A four-element based transposon system for allele specific tagging in plants--theoretical considerations

The two-element transposon constructs, utilizing either Ac/Ds or Spm/dSpm, allow random tagging of genes in heterologous model species, but are inadequate for directed tagging of specific alleles of agronomic importance. We propose the use of Ac/Ds in conjunction with Spm/dSpm to develop a four-element system for directed tagging of crop-specific alleles. The four-element based construct would include both Ds and dSpm along with relevant marker genes and would function in two steps. In the first step dSpm(Ds) stocks (a minimum of two) would be crossed to a line containing transposases of Spm and unlinked integrations would be selected from segregating population by the use of a negative selection marker to develop stocks representing integration of dSpm(Ds) at a large number of locations in the genome. Selections would be made for a line in which dSpm(Ds) shows partial or complete linkage to the allele of interest. In the second step selected line would be crossed to a line containing Ac transposase to induce transpositions of Ds element to linked sites thereby exploiting the natural tendency of Ds element to jump to linked sites. Unlinked jumps of dSpm(Ds) and linked jumps of Ds could be monitored by appropriate marker genes. The proposed model would allow tagging of allele of interest in chromosome addition lines and also help in the efficient use of genic male sterility systems for hybrid seed production by tightly marking the fertility restorer gene with a negative selection marker.