EDGE CT: Improving and Streamlining Systems for Functional Studies of Non-Model Plants

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Exploration of fundamental processes in plants has been aided through the development of new evolutionary model systems that permit comparative analyses of genes, genomes, development, and adaptation. Although most crops and the worst weeds arise as a consequence of genome doubling (polyploidy), genetic processes in polyploids are largely unknown. The plant genus Tragopogon (also known as goatsbeard) is a member of the sunflower family and an ideal system for examining the immediate consequences of genome doubling. Likewise, new evolutionary models are required for comparative analyses of morphology, development, and other phenotypes in flowering plants. Amborella trichopoda, the sister to all other living flowering plants, is a tractable genomic model that is providing new insight into patterns of genome and morphological evolution in flowering plants. Despite being excellent evolutionary models used by global research communities, neither Tragopogon nor Amborella is considered a functional model, limiting their usefulness for studying gene function. The goal of this project is to optimize transformation and gene editing methods for use in both Tragopogon and Amborella. Broader impacts include wide dissemination of methods and resources to a broad community of plant biologists and the training of students and postdoctoral scientists with emphasis on team science, collaboration for innovation, and the value of melding plant diversity and comparative genomics for understanding the connection between genome and phenome. This project aims to refine and make widely available two new genetic systems in the non-model plants Tragopogon and Amborella trichopoda. Specifically, efforts will focus on establishing efficient CRISPR/Cas9 and CRISPR/Cpf1 gene editing platforms that are tailored for both Tragopogon and Amborella and are sufficiently flexible to be applicable to other related plant species. Project objectives include: (1) developing and improving genetic transformation methods to enable genome editing in Tragopogon and Amborella; (2) establishing a CRISPR/Cas9 gene editing toolkit that will then be extended to include CRISPR/Cpf1 using protoplast transient assays and stable transgenics in Tragopogon and Amborella; and, (3) further developing the CRISPR systems for optimal gene editing of both specific candidate gene loci and genes of unknown function, enabling phenotypic analyses of gene-edited versus unedited plants to directly link genotype to phenotype. If successful, this research will provide an easy-to-use gene editing/genetic transformation system for Tragopogon that will enable further analyses of the gene functional drivers of whole-genome duplication (WGD), as well as a wide range of novel research on gene function and phenotype in Asteraceae independent of WGD. Likewise, a feasible editing system in Amborella, the sister to all other extant angiosperms, will enable integrative analyses of gene function for comparative study of floral morphology and development, sex expression, habitat specialization, and other processes. All protocols and resources will be made freely available and accessible to the public through a project website and an interconnected series of fellowships, workshops, and cross-training. This project is co-funded by the Enabling Discoveries through Genomic Tools (EDGE) and the Plant Genome Research Program (PGRP) in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.