Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Abstract

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC1 progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5' untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2':GUS marker conferring β-glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F1D, were crossed individually as seed parents to wild-type plants to generate TC1 progenies. TC1 seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked β-glucuronidase activity. From segregation ratio in TC1 the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F1 plants. A total of 14560 TC1 seedlings of 1481J and 16195 TC1 seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF1481J = 24 %, GEF1501D = 25 %, FR1481J = 42 %, FR1601D = 46 %.