T. E. Samatadze

Comparison of genomes of eight species of sections Linum and Adenolinum from the genus Linum based on chromosome banding, molecular markers and RAPD analysis.

Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2nxa0=xa016) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2nxa0=xa016. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2nxa0=xa016, 2nxa0=xa030 and 2nxa0=xa018 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2nxa0=xa016, 2nxa0=xa018 and 2nxa0=xa030 originated from a 16-chromosome ancestor.

The unique genome of two-chromosome grasses Zingeria and Colpodium, its origin, and evolution

Chromosome C-banding and two-color fluorescent in situ hybridization (FISH) were used to compare the chromosomes, to identify the chromosomal localization of the 45S and 5S rRNA genes, and to analyze the sequences of internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the 45S rRNA genes in the genomes of grasses Zingeria biebersteiniana (2n = 4), Z. pisidica, Z. trichopoda (2n = 8), Colpodium versicolor (2n = 4), and Catabrosella variegata (syn. Colpodium variegatum) (2 n = 10). Differences in C-banding pattern were observed for two Z. biebersteiniana accessions from different localities. Similar C-banding patterns of chromosomes 1 and 2 were demonstrated for the Z. pisidica and Z. biebersteininana karyotypes. Chromosome C banding and localization of the 45S and 5S rRNA genes on the chromosomes of the two Zingeria species confirmed the assumption that Z. pisidica is an allotetraploid with one of the subgenomes similar to the Z. biebersteiniana genome. ITS comparisons showed that the unique two-chromosome grasses (x = 2)—Z. biebersteiniana (2n = 4), Z. trichopoda (2n = 8), Z. pisidica (2n = 8), and C. versicolor (2n = 4), which were earlier assigned to different tribes of subtribes of the family Poaceae—represent two closely related genera, the genetic distance (p-distance) between their ITSs being only 1.2–4.4%. The Zingeria species and C. versicolor formed a common clade with Catabrosella araratica (2n = 42, x = 7) on a molecular phylogenetic tree. Thus, the karyotypes of Zingeria and Colpodium, which have the lowest known basic chromosome number (x = 2), proved to be monophyletic, rather than originating from different phylogenetic lineages.

Karyogenomics of species of the genus Linum L.

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were discovered and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.

Genetic polymorphism of flax Linum usitatissimum based on the use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flax) and in the k-37 × Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosome rearrangements (chromosome 3 inversions) were detected in the variety Luna and in the k-37 × Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosome analysis in the fiber and oil flax confirm their very close genetic similarity. In spite of this, the combined use of the chromosome and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

Comparative cytogenetic study of the forms of Macleaya cordata (Willd.) R. Br. From different localities

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of 26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.

Comparative Cytogenetic Study of the Tetraploid Matricaria chamomilla L. and Matricaria inodora L.

A comparative cytogenetic study of the autotetraploid breed of Matricaria chamomilla L. (M. recutita L.) and Matricaria inodora L. was carried out by DAPI-banding, fluorescent hybridization in situ (FISH) with 26S and 5S rDNA probes, and analysis of meiosis. All chromosomes were identified in both karyotypes on the basis of DAPI-banding images and 26S and 5S rDNA distribution, and species-specific idiograms were composed for both M. chamomilla and M. indora taking into account the polymorphous variants of DAPI-banding images, showing the location of the 26S and 5S rDNA sites.

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.

Total area of C-heterochromatin in nuclei and chromosomes as a measure of cultivated flax genome variability

Comparative statistical analysis of total C-heterochromatin content (total area of darkly stained C-bands) in prophase nuclei and metaphase chromosomes of eight flax (Linum usitatissimum L.) varieties, viz., three oil flax varieties (Shafir, Oliver, Linotte), three modern (Orshansky-2, Belinka, Baltuchai) and two ancient fiber flax varieties (Zaretsky Kryazh, K-37) has been carried out. Karyotypes of flax varieties selected for different agronomical traits differed from one another in C-heterochromatin content. The total C-band area of oil flax varieties exceeded that of fiber flax varieties. Genomic polymorphism in C-heterochromatin markers is a testimony of different breeding directions. A simple test for determining total C-heterochromatin content in prophase nuclei is recommended as a useful tool for screening specimens with predetermined characteristics during selection of new varieties.

Features of development and reproduction of transgenic flax

Primary transformants carrying a genetic construct with the chimeric gfp-tua6 gene were obtained using biolistic transformation of hypocotyl explants of flax variety Vasilek. Viable modified plants were used as a basis for the production of inbred lines with confirmed inheritance of introduced genetic construct in three generations. The characteristics of phenological growth stages, plant height, number of bolls and meiosis were studied for transgenic plants. A comparison of transformed lines based on reproduction years revealed a significant decrease of seed production in one line. Meiotic analysis of this line at metaphase I and anaphase I stages was conducted. The percentage of cells with impaired meiosis was highest in transgenic plants of the line with the lowest seed production. Thus, the nonspecific incorporation of genetic construct into the flax genome using biolistic transformation impairs meiosis to a different extent and it is the main reason for unequal reproducibility of transgenic flax. The production of stably reproducing transgenic lines requires systematic analysis of meiosis.

Molecular cytogenetic characterization, leaf anatomy and ultrastructure of the medicinal plant Potentilla alba L.

Potentilla alba L. is a valuable medicinal plant widely used in folk and traditional medicine and particularly promising in complex treatment of thyroid pathology. Natural resources of this species are insufficient due to ever-growing use in contemporary medicine. Comprehensive investigations of different P. alba populations are essential for the successful extension of P. alba plantings. Aiming for a better understanding of karyotype structure, chromosome behaviour in meiosis and developing new diagnostic characters, we performed molecular cytogenetic characterization and leaf structure and ultrastructure analyses of two introduced P. alba samples originating from different habitats. Based on chromosome morphology, distribution of 45S/5S rDNA and DAPI-banding patterns, all chromosomes in the karyotypes were identified and the P. alba chromosomal idiogram was constructed. Our findings confirmed P. alba karyotype stability and also revealed several diagnostic characters of this species: the features of cells of upper and lower leaf epidermis, the presence of calcium oxalate druses and three types of leaf indumentum, essential for evaluation of genetic diversity in different populations, validation of raw materials and further selection progress. The meiotic abnormalities were detected probably related to low pollen activity and indicated the advantages of vegetative propagation in the development of a P. alba plantation system.