project is to develop a thorough understanding of the frequency of gene flow and hybrid fitness between crop sorghum and johnsongrass when johnsongrass serves as the female parent, to provide necessary scientific data for federal regulatory agencies to assist with decision making and also help identify avenues for mitigating gene flow. The specific objectives are: 1. Determine the frequency of 2n gamete production in S. bicolor pollen as influenced by genotype (both pollinators and hybrids); 2. Determine the frequency of S. halepense x S. bicolor hybridization as influenced by distance from pollen source; 3. Elucidate backcrossing potential of F1 progeny with either parents (S. halepense and S. bicolor); 4. Understand hybrid characteristics and fitness of F1 and BC1F1 progeny of S. halepense x S. bicolor: a) with fitness-neutral herbicide-resistant traits under field conditions, b) with transgenic compositional traits under greenhouse conditions; and 5. Understand hybrid characteristics and fitness of F1 and BC1F1 progeny of shattercane (S. bicolor ssp. drummondii) x transgenic S. bicolor under greenhouse conditions. Overall, findings will help improve the existing risk assessment frameworks by providing valuable information regarding the risk of gene flow from sorghum to johnsongrass (both non-transgenic herbicide resistance and transgenic lignin modification traits), provide insights on the distance of pollen-mediated gene flow, and illustrate fitness characteristics of the F1 and BC1F1 progeny obtained with johnsongrass and shattercane.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
213	2300	1140	30%
216	2300	1070	35%
201	2499	1060	35%

Knowledge Area
216 - Integrated Pest Management Systems; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 213 - Weeds Affecting Plants;

Subject Of Investigation
2300 - Weeds; 2499 - Plant research, general;

Field Of Science
1060 - Biology (whole systems); 1070 - Ecology; 1140 - Weed science;

Keywords

crop-wild hybridization

gene flow

johnsongrass

risk assessment

shattercane

stewardship

Goals / Objectives
The overarching goal of this project is to develop a thorough understanding of the frequency of gene flow and hybrid fitness between crop sorghum and johnsongrass when johnsongrass serves as the female parent, to provide necessary scientific data for federal regulatory agencies for decision making and also help identify avenues for mitigating gene flow.Supporting objectives:Determine the frequency of 2n gamete production in S. bicolor pollen as influenced by sorghum genotypeDetermine the frequency of S. halepense x S. bicolor hybridization as influenced by distance from pollen sourceElucidate backcrossing potential of F1 progeny with either parents (S. halepense and S. bicolor)Understand hybrid characteristics of F1 and BC1F1 progeny of S. halepense x S. bicolora) With fitness-neutral herbicide-resistant traits under field conditionsb) With a transgenic compositional trait under greenhouse conditions 5. Understand hybrid characteristics of F1 and BC1F1 progeny of shattercane (S. bicolor ssp. drummondii) x transgenic S. bicolor under greenhouse conditions

Project Methods
Objective 1: Determine the frequency of 2n gamete production in S. bicolor pollen as influenced by sorghum genotypeWe will use 10 to 15 different sorghum hybrid genotypes with herbicide-resistance traits as the male parents and a naturally occurring johnsongrass susceptible to ALS- and ACCase-inhibiting herbicides as the female parent. The experiment will be arranged in a randomized complete block design with 4 replications (plot size: 8 rows x 15 m), conducted over two growing seasons (2022, 2023). To prevent any outside pollen, each plot will be established as a small isolation block by planting photoperiod-sensitive biomass sorghum that does not flower under our environment. The johnsongrass plants within one half of the isolation block (i.e. plot) will be sterilized with the use of the chemical sterilant trifluoromethanesulfonamide (TFMSA), which was already standardized by our team for Sorghum spp. (Hodnett and Rooney 2018) to facilitate crossing under male sterile conditions. Flowering synchrony is typically achieved due to the tillering and indeterminate habit of johnsongrass. Upon seed maturity, johnsongrass panicles will be harvested before shattering begins. The seedlings will be sprayed with the respective herbicide and survivors (i.e. hybrids) will be selected and subjected to ploidy analysis using flow cytometry. Further, chromosome number of the hybrids will be determined following a standard chromosome spreading technique.Objective 2: Determine the frequency of S. halepense x S. bicolor hybridization as influenced by distance from pollen sourceA field study will be conducted at Texas A&M University field research farm near College Station, TX in 2023 and 2024 field seasons, using a Nelder-wheel design. For this study, two herbicide-resistant grain sorghum lines with low and high frequency of 2n male gamete production will be utilized as the pollen source (male). Two separate Nelder-wheel experiments will be established to accommodate the two sorghum pollen donor lines, which will be planted at the center of the wheel (25 m diameter). Known herbicide susceptible johnsongrass (for both ALS- and ACCase-inhibitors) will be established at 9 different distances from the pollen source (0.5, 1, 5, 10, 15, 25, 50, 75, 100 m) at 8 different directions (N, NE, E, SE, S, SW, W, NW). The study will be established in a site that already has good and uniform infestation of naturally occurring johnsongrass. Johnsongrass panicles that attained flowering synchrony with sorghum will be tagged and harvested at maturity. A weather station will be installed at the experimental site to record weather parameters. The progeny will be treated at the 2 to 3 leaf stage using a lethal dose of the appropriate herbicide. At 28 days after herbicide application, total number of seedlings survived in each flat will be counted. Gene flow frequency at each direction and distance is calculated as the total number of survivors out of the total seedlings treated with the herbicide times 100.Objective 3: Elucidate backcrossing potential of F1 progeny with either parents (S. halepense and S. bicolor)To understand backcrossing potential of the F1 progeny with either of the parents (sorghum and johnsongrass), four F1 progeny originating from crosses with a range of 2n male gamete production across the 10-15 sorghum genotypes will be used. A total of 25 F1 plants will be established in an F1 hybrid nursery (2 m x 2 m spacing). The four sorghum lines with a range of 2n gamete production potential identified above (sorghum parents) and the wild johnsongrass parent will be established in the nursery area to provide sufficient pollen for conducing backcrosses. To allow for flowering synchrony, the parents will be planted thrice in a staggered fashion at weekly intervals. Upon flowering of the F1 progeny and the parents, crosses will be carried out manually following standard procedures. Upon seed maturity, the panicles will be harvested, threshed, cleaned and the number of seed produced in each panicle will be counted. The seed will then be stored under 4C until used further for progeny characterization. The backcrossing experiment will be conducted over two field seasons (2023, 2024).Objective 4: Understand hybrid characteristics of F1 and BC1F1 progeny of S. halepense x S. bicolor A host of phenotypic characteristics will be measured with a) fitness-neutral herbicide-resistant traits under field conditions and b) a transgenic compositional trait under greenhouse conditions. A total of twenty-three morphological characteristics will be evaluated, including plant height, stem diameter, internode length, number of tillers, leaf width, days to flowering, length and width of the flag leaf, number of panicles/plant, length of panicle, number of seeds per panicle (% seed set), seed color, size and shape, 100-seed weight, seed shattering, seed dormancy, aboveground biomass, and below-ground characteristics such as rhizome production, length, diameter, internode length and vigor of the rhizomes. All morphological observations will be conducted after initiation of flowering. Individual lines of both parents used for crossing will also be included in the evaluations for comparison.Objective 5: Understand hybrid characteristics of F1 and BC1F1 progeny of shattercane (S. bicolor ssp. drummondii) x transgenic S. bicolor under greenhouse conditionsGiven regulatory approval (to be requested), five shattercane lines sourced across the major US sorghum production regions will be grown at the Texas A&M greenhouse and used as female parents. The transgenic lignin modification trait in the Tx430 sorghum background will be used as the pollinator. Plants will be established in 9.5-liter pots and at the booting stage, the shattercane plants will be sterilized with the chemical sterilant TFMSA to allow for hybridization. All crosses will be carried out under regulated greenhouse conditions. Each cross will be conducted on 10 biological replicates. Likewise, backcrosses will also be carried out on the F1 progenies with unique parental combinations as described earlier, again with 10 biological replicates. The panicles will be bagged after the crosses were made. Upon maturity, seed will be harvested, cleaned and counted. Hybrids among the F1 progenies will be determined based on the site-specific markers associated with the transgenic trait. Crosses will also be made between the six shattercane lines and the non-transgenic Tx430 sorghum line for use as a standard. The procedure for progeny characterization will be similar to that of objective 4.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The key target audiences reached during this reporting period include sorghum industry stakeholders (United Sorghum Check-off Program, National Sorghum Producers), agribusiness industry (Corteva, Advanta, S&W), federal regulatory agencies (EPA, APHIS), and plant science research community. Our outreach activities also reached crop consultants and sorghum producers. Changes/Problems:The 2024 spring was an extremely wet season at College Station, TX with several weeks of continuous rainfall. Nevertheless, we were able to successfully complete the field experiments without much negative impact. No major changes are necessary as of yet. What opportunities for training and professional development has the project provided?The project has provided training on ploidy determination, cytology, and plant phenotypic characterization for a graduate research assistant. This project also provided professional development to two research scientists, one research assistant, and one postdoctoral research associate who were trained in plant biology and ecology research. Two undergraduate students were trained in field crossing, herbicide resistance assessment, plant phenotyping, and flow cytometry analysis. How have the results been disseminated to communities of interest?Results were communicated to the scientific community through participation at various professional meetings, including the Weed Science Society of America, Southern Weed Science Society, and Crop Science Society of America. Additionally, a presentation was made at the Texas Plant Protection Association annual meeting held at Bryan, TX. Informal discussions were carried out with the United Sorghum Checkoff Program and the sorghum seed industry. What do you plan to do during the next reporting period to accomplish the goals?The field experiments have already been concluded by the summer of 2024. In 2025, the remaining greenhouse screenings will be completed, and then proceed with data analysis and manuscript preparation.

Impacts
What was accomplished under these goals? The Nelder wheel gene flow field experiment was repeated during the 2024 field season to understand the nature and characteristics of pollen movement from sorghum (as the male parent) to johnsongrass (female parent). In the Nelder-wheel experiment, a known quizalofop-resistant grain sorghum line was planted at the center of the wheel, and the distance of pollen-mediated gene flow was determined on a natural population of johnsongrass present at the experimental site at various distances in 8 different directions. At maturity, the seeds were harvested, threshed and cleaned. They are currently being screened in the greenhouse for the presence of the quizalofop-resistance trait.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Pedireddi U, Subramanian N, Hodnett G, Klein P, Bagavathiannan M, Rooney W (2024). Studying how genes influence crossbreeding between Sorghum bicolor and S. halepense with the help of Sorghum Recombinant Inbred Lines. In: Proceedings of the Joint Annual Conference of Weed Science Society of America (WSSA) and Southern Weed Science Society (SWSS), San Antonio, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mundt T, Elizarraras G, Valiati C, Arik H, Hodnett G, Rooney W, Bagavathiannan M, Subramanian N (2024). Understanding pollen-mediated gene flow from grain sorghum (Sorghum bicolor) to johnsongrass (S. halepense). In: Proceedings of the Joint Annual Conference of Weed Science Society of America (WSSA) and Southern Weed Science Society (SWSS), San Antonio, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Pedireddi U, Subramanian N, Hodnett G, Rooney W, Bagavathiannan M (2024). Fitness of interspecific Sorghum bicolor x S. halepense F1 hybrid progeny as influenced by ploidy. In: Proceedings of the Joint Annual Conference of Weed Science Society of America (WSSA) and Southern Weed Science Society (SWSS), San Antonio, Texas.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The key target audiences reached during this reporting period include sorghum industry stakeholders (United Sorghum Check-off Program, National Sorghum Producers), agribusiness industry (Corteva, Advanta, S&W), federal regulatory agencies (EPA, APHIS), and plant science research community. Our outreach activities also reached crop consultants and sorghum producers. Changes/Problems:The 2023 field season experienced an extremely hot and dry environment at College Station, TX. Nevertheless, we were able to successfully complete the field experiments without much negative impact. No major changes are necessary as of yet. What opportunities for training and professional development has the project provided?The project has provided training on ploidy determination, cytology, and plant phenotypic characterization for a graduate research assistant. This project also provided professional development to two research scientists and one research assistant who were trained in plant biology and ecology research. Two undergraduate students were trained in field crossing, herbicide resistance assessment, plant phenotyping, and flow cytometry analysis. How have the results been disseminated to communities of interest?Results were communicated to the scientific community through participations at various professional meetings, including the Weed Science Society of America, Southern Weed Science Society, and Crop Science Society of America. The PI also presented the findings at the International Symposium for Biosafety Research (ISBR), St Louis, MO. Additionally, a presentation was made at the Texas Plant Protection Association annual meeting held at Bryan, TX. Informal discussions were carried out with the United Sorghum Checkoff Program and the sorghum seed industry. What do you plan to do during the next reporting period to accomplish the goals?We will continue to make progress on all project objectives, building on the great momentum generated over the past few years in this line of research.

Impacts
What was accomplished under these goals? Field experiments were conducted during the 2023 field season to understand the nature and characteristics of pollen movement from sorghum (as the male parent) to johnsongrass (female parent). Two specific field studies were conducted. In the first experiment, different herbicide-resistant sorghum lines (with resistance to the acetyl coenzyme-A carboxylase inhibitor quizalofop) were planted in a field naturally infested with johnsongrass. The sorghum pollen was allowed to naturally pollinate johnsongrass plants in the experimental plot. The johnsongrass panicles that achieved flowering synchrony with sorghum were tagged and harvested at maturity. To prevent pollen movement and contamination of the adjacent plots, a large buffer was established and planted with a tall-growing biomass sorghum line. The johnsongrass seed harvested from these plots were threshed, cleaned and are currently being screened for the presence of the quizalofop-resistance trait through a herbicide assay conducted in the greenhouse. This evaluation is currently ongoing. In the second experiment, a Nelder-wheel study was conducted where a known quizalofop-resistant grain sorghum line was planted at the center of the wheel, and the distance of pollen-mediated gene flow was determined on a natural population of johnsongrass present at the experimental site at various distances in 8 different directions. At maturity, the seed were harvested, threshed and cleaned. They are currently being screened in the greenhouse for the presence of the quizalofop-resistance trait. The survivors from the experiment 1 will be transplanted in a field nursery to determine the phenotypic characteristics of the F1 progeny.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pedireddi U, Subramanian N, Hodnett G, Klein P, Bagavathiannan M, Rooney W (2024). Studying how genes influence crossbreeding between Sorghum bicolor and S. halepense with the help of Sorghum Recombinant Inbred Lines. In: Proceedings of the Joint Annual Conference of Weed Science Society of America (WSSA) and Southern Weed Science Society (SWSS), San Antonio, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pedireddi U, Subramanian N, Hodnett G, Klein P, Bagavathiannan M, Rooney W (2023). Sorghum recombinant inbred lines aid in studying the genetic control of hybridization between Sorghum bicolor x S. halepense. In: Proceedings of the 2023 ASA CSSA SSSA Tri-societies Annual meeting, St. Louis, Missouri.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pedireddi U, Subramanian N, Hodnett G, Rooney W, Bagavathiannan M (2023). Backcrossing frequency in F1 progeny of Sorghum bicolor x S. halepense hybridization. In: Proceedings of the 2023 ASA CSSA SSSA Tri-societies Annual meeting, St. Louis, Missouri.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pedireddi U, Subramanian N, Hodnett G, Rooney W, Bagavathiannan M (2023). Comparison of Phenotypic characteristics of F1 hybrids resulting from Sorghum (S. bicolor) x Johnsongrass (S. halepense) hybridization. In: Proceedings of the 2023 ASA CSSA SSSA Tri-societies Annual meeting, St. Louis, Missouri.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Subramanian NK, Hodnett G, Rooney W, Bagavathiannan M (2023). Understanding pollen-mediated gene flow from grain sorghum (Sorghum bicolor) to johnsongrass (S. halepense). In: Proceedings of the 2nd International Weed Genomics Consortium (IWGC) Meeting, Arlington, VA.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The key target audiences reached during this reporting period include sorghum industry stakeholders (United Sorghum Check-off Program, National Sorghum Producers), agribusiness industry (Corteva, Advanta, S&W), federal regulatory agencies (EPA, APHIS), and plant science research community. Our outreach activities also reached crop consultants and sorghum producers. Changes/Problems:The weather conditions during the 2022 field season were extreme in the study location, which severely affected the field experiment. Gaining access to the herbicide-resistant sorghum germplasm from seed companies is a challenge, but we are trying our best to obtain as many genotypes as possible. No major changes are necessary as of yet. We are hoping to gain access to some of this material again next year and repeat the field study. What opportunities for training and professional development has the project provided?The project has provided training on ploidy determination, cytology, and plant phenotypic characterization for a graduate research assistant. This project also provided professional development to two research scientists and two research assistants who were trained in gene flow research. Two undergraduate students were trained in field crossing, plant phenotyping, and flow cytometry analysis. How have the results been disseminated to communities of interest?Results were communicated to the field managers through presentations of preliminary findings at the Texas Plant Protection Association annual meeting held at Bryan, TX. Scientific presentations are planned for the next reporting year. Informal discussions were also made with the United Sorghum Checkoff Program and the sorghum seed industry on the preliminary research findings. What do you plan to do during the next reporting period to accomplish the goals?We will continue to make progress on all project objectives, building on the great momentum generated over the past few years in this line of research.

Impacts
What was accomplished under these goals? Field experiments were conducted during the summer of 2022 to understand the nature and characteristics of pollen movement from sorghum to johnsongrass in replicated field experiments. In the first experiment, controlled crosses were carried out with pollen from male fertile sorghum dusted on male-sterile johnsongrass to determine the maximum range of hybridization potential between the two species. In the second experiment, the influence of sorghum genotype on the rate of inter-specific hybridization (S. halepense as the female parent) was determined under the field conditions, using a range of sorghum genotypes endowing a herbicide resistance trait as the selectable marker. The frequency of hybridization in this experiment is expected to provide the frequency of 2n gamete production in sorghum pollen, through validation of the ploidy status of the F1 progeny (progeny ploidy ≥4X is a result of 2n gamete production in S. bicolor). This experiment was affected by a severe heat spell and extremely dry conditions experienced in College Station during the season. In the third experiment, preliminary F1 hybrids (S. halepense as the seed parent) that were made in a previous experiment were planted in a field nursery and their phenotype was observed in comparison with a range of S. halepense and S. bicolor genotypes.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Buenaventura K, Pedireddi U, Subramanian N, Hodnett G, Rooney W, Bagavathiannan M. 2022. Comparison of phenotypic characteristics of F1 hybrids resulting from sorghum (Sorghum bicolor) x johnsongrass (Sorghum halepense) crosses. In: Texas Plant Protection Association Meeting, Bryan, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Afshang R, Pedireddi U, Subramanian N, Hodnett G, Rooney W, Bagavathiannan M. 2022. Studying the genetic introgression of traits and genes into the F1 offspring of grain sorghum and johnsongrass. In: Texas Plant Protection Association Meeting, Bryan, TX.