SGER: Gene Enrichment Sequencing of the Maize Genome with Hypomethylated Partial Restriction Libraries

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The long-term goal of this Small Grant for Exploratory Research project is to test a new approach to sequencing the gene-rich portions of higher plant genomes, using maize as the model species. Like many other higher plant genomes, the nuclear DNA of maize is primarily composed of repetitive sequences. Most of these repeated sequences are transposable elements of various types, with over 60% of the nuclear DNA composed of retrotransposons. Many of the retrotransposons in maize are large in size and highly similar in sequence, making it very difficult to distinguish between individual elements of the same family when trying to assemble a shotgun genome sequence. In order to avoid the inefficiency of sequencing retrotransposons and other repeats that cannot be assembled, various gene-enrichment technologies have been proposed to improve the efficacy of a shotgun sequence approach. This project will test a new technology that relies on the fact that most maize retrotransposons are modified (methylated) at cytosines in the sequences 5'-CG-3' and 5'- CNG-3' in most or all tissues. This technique will use partial digestion with restriction enzymes that are sensitive to 5'-CG-3' methylation to create hypomethylated partial restriction (HMPR) libraries that will permit high efficiency shotgun sequencing of the maize genome, and of any other plant, animal or microbial genome that has a large component of repetitive DNA that is 5-methylated at 5'-CG-3' sequences.Six libraries each will be produced using two different restriction enzymes (HpaII and HpyCH4IV) that recognize and cut specific sites in a methylation-dependent manner. A total of 3600 sequences will be generated using clones from these libraries. Sequences will be compared against the full plant genome databases to see the frequency of genes, transposable elements, and organellar DNA. These comparisons will determine the degree to which the enrichment technology has been successful. The clones and information generated will be available to all interested parties, providing a valuable resource and tool for the future study of higher eukaryotic genomes.