Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
            

NEW AND OLD APPROACHES TO SELECTIVE SEQUENCE ANALYSIS OF GENE-CONTAINING REGIONS OF THE MAIZE GENOME

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907,USA

² Purdue University Genomics Core, S004 WSLR, Purdue University, West Lafayette, IN47907, USA

Maize has a moderately large genome of 2.3~2.7 X 10⁹ bp, and most of it is composed of retrotransposons. In order to understand and characterize maize genome structure and organization without sequencing the entire genome, we have utilized two technologies to selectively sequence the genic regions of the maize genome. One approach employed BAC end sequencing of clones that were derived from a series of libraries constructed with two restriction enzymes, Sal I and Hpa II, that are sensitive to 5 methyl cytosine in the sequences CG or CNG. Limited Sal I BAC library sequencing demonstrated that 18% of the end sequences were homologous to retrotransposons, compared to 55% from a maize BAC library made with EcoR I. More Sal I and Hpa II libraries are being sequenced. Another approach is based on DNA renaturation (Cot) analysis. Maize genomic DNA was reassociated to a Cot value at which most repetitive DNA should reassociate, but not the unique genes or other low-copy-number regions. These unrenatured sequences were cloned and sequenced. Our preliminary experiment showed that, out of 140 reads, 25% had high homology to retrotransposons. This compares with 57% retrotransposons from an unnormalized maize shotgun genomic library. As expected, the high Cot ssDNA fraction was also enriched in gene-homologous sequences. We believe that these two approaches will help identify all maize genes and also help determine how these genes are organized relative to repetitive DNAs.