Gene Discovery, DNA Sequencing and Phenotypic Analysis
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
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
Dr. Virginia Walbot
(Kahled Sarsour and Gurpreet Randhawa have left to pursue graduate degrees.)