Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km(r)) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km(r) by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km(r); then the Km(r) progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km(r) gene. Initial tests for TMV resistance indicated possible linkage between Km(r) and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30-40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km(r) and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km(r) gene. Linkage between Km(r) and the N gene in this plant line has been verified in each of two additional backcross generations.