Progress 03/01/24 to 02/28/25

Outputs
Target Audience:During the reporting period, the primary target audiences for this project included: cotton breeders, agricultural scientists, graduate students and postdoctoral researchers, industry stakeholders (particularly those affiliated with fiber quality and cotton improvement), and public-sector collaborators involved in cotton genomics and breeding. We engaged with breeders in both the public and private sectors to inform them of advancements in high-resolution phenotyping methods and fiber quality trait discovery. Our work directly supports their needs by generating a scalable fiber phenotyping protocol and identifying diagnostic markers associated with fiber quality. We worked closely with industry professionals such as those at Cotton Incorporated who are directly involved in translating research into field-level applications. These stakeholders were targeted due to their central role in assessing fiber quality for commercial applications and driving innovation in cotton production and processing. Their feedback has been instrumental in aligning our research outputs with practical needs and economic priorities. Our research team actively disseminated new methodologies and findings through scientific meetings, and software repositories such as GitHub. Graduate and postdoctoral trainees played a central role in executing the research activities, including microscopy, image analysis, genetic mapping, and bioinformatics. The project provided transdisciplinary training spanning plant breeding, functional biology, data science, and machine learning. This audience was targeted intentionally to build capacity in agricultural research and to cultivate the next generation of plant scientists with cross-cutting expertise in phenomics and computational biology. Although broader outreach to growers and extension networks will intensify in later project stages, preliminary engagement occurred with extension professionals through our collaboration with Dr. Kothari, who serves as a conduit to the Cotton Incorporated Board of Directors and U.S. grower networks. We focused on this audience during the reporting period to align research outputs with future communication and delivery strategies, especially in anticipation of field level deployment of phenotyping technologies and cotton lines with improved traits. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Importantly, the project is also contributing to workforce development in agricultural science and engineering. During this period, we trained five undergraduate students in advanced microscopy, fiber histology, machine learning, data analysis and image annotation. Additionally, students received guidance in science communication. These hands-on research experiences foster transdisciplinary learning and prepare students to address complex challenges in crop improvement. A Ph.D. student on the project was competitively selected as a Fellow in the prestigious FFAR Fellowship - Future Leaders for Food and Agriculture program. As part of this fellowship, the student completed a structured professional development curriculum designed to foster leadership in agricultural science. The training included webinars and interactive sessions covering self-assessment, emotional self-regulation, effective advisor-advisee relationships, scientific communication, interpersonal collaboration, time management, and career exploration. Additionally, the student created an individualized professional development plan aligned with their research objectives and long-term career aspirations, ensuring targeted skill growth that supports both academic and professional success. At the undergraduate level, students actively participated in research activities that culminated in presentations, written reports, and participation in a college-wide poster competition. These experiences helped students strengthen their communication and critical thinking skills. Notably, one undergraduate earned first place in the competition, reflecting the quality and impact of their engagement in the research project. The Ph.D. student also demonstrated a strong commitment to institutional leadership and advocacy through active service in the Clemson University Graduate Student Government (CU-GSG). During the reporting period, the student held the role of Cabinet Member and Deputy Director of the Research Initiatives Committee (RIC), beginning in May 2024. Prior to this, they served as a committee member of the RIC from September 2023 to April 2024. In addition, they continue to represent the Plant and Environmental Sciences program as an elected Senator through September 2024. These roles have provided valuable opportunities to influence university policy, support fellow graduate students, and gain experience in shared governance and leadership. How have the results been disseminated to communities of interest?During the reporting period, project results were actively disseminated to multiple scientific, stakeholder, and industry audiences to foster knowledge exchange and stakeholder engagement in cotton improvement. Research findings were presented at the National Cotton Beltwide Meeting, a key forum for researchers, breeders, and industry partners in the cotton sector. The presentation, titled "Advancing Cotton Breeding: High-Resolution Genetic Mapping and Fiber Quality Improvement through Individual Fiber Phenotyping," highlighted the project's advancements in genetic mapping and novel phenotyping methodologies, sparking productive dialogue among attendees. In addition to academic venues, ongoing results and project updates were communicated directly to growers and industry decision-makers. Dr. Kothari, a key collaborator, provided multiple briefings throughout the year to the Cotton Incorporated Board of Directors, which includes more than 70 growers from across the U.S. These updates focused on strategic project outcomes and provided transparent insight into the practical implications, successes, and challenges associated with the research. These briefings are critical for ensuring that project goals remain aligned with industry priorities and that stakeholders remain engaged in the development and future deployment of new technologies. At the Tri-Society Annual Meeting (ASA-CSSA-SSSA), the project was featured through a rapid five-minute talk and a poster presentation titled "Dissecting the Genetic Basis of Cotton Fiber Quality Traits Using Biparental Mapping Populations and Revolutionized Phenotyping Technique." This venue facilitated communication with plant breeders, agronomists, and soil scientists, offering a platform to share innovative methodological advancements in phenotyping and genetic mapping. Additional dissemination took place at the National Association of Plant Breeders (NAPB) Annual Meeting, where a poster titled "Revolutionizing Cotton Fiber Quality: A Microscopy-Based Phenotyping Platform for Enhanced Precision and Accuracy" was presented. This presentation attracted interest from plant breeding professionals focused on precision phenotyping and trait improvement in crop plants. Finally, the project was showcased at the Clemson University College of Agriculture, Forestry and Life Sciences Graduate Research Symposium, where a poster titled "Enhancing Cotton Fiber Quality: A Microscopy-Based Phenotyping Platform for Enhanced Precision and Accuracy" was presented. This venue provided a valuable opportunity to engage with fellow researchers, faculty, and students at the institutional level, reinforcing the project's integration with graduate education and academic training. Through these varied dissemination efforts, the project successfully reached scientific, industry, and academic communities with interest in cotton research, fiber quality improvement, and translational breeding technologies. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, our efforts will focus on completing critical analyses, disseminating results, and advancing tool development to support fiber quality research and breeding applications. We will finalize the QTL analysis of the year 1 field trial data and proceed with the design and validation of molecular biomarkers associated with key cotton fiber quality traits. These validated markers will form the foundation for downstream marker-assisted selection in breeding programs. The findings will be prepared for publication in a peer-reviewed scientific journal. To complement this work, we will analyze the year 2 dataset using a targeted bulk-segregant analysis approach. This analysis will allow us to identify QTLs that are stable across multiple environments and years, thus improving the robustness of our trait associations and genetic models. We also plan to complete and submit a manuscript detailing the development and implementation of our custom web-based image analysis tool. This tool, designed to automatically quantify fiber cross-sectional traits, will be made accessible to the research community and represents a critical step toward democratizing high-resolution phenotyping capabilities. In addition, we will finalize the data analysis and prepare a manuscript for publication describing our controlled-environment greenhouse study, which examined within-plant variation in fiber quality across two Upland cotton genotypes. This work provides foundational insight into sampling strategy optimization for field-based phenotyping. Finally, we will present a research update at the 2025 National Association of Plant Breeders (NAPB) Annual Meeting, further engaging with the plant breeding community and sharing progress on our phenotyping platform, genetic discoveries, and biomarker development.

Impacts
What was accomplished under these goals? During this reporting period, we made significant progress toward achieving our overarching goal of developing a scalable, microscopy-based cotton fiber phenotyping platform capable of capturing a wide range of quantitative fiber traits with high precision. Our work advances the field of fiber quality research by minimizing subjectivity and reducing type II errors inherent in traditional phenotyping techniques. Ultimately, this research lays the groundwork for improving the profitability, resilience, and sustainability of cotton production by enabling the development of cotton lines that combine the yield potential of Upland cotton with the superior fiber quality of Pima cotton. To support these aims, we planted an F8 recombinant inbred line (RIL) population segregating for key fiber quality traits across six geographically diverse field sites: South Carolina, North Carolina, Mississippi, Arizona, Arkansas, and Georgia. These multi-environment trials provide essential replication to account for environmental variation and increase the resolution of downstream genetic mapping efforts. We completed fiber harvests from all six sites and collected high volume instrument (HVI) fiber quality data to establish baseline trait profiles across the RIL population. In parallel, we optimized our histology protocols to support high-throughput sample preparation and developed an efficient light microscopy pipeline for consistent image capture and stitching. To facilitate automated, quantitative phenotyping, we constructed an expansive, hand-annotated training library using the VGG Image Annotator tool. This image library was used to train a computer vision model capable of extracting key anatomical features from fiber cross-sections, including lumen area and perimeter, fiber perimeter, true fiber area, and fiber circularity. This model has been successfully integrated into a custom-built web application that allows for rapid, accurate measurement of individual fiber traits, providing an accessible platform for broader community use. As part of our early data collection efforts, we generated a deep phenotyping dataset consisting of approximately 500 fiber cross-sections from the Florence, SC field site, covering three replicate samples from ~240 RIL entries. This dataset is being used to identify quantitative variation in fiber traits and inform high-resolution mapping efforts aimed at discovering functional alleles and diagnostic biomarkers of fiber quality. In addition to field and computational advances, we established a controlled-environment greenhouse experiment to investigate within-plant variation in fiber traits. This study focused on two Upland cotton genotypes, with boll samples collected sequentially from the first to the tenth sympodial branch. The goal is to understand positional variation in fiber quality, a critical yet understudied source of trait variability.

Publications