Generating novel allelic variation through Activator insertional mutagenesis in maize.
The maize transposable element Activator (Ac) has been exploited as an insertional mutagen to disrupt, clone, and characterize genes in a number of plant species. To develop an Ac-based mutagenesis platform for maize, a large-scale mutagenesis was conducted targeting the pink scutellum1 locus. We selected 1092 Ac transposition events from a closely linked donor Ac, resulting in the recovery of 17 novel ps1 alleles. Multiple phenotypic classes were identified corresponding to Ac insertions in the 5'-UTR and coding region of the predicted Ps1 gene. To generate a stable allelic series, we employed genetic screens and identified 83 germinally heritable ps1 excision alleles. Molecular characterization of these excision alleles revealed a position-dependent bias in excision allele frequencies and the predominance of 7- and 8-bp footprint products. In total, 19 unique ps1 excision alleles were generated in this study, including several that resulted in weak mutant phenotypes. The analysis of footprint alleles suggests a model of Ac excision in maize that is consistent with recent in vitro studies of hAT element excision. Importantly, the genetic and molecular methods developed in this study can be extended to generate novel allelic variation at any Ac-tagged gene in the genome.
Bai
Ling
L
Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
Singh
Manjit
M
Pitt
Lauren
L
Sweeney
Meredith
M
Brutnell
Thomas P
TP
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D018983:DNA Footprinting; D004251:DNA Transposable Elements / Q000235:genetics*; D005787:Gene Frequency; D017343:Genes, Plant / Q000235:genetics*; D014644:Genetic Variation; D018525:Germination / Q000235:genetics; D019753:Intramolecular Lyases / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017422:Sequence Analysis, DNA; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase