RII Track-2 FEC: Functional Analysis of Nitrogen Responsive Networks in Sorghum

现代农业的高产依赖于氮肥，但合成氮肥的制造是极其能源密集型的，对农民来说是一项主要的成本。此外，肥料径流还产生了许多次要问题，如含氧量低的水生死区和水质下降，增加了该国农业地区提供安全饮用水的成本。植物氮素利用效率（NUE）的遗传增益将有助于保护产量，同时减轻农业中高施肥率带来的成本和负面环境影响。通过这个Track-2重点EPSCoR合作（RII Track-2 FEC）项目，阿拉巴马州哈德逊-阿尔法生物技术研究所和内布拉斯加州大学的研究人员将合作开展尖端的植物基因组学研究，以更好地了解氮如何影响植物的生长和发育。哈德逊-阿尔法生物技术研究所将为合作项目带来其生物技术教育和农业基因组学研究专业知识，而内布拉斯加大学林肯分校将利用其最先进的大型植物成像自动化温室系统，贡献其在植物转化和自动化表型分析方面的专业知识。研究人员将通过各种遗传、生物技术和观察方法，在广泛使用的粮食作物高粱中收集植物如何对氮水平作出反应的信息。高粱在许多粮食作物难以生存的气候条件下茁壮成长，而且在利用水和氮等资源方面效率更高。预计到2050年全球粮食需求将翻一番，这是一种理想的改良目标作物。此外，该项目将包括一个教育部分，这将训练和激励学生从事基于遗传和生物技术的农业研究。为了实现这一目标，hudson - alpha将为高级高中学生开发一个为期三周的暑期课程，名为“农业基因组学学院”。其他活动包括招收三名本科生，他们将在哈德逊-阿尔法大学和内布拉斯加大学林肯分校完成暑期实习，学习先进技术，并支持启动有抱负的生物技术学生（实验室）计划，该计划向低收入和中等收入的学生介绍生物技术。该项目还将指导四名早期职业教师。除了氮利用效率之外，这两个机构的联合努力将在理解其他复杂农艺作物性状的生物学方面取得重大进展。该项目的目标是通过结合哈德逊阿尔法生物技术研究所和林肯内布拉斯加州大学的专业知识，利用基因组学、生物技术和自动表型相结合的方法，了解基因在高粱氮(N)响应性共表达网络中的作用。我们将通过三个主要目标来实现这一目标：(i)生成转录组学和表型数据，以表征高粱对氮有效性的分子响应；（ii）通过组合CRISPR/Cas9基因组编辑和过表达N网络模块中的关键基因来干扰N响应；（iii）描述N网络组分有针对性变化的植物。基因组学和转录组学数据将用于鉴定影响氮素利用效率（NUE）的关键调控基因序列，通过植物代谢氮同化对氮素利用效率组成性状的影响。这些预测的目标调控序列和基因将在高粱中得到验证，使用创新的CRISPR/Cas9基因组编辑协议来操纵高优先基因靶点的启动子，从而导致这些基因的表达变化。确认携带错误表达等位基因的株系将使用非破坏性高通量表型技术在不同水平氮亏缺胁迫下的发育过程中进行表征。研究人员将分析在氮亏缺胁迫下表现出表型变化的植株基因表达网络的变化，以了解它们在调节高粱对氮亏缺胁迫反应中的作用。这一项目的结果具有很大的潜力，可以扩展我们对高粱和与其密切相关的C4草的氮响应基因调控网络和相关基因功能的认识。它也可以作为通过基因网络操作来理解其他复杂农艺性状的模型。该项目将通过交叉培训和激励学生和博士后探索提高作物遗传增益率的策略，了解如何使用尖端的基因组、分子和全植物工具整合基因组和表型方法，从而促进研究基础设施能力建设。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。

The high yields typical of modern agriculture rely upon nitrogen fertilizer, yet the manufacture of synthetic nitrogen fertilizer is extremely energy intensive and represents a major cost for farmers. Additionally, fertilizer runoff produces many secondary problems such as aquatic dead zones with low oxygen contents, and reduced water quality, increasing the costs of providing safe drinking water in agricultural regions of the country. Genetic gains in nitrogen use efficiency (NUE) by plants will aid in protecting yield while mitigating both costs and negative environmental impacts associated with high rates of fertilizer application in agriculture. Through this Track-2 Focused EPSCoR Collaborations (RII Track-2 FEC) project, researchers at the HudsonAlpha Institute for Biotechnology in Alabama and the University of Nebraska will partner to conduct cutting-edge plant genomics research to better understand how nitrogen affects plant growth and development. HudsonAlpha Institute for Biotechnology will bring its biotechnology education and agricultural genomics research expertise to the collaborative project while the University of Nebraska-Lincoln will contribute its expertise in plant transformation and automated phenotyping with their state-of-the-art automated greenhouse system for imaging large plants. Researchers will collect information of how plants respond to nitrogen levels through a variety of genetic, biotechnological, and observational methods in the widely-used grain crop sorghum. Sorghum thrives in climates where many food crops struggle and is more efficient at utilizing resources such as water and nitrogen. It is an ideal crop to target for improvement to meet the predicted doubling of global food demand by 2050. In addition, the project will include an educational component, which will train and inspire students to pursue genetic and biotechnology-based research for agriculture. To accomplish this, HudsonAlpha will develop a three-week summer course for advanced high school students called the "AgriGenomics Academy." Additional activities include the recruitment of three undergraduate students who will complete summer internships at both HudsonAlpha and University of Nebraska-Lincoln to learn advanced techniques, and support for the Launching Aspiring Biotechnology Students (LABS) program, which introduces low and moderate-income students to biotechnology. The project will also mentor four early career faculty. In addition to nitrogen use efficacy, the combined efforts of these two institutions will make significant progress toward understanding the biology of other complex agronomic crop traits.The goal of this project is to understand the role of genes in nitrogen (N) responsive co-expression networks in sorghum using a combination of genomics, biotechnology and automated phenotyping by combining the expertise at the Hudson Alpha Institute of Biotechnology and the University of Nebraska, Lincoln. We will achieve this goal through three main objectives: (i) Generate transcriptomic and phenotypic data to characterize the molecular responses of sorghum to N availability; (ii) perturb the N response by combinatorial CRISPR/Cas9 genome editing and overexpression of key genes in N network modules; and (iii) characterize plants with targeted changes in components of the N network. Genomic and transcriptomic data will be used to identify key regulatory sequences controlling genes predicted to affect nitrogen use efficiency (NUE), through effects on component traits of NUE from nitrogen assimilation through in planta metabolism. These predicted target regulatory sequences and genes will be validated in sorghum using innovative protocols for CRISPR/Cas9 genome editing to manipulate the promoters of high-priority gene targets, resulting in expression changes of these genes. Lines confirmed to carry misexpressing alleles will be characterized across development under different levels of nitrogen deficit stress using nondestructive, high throughput phenotyping techniques. Plants exhibiting phenomic changes under N deficit stress will be analyzed for variation in their gene expression networks to understand their roles in regulating sorghum's responses to N deficit stress. The outcome of this program holds great potential to expand our knowledge on N responsive gene regulatory networks and associated gene function in sorghum and closely related C4 grasses. It can also serve as a model for understanding other complex agronomic traits through gene network manipulation. This project will contribute to research infrastructure capacity building by cross-training and motivating students and post-docs to explore strategies to improve the rate of genetic gains in crops by understanding how genomic and phenomic approaches can be integrated using cutting edge genomic, molecular, and whole-plant tools.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.