DEVELOPING HIGH-THROUGHPUT PHENOTYPING CAPACITY AT FORT VALLEY STATE UNIVERSITY FOR THE GENETIC ENHANCEMENT OF SUGARCANE APHID RESISTANCE IN SORGHUM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1018112
• Grant No.: 2019-38821-29057
• Cumulative Award Amt.: $499,997.00
• Proposal No.: 2018-04866
• Project Start Date: Feb 15, 2019
• Project End Date: Feb 14, 2024
• Grant Year: 2019
• Program Code: [EQ]- Research Project

Recipient Organization
FORT VALLEY STATE UNIVERSITY
1005 STATE UNIVERSITY DRIVE
FORT VALLEY,GA 31030

Performing Department
Agriculture

Non Technical Summary
Sorghum is an important crop in southern cropping systems as it is used as feed for the broiler industry, forageandsilage for livestock, and for bioethanol production. Sorghum is mostly used for animal feed in Georgia and the U.S. is the number one exporter of sorghum grain in recent years. Since 2013, the U.S. southern states have witnessed a new invasive and devastating pest known as the sugarcane aphid (SCA) (Melanaphissacchari) which severely reduces sorghum crop yields, forage quality, and feed consumption. Currently, we are using visual scoring for sugarcane aphid plant damage symptoms, which are extremely, labor and time intensive. Therefore, these constraints in field phenotyping have necessitated the use of machines for high-throughput phenotyping that can increase precision in phenotyping for sugarcane aphid damage. The development of suitablehigh-throughput (HTP)platforms such as Robotic-based systems can be very powerful andcurrently,ground-based fleets of robots allow us to measure the traits of interest (esp. aphid number of which aphids are found under the leaves) for sugarcane aphid infestation. We aim to use field-based robotic systems to facilitate accelerated plant breeding in this project.This project is formulated on hypotheses that address basic research questions involved in aphid-sorghum interactions. It will investigate the underlying genetic causes of plant resistance to the sugarcane aphid. Therefore, we aim to use a diverse set of lines that utilize the allelic diversity in sorghum to enhance genetic resources for the development of insect resistant varieties using genome-wide association studies. Currently, only one region of the sorghum genome on chromosome 6 has been identified to confer resistance from the line Tx2783. Pyramiding of multiple resistance genes into sorghum cultivars is needed for durable sugarcane aphid resistance. In this proposal, we aim to identify new regions of the sorghum genome that are associated with resistance to the sugarcane aphid. These associated regions will be made publicly available so that new cultivars of sorghum with multiple resistance genes can be created by private and public breeding programs.In summary, this project will strengthen FVSU's research and education in the fields of plant genomics and plant protection. The long-term goals of this project areto:1) establish a sorghum core collection at FVSU by obtaining accessions from several sources collected in the country and, potentially, abroad available at GRIN; 2) improve FVSU's research capacity by enhancing laboratory and greenhouse facilities for insect rearing to enhance plant breeding for insect resistance, and in the process strengthen the university's academic and research capabilities which will serve as a model for other HBCUs; 3) use robotics forHTPof field grown plants, which will allow the incorporation of robotics in FVSU breeding programs; 4) evaluate and identify accessions that are resistant to the sugarcane aphid; 5) Identify genetic markers associated with sugarcane aphid resistance; 6) Provide opportunities for academic development to faculty and students in FSVU. Thus the end result of this project is to have sorghum cultivars with long-term sugarcane aphid resistance being grown in the United States.

Animal Health Component

30%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1520	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1520 - Grain sorghum;

Field Of Science
1081 - Breeding;

Keywords

agriculture

research

Goals / Objectives
Goals: This project will build the capacity of Fort Valley State University (FVSU) to conduct cutting-edge research in plant breeding using high-throughput phenotyping and genotyping technology platforms.The primary focus of this research is the development of new sorghum varieties with resistance to the sugarcane aphid, a new and highly damaging pest of sorghum in the United States.A collection of diverse sorghum lines (Sorghum Association Panel; SAP) will be acquired, for which a walk-in seed storage cooler and seed thresher will be needed to process and maintain this collection.A robotic phenotyping platform with multiple types of sensors will be developed for use in the field and greenhouse to quantify aphid population and damage, and to identify aphid-resistant lines.Personnel capacity at FVSU will be enhanced by allowing students to assist in the development of this phenotyping platform, and by training additional personnel on its use.This study will identify genetic markers associated with sugarcane aphid resistance and will provide additional training opportunities for students in MAS and bioinformatics. Objectives:Objective 1. To obtain seed for the sorghum association panel and increase it in the greenhouseThis objective will be to obtain the Sorghum Association Panel and increase the number of seed for each accession in a greenhouse . The Sorghum Association Panel (SAP) is a diverse collection of 377 photoperiod-insensitive breeding lines that are racially, geographically, and phenotypically diverse. All of the accessions in the SAP are available through GRIN as a public resource.Objective 2. Identify SAP lines that are resistant to sugarcane aphids by phenotyping this population in field trials using roboticsThis objective will use robotics to obtain phenotyping data for the association study. Aphids elicit different responses in plants by feeding on phloem sap through their probing behavior. Therefore, phenotyping is an important and difficult component for QTL mapping and association studies. The effects of the sugarcane aphid on sorghum are usually evaluated by estimating aphid population number and plant damage on a weekly or biweekly basis. As the number of accessions to be screened increases, phenotyping accuracy could be increased by using HTP methods such as field-based robotics. Since the SAP consists of 377 lines which will be replicated two times in two locations, two robotics phenotyping platforms will be utilized to aid in phenotyping and to identify resistant lines. Our aim is to phenotype the plants using robots and compare with visual damage score and identify the lines that are resistant to sugarcane aphid.Objective 3. Identify SNPs associated with sugarcane aphid resistance using Genome-Wide Association studiesA genome-wide association study (GWAS) uses largely unrelated individuals as the mapping population. This population is genotyped and then phenotyped in multiple environments and years to identify correlations between genetic markers and phenotypes. Genome-wide association mapping has higher mapping resolution than linkage mapping due to the larger number of recombination events in a sample. GWAS has not been utilized to identify SNPs associated with sugarcane aphid resistance in sorghum. The SAP will be used as the population, with genotypes coming from the public genotyping-by-sequencing data available for this panel. SNPs that are associated with sugarcane aphid number and plant damage will be identified. In future studies, SNPs associated with sugarcane aphid resistance can be moved into elite lines using MAS.

Project Methods
Objective 1: The SAP lines will be requested from the U.S. National Plant Germplasm System and seed will be increased in a temperature-controlled greenhouse. Self-pollination will be done by using the paper bag method at head emergence. The resulting seed will be dried and threshed. The sugarcane aphid resistant lines Tx2783, TAM428, and SC110 which all exist in the SAP will serve as sugarcane aphid resistant controls. TAM428 and Tx2783 share SC110 as a common parent and perhaps is the source of sugarcane aphid resistance. Line Tx2737 which has been shown to be highly susceptible to sugarcane aphids also exists in the SAP and will be used as the susceptible control. The association mapping experiment requires a large number of accessions to be phenotyped for which we require a large number of supplies and related equipment which are currently unavailable at FVSU. Thus a seed thresher will be purchased as well as related supplies (paper pollinating bags, etc). Additionally, as part of initial trial and well preparedness, we will test the robotics equipment in sorghum field plots grown yearly in Tifton to ensure adequate data can be captured. Dr. Joshua Peschel will also conduct a training session in a workshop at FVSU for the investigators as well as FVSU personnel on the proper use of the robotics equipment.Objective 2: The 377 SAP lines will be grown in a RCBD with 2 replications in field trials in Tifton, GA and Fort Valley, GA. Two identical robotic platforms will be used to measure sugarcane aphid number and plant damage at both locations (Tifton and FVSU). Additionally, the robotic platform can measure other plant traits such as plant height, stalk diameter, and leaf area index (LAI ), leaf temperature, plant canopy etc. and correlations between aphid number and plant damage with these traits will be examined. Additionally, we will manually estimate aphid number and plant damage to determine the strength of the correlation between human and robotic values.Robot Platform and Data Collection: The robot platform consists of a simple navigable basewhich will allow for weight distribution over a large proportional surface area to allow for high stability and minimal soil compaction. This design approach will allow the robot to operate regardless of environmental conditions (i.e., mud). Phenotyping will begin when aphids are first seen on the sorghum plots and will continue until the aphid populations' crash. Aphid populations often crash due to fungi such as Lecanicillium lecanii that feed on aphids. This window period will likely include different physiological growth stages of crop growth such as booting, flowering, soft dough stage and full maturity based on the resistance/tolerance level expressed by the variety. The raw data will include discrete measurements in each plot of up to 10 environmental factors (e.g., temperature, soil moisture), height, stalk diameter, and leaf area index (LAI) measurements in at least 10 plants per plot, thermal and multispectral imaging, and location information. Raw image and climate data will be collected and stored initially onboard, but ultimately will integrate on-board processing to reduce data into manageable data streams transferred wirelessly after Year 1.Objective 3: The sorghum genome sequence completed in 2008 accelerated genomics-based studies for improvement of several physiological and agronomic traits in the crop. It is a small, diploid genome that is amenable for genomic studies. The GBS data from 377 SAP lines will be obtained from the supplementary section of the Morris et al. (2013) manuscript. Dr. Jason Wallace (The University of Georgia) will assist us during the bioinformatics analysis. He will also lead a bioinformatics training session for the lead investigators and FVSU faculty and students. SNP calling, bioinformatics, and association analysis will be performed by Dr. Punnuri (FVSU) using the sorghum reference genome available from the Phytozome (https://phytozome.jgi.doe.gov). SNPs will be filtered using TASSEL software and SNPs will be filtered for only those with minor allele frequencies ≥ 0.05 and coverage across at least 70% of samples. The population structure of the panel will be determined using principal coordinate analysis. The genotype and phenotype data will be imported into TASSEL to perform GWAS analysis. GWAS will then be performed for the different insect-resistance traits using mixed linear models (MLM) that includes both population substructure and kinship matrices to correct for population stratification. The p-value threshold for significance will be determined empirically by performing permutation tests of the data. Those markers with the highest association (significant p values) to phenotypic traits will be used in candidate gene analysis after querying the sorghum genome database using BLAST software. Hence, the current work will expand the ongoing genomics-based research at FVSU's MAS lab. SNPs associated with low aphid number and plant damage can be used for breeding, or even help identify underlying resistance genes. The results and personnel trained on bioinformatics will enhance our ability to conduct high-throughput marker-assisted research for future plant breeding programs.5) Evaluation Plans: Generally, the milestones framed in the timetable will serve as checkpoints to assess development and progress in this project with phenotyping and image processing activities performed in a timely manner. The first objective will be to increase the seed of the SAP. We will request the SAP at the time of proposal submission to ensure that the seed will be available at the start of the project. Our second objective will be evaluated by confirming that known resistant and susceptible lines are identified as such in field-based trials. Additionally, we hope to identify new sources of resistance. Our third objective will be evaluated by identifying SNPs that are associated with aphid population number, plant damage, and other morphological traits by relevant candidate gene analysis and MAS. We understand that not all of the traits will have associated SNPs. The workshops to be conducted during the project will be participated by students, professionals, and researchers interested in this study. We will record the number of attendees and measure the effectiveness of the workshops by pre-test and post-test survey evaluations to determine how much each attendee learned from the workshop(s). The number of students participating in workshops provides a partial estimate of our success in training minority students. Earned diplomas by student assistants hired through this project as well as the number of undergraduate students who participated in the various facets of this project are good indicators of the project's success. The progress of undergraduate and graduate students will be monitored continually through an undergraduate advisor or thesis advisor, and students will be required to do posters and presentations in conferences, annual meetings and publish final results upon thesis completion in peer-reviewed scientific journals. Students will be encouraged to participate in graduate student competitions at annual international and regional meetings. The lead investigator will monitor the progress of the project quarterly. We will conduct initial meetings during the first quarter of the first year to review progress and later set-up bi-annual meetings as needed. Field experiments may be adversely affected by the weather, if this occurs they can be repeated in Year 3. Follow-up meetings will include early identification of potential problems so that the work plan can be adjusted and new solutions are created to accomplish the objectives in a timely manner.

Progress 02/15/19 to 02/14/24

Outputs
Target Audience:Sorghum research community and sorghum farmers were the man targets but also involved research professionals, extension personnel and students as immediate target audience in this project period. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several workshops were conducted in year 1 and 2 of the project period. These workshops trained students, professional and farmers on how to use robots and drones in plat breeding studies. These workshops also trained students on GWAS bioinformatics training and the video and workshop materials were made availabel to audiences trained under these workshops. How have the results been disseminated to communities of interest?Yes the results are disseminated to relevant communities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We accomplished all three objectives. Objective1-A SAP panel was requested from USDA and seeds are saved and baggedfor future use. Objective 2-We identified 4 resistant lines from a collection of 287 accessions and these lines are used in developing mapping populations. Objective 3-SNP identified in GWAS studies are made public for other researchers to use in their breeding program and MAS Moreover we were able to deploy robots and drones for field phenotyping and have successfully used this data.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Thudi et al. (2024) Invasive Sorghum Aphid: A decade of research on deciphering plant resistance mechanisms and novel approaches in breeding for Sorghum resistance to Aphids-Crop Science (Review paper) submitted on Nov30 under review.

Progress 02/15/23 to 02/14/24

Outputs
Target Audience:Students and professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and professionals were trained on how to use new highthroughput phenotyping methods for plant breeding studies. How have the results been disseminated to communities of interest?Yes, the results are shared among stakeholders of sorghum research and farming community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We successfully planted SAPpanel at FVSU new farm location using new precision highthroughput single row plot planter.Around 300 accessions were planted in RCBD design. All of the accessions in the SAP were requested from USDA ARS Griffin, GRIN repository. We also took crossing among known resistant and susceptible lines that were screened from previous year studies and publication. These parents and F1 crosses were evaluated in the field using RCBD design. All these plots were phenotyped with drone and robot equipments while simultaneously collecting manual phenotyping on different physiological traits and also using hand held video recorder. Data is currently under curation and processing to be used in GWAS studies.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Punnuri S, Boatwright L, Coffin A, Tadesse H, Knoll J, Harris-Shultz, Wallace J (2023) Identification of sorghum aphid resistance loci and other related traits using cornerstone genomic resources. 2023 Sorghum in the 21st Century Global Sorghum Conference: Resiliency and Sustainability in the Face of Climate Change Montpellier (France) from June 5th to 9th, 2023
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sangireddy, M. K. R., Punnuri, S. M., Boatwright, L. (2023) Association Mapping of Aphid Resistance in Sorghum using different phenotyping approaches [Abstract]. NAPB Annual Meeting, Greenville, SC. July 16-to-20-2023
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sangireddy, M. K. R., & Punnuri, S. M. (2023) Developing Microsatellite Markers Associated with Aphid Resistance in Sorghum [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO-Oct-29-Nov 1-2023
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Punnuri S 2023- Integrating High-Throughput Phenotyping and GWAS for Sorghum Plant Height and Stem Thickness: A Comparative Analysis of Manual, Robot, and Drone-Based Measurements- Plant and Animal Genome Conference / PAG 31(January 12-17, 2024)

Progress 02/15/22 to 02/14/23

Outputs
Target Audience:Research professionals, students, technicians, farmers and scientists. Changes/Problems:Due to few resources for planting we could not do field planting in 2022 and hence we will attempt one more time during the final year of the project. We will hire relevant skilled person to help carry out the planned activities. What opportunities for training and professional development has the project provided?High-throughput field phenotyoing for various traits including aphid resistance traits were assessed using drone. We are still waiting for the robot results. How have the results been disseminated to communities of interest?Yes. Some of the SAP seeds were shared with other researchers. What do you plan to do during the next reporting period to accomplish the goals?We could not do field planting and hence same will be attempted in the coming summer season.

Impacts
What was accomplished under these goals? We have identified 4 resistant lines and 8 susceptible lines relevant to sorghum aphid infestation as a result of two year screeing in the fied and published these results in peer reviewed article.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Punnuri SM, Ayele AG, Harris-Shultz KR, Knoll JE, Coffin AW, Tadesse HK, Armstrong JS, Wiggins TK, Li H, Sattler S, Wallace JG. Genome-wide association mapping of resistance to the sorghum aphid in Sorghum bicolor. Genomics. 2022 Jul;114(4):110408. doi: 10.1016/j.ygeno.2022.110408. Epub 2022 Jun 15. PMID: 35716823.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Punnuri S M. Understanding the innate resistance in sorghum. Association of 1890 Research director⿿s meeting- Atlanta GA-March 31st -April 4th 2022-(Oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Punnuri S M. Assessing sugarcane aphid resistance in sorghum in relation to plant maturity and morphology. Georgia Entomological Society meeting- Jekyll Islands-April 13-15, 2022. -(Oral)

Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Research professionals, students, technicians, farmers and scientists. Changes/Problems:We are lagging on data processing and extraction from the robot as we lackcloud computing resources. The paperwork and data agreement is in place and we hope to collect the data soon in the coming year. Covid restrcition has impacted our field experiment plansand we hope to accomplish those goals in the coming year. What opportunities for training and professional development has the project provided?We conducted a workshop with help of Dr. Wallace to familiarize and educate attendees onsoftware resources available for GWAS. This workshop was conducted at FVSU for one day and involved 16 participants from three different institutions and organizations. How have the results been disseminated to communities of interest?A preprint and non-peer reviewed version is submitted on Research Square website and data is made public to the scientific community.The research data and outcomes were also shared with several researchers interested in sorghum breeding. What do you plan to do during the next reporting period to accomplish the goals?We have received ano-cost extension. We plan to conducta field experiment to increase seeds for future studies with the help of Drs. Knoll and Harris-Shultz. In addition, weplan to publishother data colected from the experiment.

Impacts
What was accomplished under these goals? We acoomplished all three objectives of the project. The filed phentoypic data collected from 2019 and 2020 on sugarcane aphid resistance was curated and processed for GWAS analysis. The data collected from droneand greenhouse analysis were also included. We focused on GWAS methods analysis with support from Dr. Wallace. We conducted online meetings to assess different methods to analyze our GWAS data. The paperwork and agreement for SciNet data analysis was completed with the help of USDA scientists and professionals. The data collected from robot is yet to be processed and received. The results have been presented attwo different conferences such as ASA-CSSA-SSSA and PAG meetings. Amanuscript was prepared and submitted to Genomics journal and it is under review.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Punnuri* S , Ayele A, Harris-Shultz K, Knoll J, Coffin A, Tadesse H, Armstrong S, Wiggins T, Li H, Sattler S, Wallace J. (2022). Genome-Wide Association Mapping of Resistance to the Sugarcane Aphid in Sorghum bicolor Genomics journal-Under review
• Type: Websites Status: Other Year Published: 2021 Citation: Not peer reviewed and Preprint available at Research Square Punnuri* S , Addissu Ayele, Karen Harris-Shultz et al. Genome-Wide Association Mapping of Resistance to the Sugarcane Aphid in Sorghum bicolor, 21 October 2021, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-982658/v1] under review at Genomics journal
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2021 Citation: Oral Presentation at ASA-CSSA-SSSA conference in Salt Lake city-Utah- Nov 2021. Punnuri* S , Ayele A, Harris-Shultz K, Knoll J, Coffin A, Tadesse H, Armstrong S, Wiggins T, Li H, Sattler S, Wallace J. (2021).Understanding genetic diversity and Genome- Wide Association Mapping of Resistance to Sugarcane Aphid in Sorghum bicolor. ASA-CSSA-SSSA Annual meeting-Salt lake city-Utah-Nov-2021
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2022 Citation: Poster Presentation at Plant and Animal Genome conference in San Diego-CA-Jan 8-12-2022. Punnuri* S , Ayele A, Harris-Shultz K, Knoll J, Coffin A, Tadesse H, Armstrong S, Wiggins T, Li H, Sattler S, Wallace J. (2022).GWAS Analysis for Understanding Sugarcane Aphid Resistance in Sorghum. Plant and Animal Genome conference in San Diego-CA-Jan 8-12-2022.

Progress 02/15/20 to 02/14/21

Outputs
Target Audience:Research professionals, students, technicians, farmers and scientists. Changes/Problems:Due to covid restriction, we had several problems in collecting and analyzing data. We were also not able to organize workshops for the project participants.We also faced several difficulties with regard to seed multiplication and storing due to the nature of some of these genotypes. What opportunities for training and professional development has the project provided?Post-doc, students and project scientists were trained on the use of the new robot in the field for sugarcane aphid related resistance traits. Earthsens gave us training and support on how to upload andanalyze the data. GWAS analysis training was provided by Dr. Jason Wallace to the relevant project personnel. How have the results been disseminated to communities of interest?We did present our results in SICNAThe Sorghum Improvement Conference of North America in Oct 2020 (virtual). We have also shared our filed phenotypic data from the SAP panel with Dr. Doreen Ware's lab. The same will be deposited to their newly developed database. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct one final workshop on GWAS analysis and image analysis for project participants and other beneficiaries. We plan to refine and finetune robots for better usage and address all the issues and concerns from this year so that better phenotypic results will be available for genomic aided analysis. We plan to conduct one more field phenotyping for SCA at the Fort Valley location in 2021. We mainly compile all data and analyze and publish them in relevant journals so that the research community and farmers can use this information.

Impacts
What was accomplished under these goals? Objective 1-We requested one more set of SAP Sorghum Association Panel from the GRINwebsite. We had several difficulties in increasing seed from our previous planting. We also did increase some lines in the Greenhouse (Dr. Karen's Tifton greenhouse). Objective2- We did take planting in Tifton in the 2020 Summer and evaluated these lines for sugarcane aphid resistance. A new robot was also used to collect phenotypic data. Objective 3- We did GWAS studies using our previously prepared GBS genotypic data and phenotypic data. We did identify major peaks for aphid resistance and other agronomical traits. We recruited a new postdoc who joined in for field phenotyping in 2020. We had two new collaborations this year -Dr. Alisa Coffin, USDA ARS Tifton, GA and Dr. Scott Armstrong USDA ARS Stillwater, OK. The detailed report from each location is given below. Fort Valley and Tifton The Sorghum Association Panel (SAP) panel (574 plots ) was planted by Dr. Joseph Knoll and the team on 6/9/2020 at the UGA Belflower Farm in Tifton, GA (same field as in 2019). The SAP panel consisted of 287 entries, replicated twice in a randomized complete block design. Fourteen entries did not germinate any plants. There was heavy sugarcane aphid pressure (natural) which was required for our experiment. The aphid population peaked around mid-August. Dr. Harris-Shultz's team which includes Karen Harris-Shultz, Michael Purvis (research technician), and Tyler Bailey (student worker) provided weed control, added the plot tags, bagged the plots, and took phenotypic data Visual Morphological Traits were Measured (Punnuri, Knoll, Harris-Shultz, and teams) Stand count Flowering Date ( every Monday, Wednesday, and Friday) Plant Height (3/plot) at maturity and panicle length Sugarcane aphid number on the lowest green leaf (3 plants/plot), plant damage (averaged over the entire plot), and growth-stage were assessed on Aug. 11 and Aug. 18 (peak aphid infestation dates). Average basal tillers per plant per plot Lodging Lesion Color Panicle Length (3/plot) Seed color At least 5 heads/ plot were bagged and are currently being threshed to generate seeds for next year's planting (Harris-Shultz and team). Work-study student entered all of the data into Excel Dr. Harris-Shultz team harvested, dried, and threshed the SAP seed Harris-Shultz and Punnuri recruited additional help as needed and coordinated with Dr. Alisa Coffin, who collected phenotypic data using a drone Harris-Shultz and Punnuri also coordinated with Dr. Scott Armstrong, who is phenotyping the SAP panel at the seedling stage Harris-Shultz aided in the data analysis and she along with Drs. Punnuri and Knoll collaborated with Dr. Peschel on how to use the EarthSense robot. Harris-Shultz aided in obtaining computer resources for analysis of the EarthSense robot data. Dr. Alisa Coffin and her team (USDA-ARS Southeast Watershed Research Lab) placed canopy temperature sensors in the SAP Panel field and made 7 UAV flights over the field to collect thermal (4 flights) and multispectral (7 flights) data throughout the season. These data will be complementary to those collected from the ground-based robot. Athens Dr. Jason Wallace was involved and trained us on the GWAS analysis for this project. Iowa -Ames Dr. Peschel's team At the beginning of Year 2, Dr. Peschel'sresearch team began refining and hardening one ground robot platform that is capable of operating, under manual control, for field conditions (Figure 1). The four-wheel-drive platform, capable of forward and reverse transit, weighs 13-lbs and is 17.5-in (width), 21.5-in (length), and 7.75-in (height) in physical dimension. Power is provided by two 7.4-V 3,200-mAH rechargeable lithium polymer batteries carried on board, and a 2.4-GHz radio system is used for manual control of the platform. There are three onboard cameras mounted on the robot that is capable of digital video and image capture. Data are now uploaded to the cloud for processing after missions. Dr. Peshel introduced us to a new robot from Earth Sense co which was used in field phenotyping collections. The Year 2 robot platform was tested at Iowa State University first in DR. Peschel'slab, to ensure proper mechanical and electrical functioning, and was then transported byto Fort Valley State University on August 2, 2020. On August 3, 2020, a field demonstration was given to our Project Team at the research site in Tifton, Georgia. A corpus of video data was collected, as well as still images of aphid examples on sorghum that were used to refine our algorithms. The robot platform was also used to collect more time-series of sorghum growth and aphid presence information and this has been uploaded to the project cloud on Amazon Web Services. Due to Covid-19 restrictions, DR. Peschel could notmake any additional travel beyond the August 2020 site visit. To summarize, DR. Peschel's team have delivered and/or engaged for Year 2 with the following: Refined and deployed one robot platform field use. Traveled one time to Georgia to meet with the project team and refine the platform. Collected field data to refine the on-board computer vision algorithms of the platform. Stillwater. Dr. Scott did a greenhouse evaluation of the SAP panel for sugarcane aphid resistance and provided the results of seedling tolerance to SCA.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: T. Wiggins, S. Punnuri, K. Harris-Shultz, J. Knoll, X. Ni, J. Wallace-Genome-Wide Association Studies in Sorghum for Sugarcane Aphid Resistance Sorghum Improvement Conference of North America 2018-SICNA Conference Oct 28-30-2020-Virtual Conference-Poster presentation to SICNA

Progress 02/15/19 to 02/14/20

Outputs
Target Audience:Farmers, scientific professionals, extension personnels and students. Changes/Problems:We faced several difficulties with regard to seed multiplication and sorting. There were few mixtures identified and were deleted from the list before threshing. We are requesting a new set of seeds for these germplasms that had mixtures or fewer seeds during our first planting. We also had several problems with the robotas high outside temperatures led to the shutdown of the cameras and image capture and uneven surface obstructed traverse. We are trying to address these problems. We also had a lot of shortage of labor while field phenotyping and hence we plan to recruit a research assistant for this project. What opportunities for training and professional development has the project provided?Dr. Punnuri along with other members conducted the first workshop on Oct 1st, 2019 under the theme, "Sugarcane aphid management in sorghum-capacity building project workshop". The workshop consisted of a mixed audience from students to professionals and a few extensional personnel. Sorghum farmers were busy at this time and were not able to attend. The workshop consisted of lectures in the morning from all the team members of this project and few outside team members. In the afternoon session, we basically had a field tour and knowledge exchange on sugarcane aphid management in the field and lab. Dr. Pescheldemonstrated on how to use the robot for field scouting of aphids. In the field, Dr. Peschel provided a talk on an introduction to robotics and high-throughput phenotyping in general and gave an update on the robot platform. After the talk, Dr. Peschel provided an in-field demo of the robot to the participants of the workshop and other guests. How have the results been disseminated to communities of interest?Dr. Doreen Ware USDA CSHL NY is compiling phenotypic data on the SAP panel from multiple locations. We are sharing our field data on selected traits with Dr. Doreen Ware's CSHL lab while deploying their handheld tablet. We are also sharing our results with sorghum research and farming communities in the upcoming sorghum and sugarcane aphid symposium in March (SICNA). What do you plan to do during the next reporting period to accomplish the goals?During Year 2 at FVSU 1. We will replant the 287 entries with 2 replicates in the summer of 2020. The traits collected will mostly remain the same as in 2019. We will analyze the data with the help ofDr. Jason Wallace of UGA from theGWAS studies. 2. During Year 2, Iowa State will refine the driving and visual data capture capabilities of the robot platform, and the system will be hardened for field use. Dr. Peschel and his research team will travel with the robot to deploy the robot with the Fort Valley State University research team for season-long data collection in a sorghum field. Dr. Peschel and his research team will analyze the resulting visual data that is collected during acquisition.

Impacts
What was accomplished under these goals? The Sorghum Association Panel (SAP) panel was planted by Dr. Joseph Knoll and team on 6/14/2019 at Tifton GA The SAP panel consisted of 287 entries replicated twice We obtained majority of this panel from Dr. Ni's previous planting and rest from GRIN resources Plots were hand thinned (all team members) There was heavy sugarcane aphid pressure (natural) which was required for our experiment Visual Morphological Traits were Measured (Punnuri, Knoll, Harris-Shultz and teams) Stand count Flowering Date Plant Height Sugarcane aphid number on the lowest green leaf (3 plants/plot), plant damage (averaged over the entire plot), and growth-stage were assessed on August 28 and August 14, 2019 (peak aphid infestation dates). We were able to capture at least two data points for aphid infestation visually. Average basal tillers per plant Lodging Lesion Color Panicle Length Photos were taken of each plot and head morphology DNA was extracted from 10 sorghum lines that were not in the original SAP panel. These 10 lines were sent to the University of Minnesota for GBS (Harris-Shultz) 3 heads/ plot were bagged and are currently being threshed to generate a seed for next year's planting (Harris-Shultz and team) The robot platform was tested at Iowa State University in Dr. Peschel's lab, to ensure proper mechanical and electrical functioning, and was then transported to Fort Valley State University on July 24, 2019. On July 26, 2019, a field demonstration was given to our Project Team and other personnel at the research site in Tifton, Georgia. An initial corpus of video data was collected, as well as still images of aphid examples on sorghum that were identified by the Project Team. The robot platform was also used to collect two more time series of sorghum growth and aphid presence information - on August 15, 2019 and October 8-10, 2019.

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