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


CONCLUSIONS
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.
Settles
A Mark
AM
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. settles@ufl.edu
Holding
David R
DR
Tan
Bao Cai
BC
Latshaw
Susan P
SP
Liu
Juan
J
Suzuki
Masaharu
M
Li
Li
L
O'Brien
Brent A
BA
Fajardo
Diego S
DS
Wroclawska
Ewa
E
Tseung
Chi-Wah
CW
Lai
Jinsheng
J
Hunter
Charles T
CT
Avigne
Wayne T
WT
Baier
John
J
Messing
Joachim
J
Hannah
L Curtis
LC
Koch
Karen E
KE
Becraft
Philip W
PW
Larkins
Brian A
BA
McCarty
Donald R
DR
D001483:Base Sequence; D016000:Cluster Analysis; D019295:Computational Biology; D004251:DNA Transposable Elements / Q000235:genetics; D030541:Databases, Genetic; D020868:Gene Silencing; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics; D012639:Seeds / Q000235:genetics; D017422:Sequence Analysis, DNA; D016324:Sequence Tagged Sites; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D010734:Phosphogluconate Dehydrogenase