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

由插入突变剂(如转座元件或T-DNA插入)产生的大量突变集合是植物功能基因组学的重要资源。通过对每个插入片段两侧的基因组DNA进行测序，可以构建包含在集合中的所有突变的可搜索索引。序列索引插入突变资源在功能基因组学中的价值在拟南芥T-DNA和DS资源中得到了明确的证明。为玉米创造类似的资源将为整个玉米基因组序列增加巨大的价值。然而，使用常规测序技术为玉米创建一个全面的序列索引转座子资源是昂贵的。因此，显然需要新的方法来降低插入序列索引的成本。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，对于转座子索引等应用尤其强大，1)可以受益于通过大量相对较短(100到200个碱基对)的序列读取而获得的非常深的采样覆盖，以及2)可以利用现有的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的Mu插入索引的成本降低多达两个数量级，从而使全面覆盖玉米基因组成为可能。此外，深度序列覆盖将实现有效的池策略，这将允许在多个遗传(例如近亲交配)背景下索引Mu转座子种群。在这个项目中，来自两个具有不同遗传背景的转座子种群MTM和UniformMu的DNA池中的Mu插入将被采样。此外，还将评估用于扩增Mu侧翼序列的两种不同的聚合酶链式反应和混合方案的效率。454技术在插入诱变剂的序列索引中的成功应用将可能催化植物和其他生物功能基因组学的快速和创新进展。由此产生的资源将通过基因组库基因组调查序列(http://www.ncbi.nlm.nih.gov)，MTM(mtm.cshl.edu)和UniformMu(uniformmu.org)网站公开访问。

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.