The Solanum genus, being one of the largest among high plants, is distributed worldwide and comprises about 1,200 species. The genus includes numerous agronomically important species such as Solanum tuberosum (potato), Solanum lycopersicum (tomato), and Solanum melongena (eggplant) as well as medical and ornamental plants. The huge Solanum genus is a convenient model for research in the field of molecular evolution and structural and functional genomics. Clear knowledge of evolutionary relationships in the Solanum genus is required to increase the effectiveness of breeding programs, but the phylogeny of the genus is still not fully understood. The rapidly evolving intergenic spacer region (IGS) of 5S rDNA has been successfully used for inferring interspecific relationships in several groups of angiosperms. Here, combining cloning and sequencing with bioinformatic analysis of genomic data available in the SRA database, we evaluate the molecular organization and diversity of IGS for 184 accessions, representing 137 species of the Solanum genus. It was found that the main mechanisms of IGS molecular evolution was step-wise accumulation of single base substitution or short indels, and that long indels and multiple base substitutions, which arose repeatedly during evolution, were mostly not conserved and eliminated. The reason for this negative selection seems to be association between indels/multiple base substitutions and pseudogenization of 5S rDNA. Comparison of IGS sequences allowed us to reconstruct the phylogeny of the Solanum genus. The obtained dendrograms are mainly congruent with published data: same major and minor clades were found. However, relationships between these clades and position of some species (S. cochoae, S. clivorum, S. macrocarpon, and S. spirale) were different from those of previous results and require further clarification. Our results show that 5S IGS represents a convenient molecular marker for phylogenetic studies on the Solanum genus. In particular, the simultaneous presence of several structural variants of rDNA in the genome enables the detection of reticular evolution, especially in the largest and economically most important sect. Petota. The origin of several polyploid species should be reconsidered.

Aim. The 5S rDNA represents a convenient model for studying of the molecular evolution of tandemly arranged repeated sequences. However, in many groups of angiosperms this genomic region still remains poorly studied. So far, in the family Solanaceae the 5S rDNA was described only for five genera. In order to elucidate the 5S rDNA organization in representatives of other genera of Solanaceae, we decided to explore organization of this region in Brunfelsia uniflora (tribe Petunieae). Methods. PCR amplification, cloning and sequencing of 5S rDNA. Results. Three clones of 5S rDNA of Brunfelsia uniflora were sequenced. It was found that only one class of repeats ranging in length from 343 to 347 bp is present in the genome of B. uniflora. The level of intragenomic similarity for the 5S rDNA intergeneric spacer regions (IGS) ranges from 86.7 to 96.4 %. The IGSs of B. uniflora and of other members of Solanaceae family differ significantly: the sequence homology was detected only for sequence motives required for RNA polymerase III transcription initiation and termination. Conclusions. The molecular evolution of the 5S rDNA IGS occurs at a high rate in the Solanaceae family. Accordingly, the comparison of the IGS should be used to clarify the phylogenetic relationship between taxa of low rank, in particular between species and genera of the tribe Petunieae. Keywords: 5S rDNA, intergeneric spacer, molecular evolution, Brunfelsia, Solanaceae.

Aim. The study was aimed at cloning and analysis of molecular organization of 5S rDNA intergenic spacer (IGS) in two Gentiana species of Ukrainian flora, G. pneumonanthe L. and G. punctata L. Methods. 5S rDNA IGS sequence was amplified using polymerase chain reaction (PCR) with a pair of primers specific for the gene coding region. The produced PCR products were fractionated by gel-electrophoresis, isolated, ligated into plasmid pUC18, cloned into E. coli, and then sequenced. Nucleotide sequences were aligned using the Muscle algorithm and analyzed in the Unipro UGENE software. Results. The intergenic spacer region of the 5S rRNA genes was cloned and sequenced for two Gentiana species of Ukrainian flora, G. pneumonanthe and G. punctata. Based on the analysis of the alignment of the IGS sequences of five Gentiana species from three sections, some features of molecular organization of IGS of 5S rRNA genes in the studied species were established. In particular, motifs typical for other angiosperm families were identified, such as conservative oligo-dT motif at the IGS 3'-end that served as a transcription termination site and AT-rich region preceding the coding region of 5S rRNA gene. However, in the region of transcription initiation, conservative GC-element in position -13 is changed to AC. Conclusions. The interspecific variation of molecular organization of 5S rDNA IGS was identified among Gentiana species that can be used to clarify the phylogenetic relationships between members of this genus.Keywords: Gentiana species, 5S rDNA intergenic spacer, molecular organization, phylogeny.

Aim. The region encoding 5S rRNA (5S rDNA) is present in the genome of all eukaryotic organisms. The 5S rDNA represents a universal model for studying the molecular evolution of the tandemly arranged repeated sequences. However, in the family Solanaceae, the molecular organization of 5S rDNA has been investigated only for few genera. In this regard, we decided to characterize the molecular structure of the 5S rDNA of Mandragora autumnalis, a representative of the Mandragoreae tribe, which occupies an isolated position in the nightshade family. Methods. PCR amplification, cloning and sequencing. Results. Two 5S rDNA clones of M. autumnalis were sequenced. It was found that in the genome of this species only one class of the 5S rDNA repeats possessing the 103 bp-long intergenic spacer (IGS) is present. This is the smallest size of IGS known to date for the Solanaceae family. The 5S rDNA IGS of M. autumnalis demonstrates a moderate level of sequence-similarity with the IGS of other representatives of the subfamily Solanoideae. Conclusions. The results support the current opinion about the isolated taxonomic position of the genus Mandragora within the subfamily Solanoideae. The increased level of similarity was found in the IGS regions, which contain potential external elements of the RNA polymerase III promoter and terminator. The mutations occurred in the part of the IGS up-stream of the coding region had a compensatory nature, which ensured that the external elements of the promoter were preserved during the evolution. Keywords: 5S rDNA, intergeneric spacer, molecular evolution, Mandragora autumnalis, Solanaceae.

The genus Muscari is represented in Ukraine by three species. One of them is M. botryoides, listed in the Red DataBook of Ukraine. M. botryoides is a morphologically polymorphic species that has been trying to be divided into several separate species for a long time. One of the arguments in favor of this division is the karyological differences between populations from different parts of the range. In particular, in the Transcarpathian region of Ukraine, dip-loid populations were found in contrast to the typical tetraploid ones for this species. However, molecular genetic studies are needed to confirm genetic polymorphism. In this work, we studied the possibility of using the chloroplast genome region psbA-trnH for DNA barcoding of Ukrainian populations of M. botryoides. We amplified and se-quenced the psbA-trnH for M. botryoides accessions from three different geographically distant Ukrainian popula-tions and compared them with the psbA-trnH sequences available in the GenBank database for M. botryoides acces-sions from Southwestern Europe and M. armeniacum. Twelve nucleotide substitutions at the beginning of the spacer were shown to be a result of a single evolutionary event, the inversion of the region corresponding to the stem-loop region in the 3΄ UTR of psbA mRNA. The unification of the position of this inversion leads to a significant change in the similarity index. In addition to this insertion, the analyzed psbA-trnH sequences also differ by one SNP and two indels of 1 bp and 7 bp in length. The inversion and the 7-bp indel are polymorphic at the intraspecific level in M. botryoides, which limits their phylogenetic use, but may be useful for microevolutionary analysis. Thus, all three ac-cessions of M. botryoides from three different regions of Western Ukraine differ in the combination of these charac-ters. These data allow us to talk about the genetic polymorphism of Ukrainian populations of M. botryoides.