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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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Sequence similarity tree of 6PGDH enzymes. Sequence similarity tree of 6PGDH enzymes from maize (Zm), rice (Os), Arabidopsis (At), spinach, Chlamydomonas reinhardtii, and Synechocystis sp. PCC 6803. Rice and Arabidopsis proteins are identified by their locus numbers. The protein sequences used to generate the ClustalW alignment and tree were from the ORF of the Pgd3 locus and Genbank accessions: AAC27702, AAC27703, AAK49897, AAK51690, AAL76323, AAO42814, ABA93694, NP_198982, NP_442035, NP_850502, NP_910282.
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Figure 5: Sequence similarity tree of 6PGDH enzymes. Sequence similarity tree of 6PGDH enzymes from maize (Zm), rice (Os), Arabidopsis (At), spinach, Chlamydomonas reinhardtii, and Synechocystis sp. PCC 6803. Rice and Arabidopsis proteins are identified by their locus numbers. The protein sequences used to generate the ClustalW alignment and tree were from the ORF of the Pgd3 locus and Genbank accessions: AAC27702, AAC27703, AAK49897, AAK51690, AAL76323, AAO42814, ABA93694, NP_198982, NP_442035, NP_850502, NP_910282.

Mentions: 6PGDH enzymes are found in both the cytosol and chloroplast in plants (reviewed in [32]). To predict the likely subcellular localization of the PGD3 protein, a complete Pgd3 locus was assembled from EST and MAGI sequences. The predicted peptide for the Pgd3 locus contains an N-terminal extension that is absent from the predicted PGD1 and PGD2 proteins in a multiple sequence alignment (data not shown). PGD3 clusters with a known chloroplast-localized 6PGDH protein from spinach in a ClustalW sequence similarity tree ([33,34]; Figure 5). Furthermore, the PGD3 protein is predicted to be targeted to the chloroplast by the protein localization programs: TargetP, PSORT, and Predotar [35-37]. These sequence analyses suggest that the pgd3-umu1 allele disrupts a chloroplast-localized 6PGDH in maize. This prediction is consistent with previous observations that the remaining 6PGDH activity in pgd1; pgd2 double mutants is entirely plastid-localized [29].


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)

Sequence similarity tree of 6PGDH enzymes. Sequence similarity tree of 6PGDH enzymes from maize (Zm), rice (Os), Arabidopsis (At), spinach, Chlamydomonas reinhardtii, and Synechocystis sp. PCC 6803. Rice and Arabidopsis proteins are identified by their locus numbers. The protein sequences used to generate the ClustalW alignment and tree were from the ORF of the Pgd3 locus and Genbank accessions: AAC27702, AAC27703, AAK49897, AAK51690, AAL76323, AAO42814, ABA93694, NP_198982, NP_442035, NP_850502, NP_910282.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Sequence similarity tree of 6PGDH enzymes. Sequence similarity tree of 6PGDH enzymes from maize (Zm), rice (Os), Arabidopsis (At), spinach, Chlamydomonas reinhardtii, and Synechocystis sp. PCC 6803. Rice and Arabidopsis proteins are identified by their locus numbers. The protein sequences used to generate the ClustalW alignment and tree were from the ORF of the Pgd3 locus and Genbank accessions: AAC27702, AAC27703, AAK49897, AAK51690, AAL76323, AAO42814, ABA93694, NP_198982, NP_442035, NP_850502, NP_910282.
Mentions: 6PGDH enzymes are found in both the cytosol and chloroplast in plants (reviewed in [32]). To predict the likely subcellular localization of the PGD3 protein, a complete Pgd3 locus was assembled from EST and MAGI sequences. The predicted peptide for the Pgd3 locus contains an N-terminal extension that is absent from the predicted PGD1 and PGD2 proteins in a multiple sequence alignment (data not shown). PGD3 clusters with a known chloroplast-localized 6PGDH protein from spinach in a ClustalW sequence similarity tree ([33,34]; Figure 5). Furthermore, the PGD3 protein is predicted to be targeted to the chloroplast by the protein localization programs: TargetP, PSORT, and Predotar [35-37]. These sequence analyses suggest that the pgd3-umu1 allele disrupts a chloroplast-localized 6PGDH in maize. This prediction is consistent with previous observations that the remaining 6PGDH activity in pgd1; pgd2 double mutants is entirely plastid-localized [29].

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