Arabidopsis 2010: Tools and Technologies to Enable Genome-Wide Screens in Arabidopsis

该项目专门针对2010年项目的一个重要目标-确定所有拟南芥基因中遗传稳定的功能丧失突变。2000年对拟南芥（Arabidopsis thaliana）初始基因组序列的计算分析提供了大约25,500个基因存在的证据。在过去的六年里，对这个基因组序列进行了大量的实验注释，发现了许多额外的蛋白质编码和非编码RNA基因，将基因列表扩展到31,128个基因。重要的是，即使是最新版本的基因组注释也不包括数百个未注释的非编码（大的和小的）RNA基因。鉴定所有拟南芥基因中的T-DNA/转座子插入突变一直是全球功能基因组学计划的持续目标。对当前序列索引整合位点的分析表明，大约6000个“新”基因的突变尚未被确定。该项目将使用特定的基因定向PCR来鉴定这6000个基因中的每个基因的两个插入突变，从而完成“单突变”的拟南芥基因注释集。此外，该项目将提供这些突变的基因分型，这些突变分离为这些T-DNA插入，以识别所有基因中的纯合突变，这些突变不会导致致命或不育。此外，将对已知的Salk、SAIL、Wisconsin和GABI-KAT T-DNA插入突变系进行大规模基因分型，以获得拟南芥基因组计划最初鉴定的25,000个基因的纯合插入突变体。这些努力结合起来，将产生一组植物核心基因的“无突变”纯合插入突变。一旦完成，该资源将启动全基因组表型筛选，从而鉴定对了解基本植物生物学至关重要的基因，以及那些功能对进一步改善食物、生物质和能源生产至关重要的基因。更广泛的影响。该项目开发的基因组资源将广泛提供给大量的研究人员，将为依赖全基因组信息的各种研究项目提供基础，并将使全基因组突变筛选任何可见的感兴趣的表型。所有的突变植物一旦通过拟南芥生物资源中心（ABRC）生产出来，就会提供给更广泛的研究界。所有DNA序列数据（T-DNA插入和基因组序列）将存储在GenBank和TAIR （http://www.arabidopsis.org）中。数据也将通过SIGnAL数据库（http://signal.salk.edu/cgibin/tdnaexpress）提供，允许浏览/检索这些数据类型，整合基因组注释，转录单位和DNA甲基化，cDNA/ORF克隆，以及所有公开插入突变序列。该项目的另一个同样重要的方面是植物基因组研究的实践培训，该培训将在不同层次上提供，包括向少数民族高中和本科生提供服务。

This project specifically addresses an important aim of the 2010 project - to identify genetically stable loss-of-function mutations in all Arabidopsis genes. Computational analysis of the initial genome sequence of Arabidopsis thaliana in 2000 provided evidence for the existence of approximately 25,500 genes. Intensive efforts during the past six years to experimentally annotate this genome sequence have identified many additional protein-coding and non-coding RNA genes, expanding the gene list to 31,128 genes. Importantly, even the most current version of the genome annotation does not include many hundreds of unannotated non-coding (large and small) RNA genes. The identification of T-DNA/transposon insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the current set of sequence-indexed integration sites reveals that mutations for ~6,000 "new" genes have not yet been identified. This project will use specific gene-directed PCR to identify two insertion mutations for each of these 6,000 genes, thereby allowing completion of the "uni-mutant" set for annotated Arabidopsis genes. In addition, the project will provide for the genotyping of these mutations which segregate for these T-DNA insertions to identify homozygous mutants in all genes that do not lead to lethality or infertility. In addition, large-scale genotyping of the known Salk, SAIL, Wisconsin and GABI-KAT T-DNA insertion mutant lines will be done as part of the effort to obtain homozygous insertion mutants in the 25,000 genes initially identified by the Arabidopsis Genome Initiative. Combined, these efforts will result in the production of a "unimutant" set of homozygous insertion mutations for the core set of plant genes. When completed, this resource will enable the initiation of whole genome phenotypic screens, allowing the identification of genes important for understanding basic plant biology as well those genes whose functions are important to further improvement of food, biomass and energy production.Broader Impacts. The genomic resources developed by this project will be widely available to a large number of researchers, will provide the basis for a variety of research projects that rely upon whole genome information and will enable genome-wide mutant screens for any visible phenotype of interest. All of the mutant plants will be available to the broader research community as soon as they are produced through the Arabidopsis Biological Resource Center (ABRC). All DNA sequence data (T-DNA insertions and genome sequences) will be deposited in GenBank, and TAIR (http://www.arabidopsis.org). The data will also be made available via the SIGnAL database (http://signal.salk.edu/cgibin/tdnaexpress), allowing browsing/retrieval of these data types, integration with the genome annotation, transcription units and DNA methylation, cDNA/ORF clones, and with all public insertion mutant sequences. An equally important aspect of the project 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.