Construction of a designer chromosome in tobacco

The tobacco (Nicotiana tabacum L.) breeding line NC 152 is a doubled haploid that possesses an addition chromosome from N. africana [Merxm. and Buttler]. A gene on this chromosome confers potyvirus resistance (Poty(R)). Our objective was to use the addition chromosome as a base on which to construct a designer chromosome containing a foreign gene linkage package. A mutant dhfr gene conferring resistance to methotrexate (Mtx) was inserted into NC 152-haploid (n = 25) leaf tissue via Agrobacterium tumefaciens-mediated transformation. After chromosome doubling, 135 NC 152dhfr transformants (2n = 50) were pollinated with the potyvirus-susceptible (Poty(S)) cultivar 'McNair 944' (2n = 48). Linkage analysis was performed in the BC1 generation. Two transformants, NC 152dhfr-996 and NC 152dhfr-1517 exhibited complete linkage between Mtx resistance (Mtx(R)) and Poty(R). Segregants from these two transformants which were Mtx(R) and Poty(R) possessed 49 chromosomes, while Mtx sensitive (Mtx(S)) and Poty(S) progeny possessed 48 chromosomes. Eighty percent of the NC 152dhfr transformants transmitted the dhfr gene as one locus. Other foreign genes can be directed to the addition chromosome through transformation followed by selection for single loci with linkage to Poty(R) or Mtx(R). The integrity of both the foreign-gene linkage package and the rest of the genome will be maintained because recombination between the N. africiana and the N. tabacum chromosomes has not been observed.