Generating novel allelic variation through Activator insertional mutagenesis in maize

Abstract

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.

Figure 1.—

Ac-induced ps1 allelic variation. (A) Ac insertions in ps1. The long horizontal black bar represents the coding and noncoding regions of the intronless Ps1 gene. Red triangles above the gene represent Ac insertions in 5′–3′ orientation in each ps1-m∷Ac allele; red triangles below the gene are Ac insertions in 3′–5′ orientation. Orientation is related to Ac transcriptional start sites. Rectangular open boxes represent Ac-induced ps1-d alleles with the signature of Ac excision. Previously identified conserved regions (Cunningham et al. 1996) are shown by colored boxes: DB, dinucleotide-binding motif; CYI, cyclase motif I; CYII, cyclase motif II; TM, predicted transmembrane helix; C, charged region. Representative seedling phenotypes that are homozygous for (B) the W22 Ps1 allele, (C) ps1-m18∷Ac, and (D) ps1-m7∷Ac are shown.

Figure 2.—

Genetic scheme II used to identify novel Ac excision alleles. (A) Genetic strategy developed to recover ps1 footprint alleles. Triangles represent Ac insertions. “+” represents the wild-type allele. Large parentheses represent putative excision alleles. Ac excision events are indicated by curved arrows. Solid arrows denote primers designed to amplify the donor Ac (dAc, bti97156∷Ac). Shaded angled arrows represent primers designed to amplify the Ac insertion site at ps1 locus. (B) A representative PCR gel image. When Ac is present, Ac-specific and locus-specific primers result in amplification products of a predicted size. If Ac is absent from the locus, no products are detected. The 1-kb DNA ladder (Promega) is labeled as M. Eight individual plants were genotyped using two PCR reactions. Odd-numbered lanes show products that result from amplification of the dAc (lanes 1, 5, 9, 13, and 15), whereas even-numbered lanes show expected products when Ac is inserted at the ps1 locus (lanes 2, 4, 8, 10, and 14). Lanes without amplification products indicate a potential Ac excision allele.

Figure 3.—

ps1 footprint alleles carrying in-frame insertions. (A) Plant lycopene-β-cyclase partial sequence alignments (Singh et al. 2003). The sequence of in-frame amino acid insertions in each ps1 footprint allele is denoted by arrows. (B) Phenotypes of seedlings homozygous for ps1 alleles induced by in-frame amino acid insertions. Seedlings homozygous for the ps1′-m6.1 allele failed to germinate.

Figure 4.—

Modified endonuclease model for ps1 footprint formation. Ac-mediated cleavage is indicated by arrows. Curved arrows represent the nucleophylic attack forming DNA hairpins at donor site. Endonuclease cleavage sites are indicated by triangles. Nonhomologous end-joining and repair synthesis results in the formation of an (A) 8-bp insertion with transversions at the junction. Limited exonucleolytic digestion results in (B) 7-bp or (C) 6-bp insertions.