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

由插入诱变剂（如转座因子或T-DNA插入物）产生的大量突变体是植物功能基因组学的重要资源。通过对每个插入的基因组DNA进行测序，可以构建集合中包含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已被拟南芥T-DNA和Ds资源清楚地证明。创建类似的玉米资源将为完整的玉米基因组序列增加巨大的价值。然而，利用传统的测序技术创建一个全面的玉米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公司开发的大规模平行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)可以从大量相对较短（100到200个碱基对）序列读取的非常深的采样覆盖中受益，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插入物的索引成本降低2个数量级，使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，这将允许在多个遗传（例如近交系）背景下索引Mu转座子群体。在本项目中，将对两个具有不同遗传背景的转座子群体MTM和UniformMu的DNA池中的Mu插入进行采样。此外，还将评估两种不同PCR方案和池化方案扩增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将促进植物和其他生物功能基因组学的快速创新发展。结果资源将通过Genbank Genome Survey Sequences （http://www.ncbi.nlm.nih.gov）、MTM （mtm.cshl.edu）和UniformMu （uniformmu.org）网站公开访问。

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.