Poster: Genomic & proteomic resources
Abs #
889: A reverse genetics approach to identifying Mu-induced knockouts of specific maize genes in the UniformMu population.
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Presenter: |
Hunter, Charles T, ibe@ufl.edu |
Authors | Hunter, Charles T (A) Lu, Chung-An (A) Huang, Li-Fen (A) Avigne, Wayne T (A) McCarty, Donald R (A) Settles, Mark (A) Hannah, Curtis (A) Carpita, Nick (B) Koch, Karen E (A) | | Affiliations: |
(A): University of Florida, Plant Molecular and Cellular Biology Program
(B): Purdue University, Botany & Plant Path Dept
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| Web Site: | http://pmcb.ifas.ufl.edu/ | |
Mu transposon insertions have been invaluable to production of stable gene knockouts. Identification of gene roles through functional analysis of knockouts is now being conducted on a genomics scale, using the unique “UniformMu” maize population from the University of Florida. These lines have been generated by introgressing Robertson’s Mutator (a Mu-active line) into a W22 background. The resulting UniformMu population has a traceable pedigree for each plant, a low mutation load, high rate of forward mutations, and a genetic mechanism for stabilizing desired mutants. Also, because this population is inbred, an essentially isogenic group of non-mutant, wildtype control plants is available for comparative analysis of mutant plants.
This material is now being utilized for a reverse genetics approach to identifying individual plants with Mu insertions in specific genes of interest. Target genes were selected for their involvement in cell wall formation. Gene-specific primers were designed to be utilized along with a Mu-specific primer in a PCR-based screen. Sixteen grids for this screening are being constructed, each with genomic DNA from 2,304 UniformMu maize families. Extracts were pooled into 48 groups of 48 plants each and arranged on x-y axes. An internal control (vp14) was successfully identified, and a new allele of this gene was discovered in the process. Screening of the first grid with 15 genes yielded at least 3 knockouts, indicating a mutation frequency of ~10-4 insertions/locus/plant. This is consistent with forward mutation rates reported for Mu-active lines. Two knockouts are putative contributors to cell wall biosynthesis (cslc9 and cslf4), and screening is in progress for additional cell wall genes.
