Functional Genomics of Endosperm Development in Maize

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AbstractThe endosperm of the maize seed is one the most economically important and scientifically interesting structures in plant biology. The goal of this project is comprehensive genetic dissection of the molecular mechanisms underlying endosperm development and metabolism. Analysis of mutations that disrupt the endosperm will allow identification of genes that control endosperm development. Molecular analysis of such mutants will in turn lead to other genes that function in the same or interacting pathways. The approach to identifying this critical initial set of genes can be viewed as passing all maize genes through a series of screens, each filtering genes by a different criterion. In the end a large set of cloned genes that confer endosperm phenotypes are identified. 1. Screen one is phenotype. In principle, all genes that have visible phenotypes in endosperm can be tagged by transposon mutagenesis. At least 2000 independent mutations may be needed to ensure tagging of all endosperm mutants (~300 genes). A Mutator transposon tagging population large enough to contain the 2000 visible endosperm mutants has been created at Florida. This population will be screened to identify a set of maize lines enriched for Mutator insertions in endosperm genes.2. Screen two is endosperm expression. The goal is to identify the subset of tagged genes whose wildtype transcripts are present in a large endosperm cDNA microarray. 20,000 new endosperm cDNA's will be sequenced and used to construct the microarray. DNA flanking Mutator insertion sites in the mutant lines is amplified by PCR. By probing the microarray with PCR products from pooled DNA samples, insertions that are in genes represented in the array can be traced to individual maize lines. cDNA hits will include candidates for the mutant gene, plus incidental gene knockouts not associated with phenotypes. Independent mutants that hybridize to the same cDNA's define a hybridization group. 3. Screen three is complementation testing. Mutant lines within each hybridization group will be systematically tested for genetic allelism. Finding independent mutant alleles of the same gene will confirm the identity of clones of mutated genes. 4. Screen four is bioinformatics and functional analysis. The mutant gene sequence and phenotype will be used to infer function and guide experimental analysis of selected genes. The participating labs have complementary interests covering the essential processes underlying endosperm development. Participants:Donald R. McCarty, PI, U. of FloridaJoachim Messing, Co-PI, RutgersBrian Larkins, Co-PI, U. of ArizonaPhilip Becraft, Co-PI, Iowa State UniversityKaren E. Koch, Co-PI, U. of FloridaL. Curtis Hannah, Co,PI, U. of Florida