Sequential mutagenesis of a maize gene, using the transposable element Dissociation

Forty unstable isolates of an R allele conferring strongly colored seed (R-sc:124) were established using a two-step procedure involving the Ac – Ds transposable element system. First, a series of full-color reversions to R-sc were isolated from two existing Ds mutable alleles, r-sc:m1 and r-sc:m3. Variegated kernels were then selected from large-scale testcrosses of the revenant strains (Ac still present) to establish new mutants. Four of the 9 R-sc revenants from r-sc:m3 gave no mutable alleles, whereas 5 produced a total of 40, in frequencies ranging from 0.8 × 10 −4 to 10.2 × 10 −4 . Upon removal of Ac, each of the 40 mutations was stabilized as colorless or pale, indicating insertion of a Ds element at the R locus. When placed in heterozygous combination with r-sc:m3 (Ac absent), all but possibly 1 of the 38 mutants tested gave R-sc recombinants, showing that insertion had occurred in sites different from that in r-sc:m3. Thirty-eight of the 40 new mutable alleles that were examined by Southern blotting contained a Ds insert of 2.1 kbp, the same size as that found for r-sc:m3. These findings are consistent with the excision of Ds from r-sc:m3, followed by its insertion into a linked site in the R-sc revertant, and the subsequent reinsertion of the element into R. The testcrosses of R-sc revenants obtained from r-sc:m3 also produced five stable mutations to colorless or pale. In contrast with the r-sc:m3 revertant series, no r-mutable or stable variant was obtained from 10 R-sc revenants of r-sc:m1. Either the Ds involved (~ 400 bp) does not transpose to linked sites preferentially or, when it does, it becomes relatively immobile.