Two Families of 5S rDNA in Allotetraploid N. tabacum
Two families of 5S rDNA coexist in N. tabacum, one S-family of 431 bp unit length and another T-family of 646 bp unit length. N. tabacum is thought to be an allotetraploid derived from the ancestors of modern N. sylvestris and N. tomentosiformis and we investigated whether the two families have their origin in these diploid progenitors. The diploid species N. sylvestris and N. tomentosiformis are shown to each have only one 5S family; N. sylvestris with a 430 bp unit length and N. tomentosiformis a 650 bp unit length. Moreover, there are no intrinsic differences in mutual homologies in units of the S-family in N. tabacum (96.4%), N. sylvestris (96.7%) and between them (96.5%). Thus N. tabacum appears to have conserved the 5S rDNA unit structure found in the progenitor species and the genes do not appear to have undergone interlocus gene conversion as is seen for the 18S-5.8S-26S rDNA units. The long tobacco 5S rDNA unit differs from short one by multiple insertions in the non-transcribed spacer. The largest insertion was cloned and used as probe. This specific fragment was found in all species of subgenus Tabacum but not in others. Thus 5S rDNA may be particularly useful for phylogenetic studies in the genus Nicotiana. The long?scale organization of 5S rRNA gene cluster was characterized using in situ hybridization, PFGE, and Southern blot hybridization. The copy numbers of the 5S units are: N. tabacum, 800-1400 short family units, 700-1600 long family units per 1C genome depending on cultivar; N. tomentosiformis, 600 5S units per 1C genome; N. sylvestris, 1400 units per 1C genome and; N. otophora, 1500 units per 1C genome. The copy number has stable inheritance, is the same in different tissues, and does not change in the course of plant development. However the high levels of copy number polymorphism between cultivars offers the possibility of a sensitive fingerprinting method for tobacco. The two families of 5S genes can be distinguished in situ as two loci on different chromosomes and show decondensation at interphase indicating that both are potentially active. DNA methylation analysis showed that both families of 5S rDNA sequences are more heavily methylated both in symmetric and non-symmetric sites than other repetitive sequences of N. tabacum.
R. Matyasek, K.Y. Lim1, J. Fulnecek, A.R. Leitch1, and A. Kovarik
Institute of Biophysics, Academy of Sciences of the Czech Republic,