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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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Phenotype of the rgh*-00S-005-14. (A) Self-pollinated ear segregating for rgh kernels (arrows). (B) Mature normal and rgh sibling kernels. The top row shows the abgerminal side of the kernels, and the bottom row shows the germinal side of the kernels. Scale bars are 5 mm. (C) Longitudinal hand sections of normal and rgh kernels. Aleurone, starchy endosperm, and embryo tissues are denoted by a, s, and e, respectively. Arrows indicate embryonic shoot and root tissues in the normal kernel. Scale bars are 1 mm. (D) Normal and rgh mutant plants 50 days after initiating tissue culture. (E) Homozygous rgh mutant ear at 35 DAP. (F) Native PAGE assay for 6PGDH activity from 25 DAP endosperm extracts. Black arrow indicates the inferred PGD3 isozyme and the open arrow indicates the PGD1 and PGD2 homo- and heterodimer isozymes.
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Figure 4: Phenotype of the rgh*-00S-005-14. (A) Self-pollinated ear segregating for rgh kernels (arrows). (B) Mature normal and rgh sibling kernels. The top row shows the abgerminal side of the kernels, and the bottom row shows the germinal side of the kernels. Scale bars are 5 mm. (C) Longitudinal hand sections of normal and rgh kernels. Aleurone, starchy endosperm, and embryo tissues are denoted by a, s, and e, respectively. Arrows indicate embryonic shoot and root tissues in the normal kernel. Scale bars are 1 mm. (D) Normal and rgh mutant plants 50 days after initiating tissue culture. (E) Homozygous rgh mutant ear at 35 DAP. (F) Native PAGE assay for 6PGDH activity from 25 DAP endosperm extracts. Black arrow indicates the inferred PGD3 isozyme and the open arrow indicates the PGD1 and PGD2 homo- and heterodimer isozymes.

Mentions: The linked rgh mutant shows highly reduced grain-fill, along with a characteristic etching or pitting at the endosperm surface (Figure 4A–B). Longitudinal hand sections of mature mutant kernels showed both reduced endosperm development as well as embryo defects, with most mutants failing to develop embryonic roots or leaves (Figure 4C). Rare embryos with a visually apparent shoot-root axis were observed in homozygous mutants. A small fraction of these seeds were able to germinate when cultured prior to seed desiccation. These mutant escapes developed into morphologically normal seedlings and plants (Figure 4D). However, the plants showed a pale green leaf phenotype that was less severe near leaf veins. The homozygous rgh individuals were also confirmed as homozygous for the pgd3-umu1 allele using the locus-specific PCR assay (data not shown). Self or sibling pollinations of these mutant plants showed all mutant seed, which confirmed that the plants were homozygous rgh individuals and not the result of hetero-fertilization (Figure 4E).


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)

Phenotype of the rgh*-00S-005-14. (A) Self-pollinated ear segregating for rgh kernels (arrows). (B) Mature normal and rgh sibling kernels. The top row shows the abgerminal side of the kernels, and the bottom row shows the germinal side of the kernels. Scale bars are 5 mm. (C) Longitudinal hand sections of normal and rgh kernels. Aleurone, starchy endosperm, and embryo tissues are denoted by a, s, and e, respectively. Arrows indicate embryonic shoot and root tissues in the normal kernel. Scale bars are 1 mm. (D) Normal and rgh mutant plants 50 days after initiating tissue culture. (E) Homozygous rgh mutant ear at 35 DAP. (F) Native PAGE assay for 6PGDH activity from 25 DAP endosperm extracts. Black arrow indicates the inferred PGD3 isozyme and the open arrow indicates the PGD1 and PGD2 homo- and heterodimer isozymes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Phenotype of the rgh*-00S-005-14. (A) Self-pollinated ear segregating for rgh kernels (arrows). (B) Mature normal and rgh sibling kernels. The top row shows the abgerminal side of the kernels, and the bottom row shows the germinal side of the kernels. Scale bars are 5 mm. (C) Longitudinal hand sections of normal and rgh kernels. Aleurone, starchy endosperm, and embryo tissues are denoted by a, s, and e, respectively. Arrows indicate embryonic shoot and root tissues in the normal kernel. Scale bars are 1 mm. (D) Normal and rgh mutant plants 50 days after initiating tissue culture. (E) Homozygous rgh mutant ear at 35 DAP. (F) Native PAGE assay for 6PGDH activity from 25 DAP endosperm extracts. Black arrow indicates the inferred PGD3 isozyme and the open arrow indicates the PGD1 and PGD2 homo- and heterodimer isozymes.
Mentions: The linked rgh mutant shows highly reduced grain-fill, along with a characteristic etching or pitting at the endosperm surface (Figure 4A–B). Longitudinal hand sections of mature mutant kernels showed both reduced endosperm development as well as embryo defects, with most mutants failing to develop embryonic roots or leaves (Figure 4C). Rare embryos with a visually apparent shoot-root axis were observed in homozygous mutants. A small fraction of these seeds were able to germinate when cultured prior to seed desiccation. These mutant escapes developed into morphologically normal seedlings and plants (Figure 4D). However, the plants showed a pale green leaf phenotype that was less severe near leaf veins. The homozygous rgh individuals were also confirmed as homozygous for the pgd3-umu1 allele using the locus-specific PCR assay (data not shown). Self or sibling pollinations of these mutant plants showed all mutant seed, which confirmed that the plants were homozygous rgh individuals and not the result of hetero-fertilization (Figure 4E).

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