ZmDB-Iowa State University   Stanford Genome Technology Center Homepage

Our project is a comprehensive effort to sequence maize genes and to develop new tools to elucidate the function of all maize genes. Because transposon insertions are the mechanism for generating genes for sequencing, all sequenced genes will be accompanied by an insertion mutation for phenotypic analysis. The project components include DNA sequencing projects, molecular and visual phenotypic analysis, and new methods of gene mapping and annotation.

This is a five year project funded by the National Science Foundation.  Please visit the ZmDB web site for the full text of the grant proposal, the complete database of sequences published to date, and contact names at the six participating universities. Below are descriptions of the sub-projects.

 

DNA Sequencing Projects

• EST Project  We have greatly exceeded our goal to sequence 50,000 ESTs drawn from libraries prepared by the project participants and donated by members of the maize genetics community. As of April 2002, we have deposited 110,000 ESTs in GenBank.

• Transposon Tagging All Maize Genes  Grids will be organized of 2304 plants (48 rows of 48 individuals each) that have been tagged with RescueMu, a derivativeof the Mu1 transposon found in Mutator lines of maize.  Pools of leaf punches will be collected from each row and from each column to generate 96 libraries of RescueMu insertions.

• Genomic DNA Sequencing  Plasmid rescue of RescueMu and the adjacent genomic DNA from tagging populations will create a permanent collection of the insertion mutations.  Virtually all Mu element insertions are into or near genes so sequencing 1.2 kb flanking >150,000 insertions should provide the genomic sequences of the expected 50,000 maize genes with 95% probability.  We will sequence from the row libraries only. As of April, 2002, we have sequenced grids G, H, and I, and we will be sequencing a new grid approximately every three months until the end of the project."

• Gene Annotation Tools Tailored To Maize A major bioinformatics goal of our project is to use new, original research on intron definition and new statistical tools to improve prediction of gene structure from genomic sequences.  EST sequences will also be important in this effort.

 
 

Phenotypic Analysis of Transposon-Tagged Mutants

• Microarray ESTs and Genomic Sequences To aid the phenotypic characterization of maize genes, all sequenced items will be microarrayed onto glass slides suitable for gene expression studies. The project team will conduct survey experiments to define the patterns of gene expression in the major organs of maize, to compare Mutator and non-Mutator lines of maize, and to compare inbred lines used in the transposon tagging populations.

• Phenotypic Analysis of Mutator Lines The RescueMu tagging individuals will be self-pollinated, and the progeny will be evaluated for novel phenotypes at the kernel, seedling, young adult, and floral stages. Phenotypic records and photographs will be provided for each tagging individual, organized into a row composed of 48 individuals. The DNA sequences of the RescueMu elements from a row of individuals will be reported with that row. As of April, 2002, we are distributing microarrays of PCR-amplified cDNAs drawn from four EST sequencing projects (~3,000 different genes on each array type) and the first Unigene arrays (~6,000 genes on Unigene1.1).

 
 

New Tools for the Maize Genetics Community

• Distribute the RescueMu Plasmid Collections in Indexed Sets Tagging populations consist of grids of 2304 plants organized into 48 rows and 48 columns. The 48 row plus 48 column libraries from one grid fit into a 96 well plate. These plates can be searched using PCR to find RescueMu insertions in genes of interest. As the genomic DNA sequencing project reports the precise sequences from the row libraries in each grid, a search of the columns for an exact match will identify which plant in the grid contains the mutation of interest (a specific row and column address corresponds to a specific plant in the field).

• Distribute Transposon Tagged Lines Individuals can request selfed seed of any line, provided they donate 1 kb of genomic sequence flanking a RescueMu insertion in that plant.

• Develop a New DNA Hybridization Gene Mapping Method Using existing stocks of maize that vary the dosage of individual chromosomes and chromosome arms, a dot blot will be distributed that allows quick placementof an unknown EST or gene to a chromosome. As many maize genes exist in small gene families, several chromosome regions may hybridize. In collaboration with researchers at the University of Minnesota, in a project managed by Ronald Phillips and Howard Rines, we will also use the oat addition lines they have built, each of which contains a single maize chromosome. In the future, a more refined hybridization blot will be developed. The Minnesota group is making radiation deletions of each maize chromosome with the goal of subdividing each of the 10 maize chromosomes into 100 segments.

 
Stanford Project Contacts

Principal Investigator

    Dr. Virginia Walbot    (walbot@leland.stanford.edu)  Web Site

Sequencing Technicians

    Brian Nakao               (bnakao@sequence.stanford.edu )
    Bret Schneider           (brets@leland.stanford.edu )
    Darren Morrow          (dmorrow@sequence.stanford.edu )

    (Kahled Sarsour and Gurpreet Randhawa have left to pursue graduate degrees.)

Bioinformatics Contact

    John Fernandes         (jfernandes@pacbell.net)