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

Settles AM, Holding DR, Tan BC, Latshaw SP, Liu J, Suzuki M, Li L, O'Brien BA, Fajardo DS, Wroclawska E, Tseung CW, Lai J, Hunter CT, Avigne WT, Baier J, Messing J, Hannah LC, Koch KE, Becraft PW, Larkins BA, McCarty DR - BMC Genomics (2007)

Bottom Line: 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.Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. settles@ufl.edu

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.

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Related in: MedlinePlus

Informatic analysis of UniformMu FSTs validated by PCR. (A) Distribution of Mu insertion sites relative to transcribed maize sequences. (B) Schematics of the locus-specific primer design as constrained by available sequences. Left and right specific primers were designed when genomic sequence was available on either side of the insertion site. Right specific primers were designed when the insertion site defined novel maize genomic sequence. A presumptive right primer was designed for FSTs without a MuTIR sequence.
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Figure 1: Informatic analysis of UniformMu FSTs validated by PCR. (A) Distribution of Mu insertion sites relative to transcribed maize sequences. (B) Schematics of the locus-specific primer design as constrained by available sequences. Left and right specific primers were designed when genomic sequence was available on either side of the insertion site. Right specific primers were designed when the insertion site defined novel maize genomic sequence. A presumptive right primer was designed for FSTs without a MuTIR sequence.

Mentions: The FSTs selected for PCR validation were compared to the maize assembled genomic islands (MAGI) database as well as the TIGR Zea mays Gene Index (ZMGI) to determine the position and identity of the insertion sites [27,28]. Both the MAGI and ZMGI databases are enriched for maize gene sequences. The insertions were categorized as being within exons, introns, promoters, or unknown sequences (Fig 1A). Sixty-three percent of the insertion sites are found within 850 bp of transcribed sequences as defined by either a match of greater than 95% sequence identity between the FST and a ZMGI sequence or a match to a MAGI sequence that shows sequence identity to a ZMGI sequence (see Table 1 and Additional file 1). The remaining 39 FSTs either matched 1–2 MAGI sequences that contained no EST support for a transcribed sequence or did not show a significant match to MAGI or ZMGI sequences.


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

Settles AM, Holding DR, Tan BC, Latshaw SP, Liu J, Suzuki M, Li L, O'Brien BA, Fajardo DS, Wroclawska E, Tseung CW, Lai J, Hunter CT, Avigne WT, Baier J, Messing J, Hannah LC, Koch KE, Becraft PW, Larkins BA, McCarty DR - BMC Genomics (2007)

Informatic analysis of UniformMu FSTs validated by PCR. (A) Distribution of Mu insertion sites relative to transcribed maize sequences. (B) Schematics of the locus-specific primer design as constrained by available sequences. Left and right specific primers were designed when genomic sequence was available on either side of the insertion site. Right specific primers were designed when the insertion site defined novel maize genomic sequence. A presumptive right primer was designed for FSTs without a MuTIR sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1878487&req=5

Figure 1: Informatic analysis of UniformMu FSTs validated by PCR. (A) Distribution of Mu insertion sites relative to transcribed maize sequences. (B) Schematics of the locus-specific primer design as constrained by available sequences. Left and right specific primers were designed when genomic sequence was available on either side of the insertion site. Right specific primers were designed when the insertion site defined novel maize genomic sequence. A presumptive right primer was designed for FSTs without a MuTIR sequence.
Mentions: The FSTs selected for PCR validation were compared to the maize assembled genomic islands (MAGI) database as well as the TIGR Zea mays Gene Index (ZMGI) to determine the position and identity of the insertion sites [27,28]. Both the MAGI and ZMGI databases are enriched for maize gene sequences. The insertions were categorized as being within exons, introns, promoters, or unknown sequences (Fig 1A). Sixty-three percent of the insertion sites are found within 850 bp of transcribed sequences as defined by either a match of greater than 95% sequence identity between the FST and a ZMGI sequence or a match to a MAGI sequence that shows sequence identity to a ZMGI sequence (see Table 1 and Additional file 1). The remaining 39 FSTs either matched 1–2 MAGI sequences that contained no EST support for a transcribed sequence or did not show a significant match to MAGI or ZMGI sequences.

Bottom Line: 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.Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. settles@ufl.edu

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.

Show MeSH
Related in: MedlinePlus