Tools and Technologies to Enable Genome-Wide Screens in Arabidopsis

智力优势：本项目旨在开发全基因组实验工具和技术，用于分析拟南芥基因调控和功能。对拟南芥初始基因组序列的计算分析于2000年完成，为大约25500个蛋白质编码基因的存在提供了证据。在过去的十年中，对基因组序列进行了大量的实验注释，目前已经鉴定出31,128个基因，其中包括额外的蛋白质编码基因和非编码RNA基因。鉴定所有拟南芥基因中的T-DNA插入突变一直是全球功能基因组学计划的持续目标。对现有TDNA突变体的分析表明，要建立一个全面的纯合突变体集合，还需要大约24,000个突变等位基因。本研究的目标是：在拟南芥所有基因中鉴定出两个遗传稳定的功能缺失突变，并完成在基因组中每个基因中分离两个纯合等位基因的目标。lumina配对末端深度测序将用于鉴定这些“缺失”基因的突变，从而完成对拟南芥基因注释集的“非突变体”。进一步开发大规模捕获和测序约100万个T-DNA插入位点的T-DNA- seq方法将允许鉴定必需基因的清单，并为T-DNA整合和相关基因沉默事件的机制提供见解。更广泛的影响：本项目开发的基因组资源将被大量研究人员广泛使用，并将为依赖全基因组信息的各种研究项目提供基础。该研究的完成将为植物生物学界提供一个新的重要资源，使各种全基因组突变筛选任何可见表型感兴趣。这项研究的一个重要特点是，所有的突变植物/群体一旦生产出来，就可以提供给研究界。该计划的受益者将是整个植物生物界，为阐明拟南芥基因的功能提供必要的试剂。此外，通过对任何植物或动物的下一代测序来快速和廉价地索引插入突变体的新技术将远远超出拟南芥研究的范围。这些使能工具和技术对农业的长期影响预计将是深远的，为构建具有优良农艺性状的植物新品种提供基础知识。我们研究项目的一个同样重要的方面是植物基因组研究的实践培训，这将在各个层面上提供，包括向少数民族高中和本科生提供服务。

Intellectual Merit: This project aims to develop genome-wide experimental tools and technologies for analyzing gene regulation and function in Arabidopsis. Computational analysis of the initial genome sequence of Arabidopsis thaliana, completed in the year 2000, provided evidence for the existence of approximately 25,500 protein-coding genes. Intensive efforts during the past ten years to experimentally annotate this genome sequence have now identified 31,128 genes, which include additional protein-coding genes as well as non-coding RNA genes. The identification of T-DNA insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the available set of TDNA mutants reveals that ~24,000 additional mutant alleles are needed to create a comprehensive homozygous mutant collection. This research specifically addresses the following goals: to identify two genetically stable loss-of-function mutations in all Arabidopsis genes and to complete the goal of isolating two homozygous alleles for every gene in the genome. llumina paired-end deep sequencing will be used to identify mutations in these "missing" genes, thereby allowing completion of the "unimutant" for the annotated set of Arabidopsis genes. Further development of the T-DNA-Seq method for large scale capture and sequencing of ~ one million T-DNA insertion sites will allow identification of the inventory of essential genes as well as provide insights into the mechanism of T-DNA integration and associated gene silencing events. Broader Impacts: The genomic resources developed by this project will be widely available to a large number of researchers and will provide the basis for a variety of research projects that rely upon whole genome information. Completion of the proposed research will provide a new important resource for the plant biology community, enabling a variety of genome-wide mutant screens for any visible phenotype of interest. An important feature of this research is that all of the mutant plants/populations will be available to the research community as soon as they are produced. The beneficiaries of this program will be the entire plant biology community, providing essential reagents necessary to elucidate the functions of the Arabidopsis genes. Additionally, the new technology developed to rapidly and inexpensively index insertion mutants by next generation sequencing in any plant or animal will have applications far beyond Arabidopsis research. The long-term impact of these enabling tools and technologies on agriculture is expected to be profound, providing fundamental knowledge for the construction of new plant varieties with superior agronomic traits. An equally important aspect of our research program is the hands-on training in plant genome research that will be provided at a variety of levels, including outreach to minority high school and undergraduate students.