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

该项目具体涉及2010年项目的一个重要目标--识别所有拟南芥基因中遗传稳定的功能丧失突变。2000年对拟南芥初始基因组序列的计算分析为大约25500个基因的存在提供了证据。在过去六年中，为对这一基因组序列进行实验性注释而进行的密集努力已经确定了许多额外的蛋白质编码和非编码RNA基因，将基因列表扩大到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(大小)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目标。对当前一组序列索引整合位点的分析表明，约6,000个“新”基因的突变尚未被识别。该项目将使用特定的基因导向的聚合酶链式反应来确定这6,000个基因中每个基因的两个插入突变，从而完成带注释的拟南芥基因的“单一突变”集。此外，该项目将对分离这些T-DNA插入的这些突变进行基因分型，以确定所有不会导致致命性或不育的基因中的纯合子突变。此外，将对已知的Salk、SAIL、威斯康星州和Gabi-Kat T-DNA插入突变系进行大规模基因分型，作为获得由拟南芥基因组倡议最初确定的25,000个基因中纯合子插入突变的努力的一部分。综合起来，这些努力将导致产生一组植物核心基因的纯合子插入突变。完成后，这一资源将能够启动全基因组表型筛选，从而能够识别对了解基础植物生物学重要的基因，以及那些功能对进一步改善食物、生物量和能源生产至关重要的基因。该项目开发的基因组资源将广泛供大量研究人员使用，将为依赖全基因组信息的各种研究项目提供基础，并将能够在全基因组范围内筛选感兴趣的任何可见表型。所有的突变植株一旦通过拟南芥生物资源中心(ABRC)培育出来，就将立即提供给更广泛的研究社区。所有的DNA序列数据(T-DNA插入和基因组序列)将保存在GenBank和TAIR(http://www.arabidopsis.org).数据还将通过信号数据库(http://signal.salk.edu/cgibin/tdnaexpress)，)提供，允许浏览/检索这些数据类型、与基因组注释、转录单位和DNA甲基化、cDNAORF克隆以及所有公共插入突变序列的整合。该项目的一个同样重要的方面是将在不同级别提供的植物基因组研究实践培训，包括面向少数族裔高中生和本科生的培训。

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.