Progress 07/15/24 to 07/14/25

Outputs
Target Audience:During this reporting period, our primary target audience has been academic plant scientists and plant breeders working in the areas of wheat and barley improvement, particularly those focused on hybrid seed production and reproductive biology. This audience includes researchers in both public and private sectors who are actively developing breeding strategies for Triticeae crops, as well as those studying male sterility and gene-environment interactions. We targeted this group through presentations at scientific conferences and lab-based discussions that discussed early-stage findings and engaged stakeholders on the potential of small RNA pathways for inducing conditional male sterility. These engagements are critical because they raise awareness of the genetic and molecular basis of hybrid sterility systems in wheat and barley, crops that historically lack simple hybridization systems compared to maize. Researchers and breeders in this field are best positioned to adopt or extend our discoveries into applied programs, making them a logical and essential audience for our outreach. In addition, we have engaged early-career scientists and students through lab-based training and mentoring, introducing them to genome editing techniques, anther phenotyping, and environmental manipulation experiments. These experiences provide a pipeline for future researchers who can carry this knowledge into both academic and industry settings. These interactions, though more limited during this period, represent an important component of our broader goal to support workforce development in translational plant biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided significant training and professional development opportunities for a second-year Ph.D. student in the UC Davis Plant Biology Graduate Group. The student has been actively involved in CRISPR construct design, molecular cloning, genotyping workflows, and phenotyping assays, gaining hands-on experience with gene editing technologies in cereal crops. Through this work, the student has developed technical skills in plant transformation, PCR-based genotyping, sequence analysis, and phenotypic evaluation of male sterility traits. These experiences have provided her with a deeper understanding of reproductive biology, small RNA pathways, and translational applications in crop breeding. The student's training was supported by close mentorship from the PI and the group's senior lab manager, providing support for independent research and in plant molecular genetics. How have the results been disseminated to communities of interest?Preliminary results from this project have been shared with the broader plant biology community through poster presentations by the participating graduate student at local and regional scientific meetings. Notably, the student presented at the Western Section Meeting of the American Society of Plant Biologists (ASPB), engaging with fellow researchers, faculty, and students to discuss findings and gather feedback. These events provided an opportunity to communicate project goals, approaches, and early outcomes to an audience with strong interest in plant reproductive biology, gene editing, and translational crop research. Additional dissemination is anticipated as the project progresses toward manuscript preparation and presentations at national meetings. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will advance our gene editing efforts by completing transformations for selected bread wheat and barley genotypes using our CRISPR constructs targeting the 24-nt phasiRNA pathway gene. We will genotype regenerated plants to identify successful edits and begin generating homozygous mutant lines. Parallel efforts will include growing these edited lines under permissive and restrictive environmental conditions to assess conditional male sterility phenotypes. We also plan to complete and refine our phenotyping protocols, including microscopy of anther development and fertility scoring under variable photoperiod and temperature regimes. Additionally, we will initiate transcriptomic and small RNA profiling of edited and control plants to examine molecular signatures associated with fertility switching. The integration of genotyping, environmental manipulation, and phenotyping will allow us to test the reproducibility of the phenotype across cultivars and optimize conditions for hybrid seed production.

Impacts
What was accomplished under these goals? During the first year of this project, we have made substantial progress toward establishing the tools, methods, and materials necessary to assess conditional male sterility in barley and bread wheat, targeting the reproductive phasiRNA pathway previously validated in durum wheat. We successfully designed and assembled CRISPR constructs targeting the DCL5 orthologs in both species. These constructs were optimized for high-efficiency gene editing and delivered into the UC Davis transformation pipelines for barley and bread wheat. In parallel, we validated our genotyping workflow by sequencing the targeted CRISPR sites across a panel of diverse cultivars to ensure the constructs were compatible with the genetic backgrounds of interest. To support downstream phenotyping, we identified and bulked seed stocks for the cultivars selected for transformation and phenotypic evaluation. Simultaneously, we tested and refined our phenotyping pipeline using previously generated dcl5 mutants in durum wheat and barley. These tests confirmed the reproducibility of the conditional male sterility phenotype and validated our environmental control strategies (e.g., growth chamber conditions) for inducing or suppressing sterility. We also completed sequencing of the CRISPR target sites in the selected genotypes to account for natural variation and to predict editing efficiencies and potential off-target effects. These efforts will guide the selection of homozygous edited lines for phenotypic assays in Year 2. This work lays the groundwork for the core objectives of the project: assessing phenotypic responses to environmental conditions in dcl5 mutants, testing for background-specific effects, and characterizing the developmental consequences of sterility at the molecular and cellular levels in anthers. We are on track to generate and assess homozygous edited lines under both permissive and restrictive conditions in the upcoming project year.

Publications