Open Access Research article

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

A Mark Settles1*, David R Holding2, Bao Cai Tan1, Susan P Latshaw1, Juan Liu3, Masaharu Suzuki1, Li Li1, Brent A O'Brien1, Diego S Fajardo1, Ewa Wroclawska1, Chi-Wah Tseung1, Jinsheng Lai4, Charles T Hunter1, Wayne T Avigne1, John Baier1, Joachim Messing4, L Curtis Hannah1, Karen E Koch1, Philip W Becraft3, Brian A Larkins2 and Donald R McCarty1

Author Affiliations

1 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA

2 Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA

3 Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA

4 Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA

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BMC Genomics 2007, 8:116  doi:10.1186/1471-2164-8-116

Published: 9 May 2007

Abstract

Background

Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.

Results

Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.

Conclusion

We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.