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United States Department of Agriculture

Agricultural Research Service

Title: Steady-State Transposon Mutagenesis in Inbred Maize

Authors
item Mccarty, Donald - UNIV. FL., GAINESVILLE
item Settles, Mark - UNIV. FL., GAINESVILLE
item Suzuki, Masaharu - UNIV. FL., GAINESVILLE
item Tan, Bao Cai - UNIV. FL., GAINESVILLE
item Latshaw, Susan - UNIV. FL., GAINESVILLE
item Porch, Timothy
item Robin, Kevin - UNIV. FL., GAINESVILLE
item Baier, John - UNIV. FL., GAINESVILLE
item Avigne, Wayne - UNIV. FL., GAINESVILLE
item Lai, Jinsheng - WAKSMAN INS, RUTGERS UNIV
item Messing, Joachim - WAKSMAN INS, RUTGERS UNIV
item Koch, Karen - UNIV. FL., GAINESVILLE
item Hannah, Curtis - UNIV. FL., GAINESVILLE

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 28, 2005
Publication Date: October 1, 2005
Citation: McCarty, D.R., Settles, M.A., Suzuki, M., Tan, .C., Latshaw, S., Porch, T.G., Robin, K., Gaier, J., Avigne, W., Lai, J., Messing, J., Koch, K.E., Hannah, C. 2005. Steady-state transposon, mutagenesis in inbred maize. The Plant Journal 44:52-61.

Interpretive Summary: Transposable elements are fragments of DNA that are able to move to different positions within the genome. The maize Mutator transposable element is characterized by its high level of activity which, when coupled with preferential insertion into genes, results in high mutation rates. This study has taken advantage of Mutator's high rate of transposition, and thus the high copy number of transposons, for the study of seed mutations in maize. A population of Mutator transposon lines, the UniformMu population, was developed and a database was created containing the genetic sequence of the sites flanking the transposon insertion sites in selected seed mutants. These flanking sequences were generated by sequencing PCR amplification products using Mutator transposon specific PCR, or MuTAIL PCR. Most insertions were found in gene rich sequences while there was a random distribution of insertion sites in the maize genome. The identification of sequences unique to specific mutants will facilitate the cloning of genes involved in seed development. The pedigree and phenotypic data for over 2,000 independent seed mutants and the analyses of 34,255 MuTAIL and 5,326 endosperm unigene cDNA sequences from specific seed mutants are publicly available (unformmu.org; endosperm.org).

Technical Abstract: We implement a novel strategy for harnessing the power of high copy transposons for functional analysis of the maize genome and present insights into the dynamics of the Mutator system. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: 1) High mutation frequencies (7% independent seed mutations) and moderation of copy number (~57 total Mu elements; 1-2 MuDR copies) were maintained by continuous backcrossing into a phenotypically uniform inbred background. 2) A bz1-m9 marker enabled selection of stable lines (loss of MuDR) inhibiting further transpositions in lines selected for molecular analysis. 3) Build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. Insertions targeted gene rich sequences and in silico mapping revealed a random distribution over the maize genome. Our results indicate that Mu populations differ markedly in occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR dosage in UniformMu lines is a key determinant of these properties. The public database (unformmu.org; endosperm.org) includes pedigree and phenotypic data for over 2,000 independent seed mutants integrated with analyses of 34,255 MuTAIL and 5,326 endosperm unigene cDNA sequences.

Last Modified: 10/21/2014
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