Relationships among Nicotiana species revealed by the 5S rDNA spacer sequence and fluorescence in situ hybridization

To investigate phylogenetic relationships in Nicotiana, the intergenic spacer sequences of 5S rDNA were analyzed in species with 2n = 18, 20 or 24, and amphidiploid species with 2n = 48. The chromosomal localization of the 5S rDNA was determined by fluorescence in situ hybridization (FISH). In species with 2n = 24 and their descendants, a major 5S rDNA-specific PCR fragment of 400-650 bp was obtained. The amphidiploid species contained similar length of 5S rDNA units derived from putative diploid progenitors. Among the five clones from each representative PCR fragment, some nucleotide exchanges and length heterogeneity were observed. The latter was due to variation in the spacer region, such as differences in the length of poly A and/or poly T tracts as well as insertions/deletions. Interspecific comparisons of each 5S rDNA sequence demonstrated that the spacer sequence could be divided into three regions. Excluding gaps from the aligned spacer sequences of 5S rDNA, phylogenetic trees were constructed. Each phylogenetic tree showed an almost identical topology even if different algorithms were applied. The chromosomal locations of the 5S rDNA in each species correlated with the phylogenetic topology. The phylogenetic trees were generally in agreement with the current classification.