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

通过新的进化模型系统的发展，可以对基因、基因组、发育和适应进行比较分析，从而有助于探索植物的基本过程。尽管大多数作物和最严重的杂草是基因组加倍（多倍体）的结果，多倍体的遗传过程在很大程度上是未知的。植物属Tragopogon（也被称为山羊须）是向日葵家族的一员，也是研究基因组加倍的直接后果的理想系统。同样，需要新的进化模型来比较分析开花植物的形态、发育和其他表型。作为所有其他现存开花植物的姐妹植物，三角Amborella trichopoda是一种易于处理的基因组模型，它为开花植物的基因组模式和形态进化提供了新的见解。尽管是全球研究界所使用的优秀的进化模型，但无论是角羚还是小角羚都不被认为是一个功能模型，这限制了它们在研究基因功能方面的有用性。该项目的目标是优化转化和基因编辑方法，适用于角羚和Amborella。更广泛的影响包括向广泛的植物生物学家社区广泛传播方法和资源，培养学生和博士后科学家，重点是团队科学，创新合作，以及融合植物多样性和比较基因组学的价值，以了解基因组和表型之间的联系。本项目旨在完善和推广两种新的非模式植物遗传系统。具体来说，我们将致力于建立高效的CRISPR/Cas9和CRISPR/Cpf1基因编辑平台，这些平台将为角羚和Amborella量身定制，并具有足够的灵活性，可适用于其他相关植物物种。项目目标包括：(1)开发和改进遗传转化方法，使角羚和大鲵基因组编辑成为可能；(2)建立一个CRISPR/Cas9基因编辑工具包，然后将其扩展到包括CRISPR/Cpf1，使用原生质体瞬态检测和稳定的转基因方法在家兔和Amborella中进行；(3)进一步开发CRISPR系统，对特定候选基因位点和未知功能基因进行最佳基因编辑，使基因编辑与未编辑植物的表型分析能够直接将基因型与表型联系起来。如果研究成功，该研究将为角雉提供一个易于使用的基因编辑/遗传转化系统，从而可以进一步分析全基因组复制（WGD）的基因功能驱动因素，以及在不依赖于WGD的情况下对菊科基因功能和表型进行广泛的新研究。同样，在Amborella（所有现存被子植物的姐妹植物）中建立一个可行的编辑系统，将能够对基因功能进行综合分析，以比较研究花的形态和发育、性别表达、栖息地专业化和其他过程。所有协议和资源都将通过项目网站和一系列相互关联的奖学金、研讨会和交叉培训免费提供给公众。该项目由生物科学理事会的“通过基因组工具实现发现”（EDGE）和植物基因组研究计划（PGRP）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.