SGER -Towards a Comprehensive, Sequence-Indexed Functional Genomics Resource in Maize

SGER - 建立全面的、序列索引的玉米功能基因组学资源

• 批准号: 0542665
• 负责人: Donald McCarty
• 金额: $ 20万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0542665&HistoricalAwards=false
• 关键词: SGER; Towards; Comprehensive; Sequence; Indexed

Large mutant collections created by insertional mutagens (e. g. transposable elements or T-DNA inserts) are an essential resource for plant functional genomics. A searchable index of all mutations contained in a collection can be constructed by sequencing genomic DNA flanking each insertion. The value of sequence indexed insertional mutation resources for functional genomics is clearly demonstrated by the Arabidopsis T-DNA and Ds resources. Creation of a similar resource for maize will add enormous value to the complete maize genome sequence. However, creation of a comprehensive sequence indexed transposon resource for maize using conventional sequencing technologies is costly. Hence, there is a clear need for new approaches that reduce the cost of sequence indexing of insertions. The massively parallel DNA sequencing platform developed by the 454 Corporation is an emerging, transformative technology that is especially powerful for applications such as transposon indexing that 1) can benefit from the very deep sampling coverage attainable with large numbers of relatively short (100 to 200 base pairs) sequence reads and 2) can leverage an existing or emerging reference genome sequence. Application of 454 technology promises to reduce the cost of indexing Mu insertions in the maize genome by as much as 2 orders of magnitude making comprehensive coverage of the maize genome feasible. Moreover, the deep sequence coverage would enable efficient pooling strategies that will allow indexing of Mu transposon populations in multiple genetic (e.g. inbred) backgrounds. In this project, Mu insertions from DNA pools made from two well-characterized transposon populations, MTM and UniformMu, having different genetic backgrounds will be sampled. In addition, the efficiency of two different PCR protocols and pooling schemes for amplifying Mu flanking sequences will be evaluated. The successful application of 454 technology to sequence indexing of insertional mutagens will likely catalyze rapid and innovative advances in functional genomics of plants and other organisms.The resulting resources will be publicly accessed through Genbank Genome Survey Sequences (http://www.ncbi.nlm.nih.gov), MTM (mtm.cshl.edu) and UniformMu (uniformmu.org) websites.

由插入诱变剂产生的大型突变体集合（e. G.转座因子或T-DNA插入片段）是植物功能基因组学的重要资源。可以通过对每个插入侧翼的基因组DNA进行测序来构建集合中包含的所有突变的可搜索索引。拟南芥T-DNA和Ds资源清楚地证明了序列索引插入突变资源在功能基因组学中的价值。为玉米创造类似的资源将为完整的玉米基因组序列增加巨大的价值。然而，使用常规测序技术创建用于玉米的综合序列索引转座子资源是昂贵的。因此，显然需要降低插入的序列索引的成本的新方法。由454公司开发的大规模并行DNA测序平台是一种新兴的变革性技术，其对于诸如转座子索引的应用特别强大，所述转座子索引1）可以受益于通过大量相对短（100至200个碱基对）的序列读取可获得的非常深的采样覆盖，以及2）可以利用现有或新兴的参考基因组序列。454技术的应用有望将玉米基因组中Mu插入的索引成本降低多达2个数量级，使得玉米基因组的全面覆盖成为可能。此外，深度序列覆盖将实现有效的汇集策略，其将允许在多个遗传（例如近交）背景中索引Mu转座子群体。在这个项目中，Mu插入从DNA池制成的两个良好表征的转座子群体，MTM和UniformMu，具有不同的遗传背景将被采样。此外，将评估两种不同PCR方案和合并方案用于扩增Mu侧翼序列的效率。454技术在插入诱变剂序列索引中的成功应用可能会促进植物和其他生物功能基因组学的快速创新进展，由此产生的资源将通过Genbank Genome Survey Sequences（http：www.ncbi.nlm.nih.gov）、MTM（mtm.cshl.edu）和UniformMu（uniformmu.org）网站公开访问。