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

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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.