Genome Studies in Four Species of Calendula L. (Asteraceae) Using Satellite DNAs as Chromosome Markers.

The taxonomically challenging genus Calendula L. (Asteraceae) includes lots of medicinal species characterized by their high morphological and karyological variability. For the first time, a repeatome analysis of a valuable medicinal plant Calendula officinalis L. was carried out using high-throughput genome DNA sequencing and RepeatExplorer/TAREAN pipelines. The FISH-based visualization of the 45S rDNA, 5S rDNA, and satellite DNAs of C. officinalis was performed on the chromosomes of C. officinalis, C. stellata Cav., C. tripterocarpa Rupr., and C. arvensis L. Three satellite DNAs were demonstrated to be new molecular chromosome markers to study the karyotype structure. Karyograms of the studied species were constructed, their ploidy status was specified, and their relationships were clarified. Our results showed that the C. officinalis karyotype differed from the karyotypes of the other three species, indicating its separate position in the Calendula phylogeny. However, the presence of common repeats revealed in the genomes of all the studied species could be related to their common origin. Our findings demonstrated that C. stellata contributed its genome to allotetraploid C. tripterocarpa, and C. arvensis is an allohexaploid hybrid between C. stellata and C. tripterocarpa. At the same time, further karyotype studies of various Calendula species are required to clarify the pathways of chromosomal reorganization that occurred during speciation.

Agro-Morphological and Cytogenetic Characterization of Colchicine-Induced Tetraploid Plants of Polemonium caeruleum L. (Polemoniaceae).

Polemonium caeruleum L. (Polemoniaceae) is a valuable medicinal herb with a wide spectrum of biological activities. Under natural conditions, the productivity of this species is rather low. In this study, colchicine-induced tetraploid plants (2n = 4x = 36) of P. caeruleum were obtained, and for the first time, their morphological and cytogenetic characterization was performed. In the tetraploid plants, raw material productivity and also the content of triterpene saponins were significantly higher than in the control diploids. The analysis of chromosome behavior at meiosis and FISH chromosome mapping of 45S and 5S rDNA generally demonstrated stability of both genomes in the tetraploid plants. Based on chromosome morphology and distribution patterns of the studied molecular cytogenetic markers, all chromosome pairs in karyotypes were identified, and chromosome karyograms and idiograms of P. caeruleum were constructed. The revealed specific microdiagnostic characteristics of P. caeruleum (strongly sinuous cells and anomocytic stomata of the leaf epidermis, and also glandular hairs along the veins) could be useful for raw material identification. In the obtained tetraploids, the predominance of large stomata on the lower leaf epidermis was determined. The studied tetraploids can be used in various breeding programs to obtain high-quality pharmaceutical raw materials of P. caeruleum.

Agro-Morphological, Microanatomical and Molecular Cytogenetic Characterization of the Medicinal Plant Chelidonium majus L.

Chelidonium majus L. is a medicinal plant well-known as a valuable source of isoquinoline alkaloids, which has a variety of pharmacological properties including anti-viral and anti-bacterial effects. However, considerable intraspecific bio-morphological variability in C. majus complicates raw material identification and verification. For the first time, we have brought into cultivation five populations of C. majus subsp. majus originated from different regions, and performed their agro-morphological, microanatomical and molecular cytogenetic characterization. All examined populations produced high seed (18.6-19.9 kg/ha) and raw material (0.84-1.08 t/ha) yields; total alkaloid contents were within 0.30-0.38%. Nevertheless, significant differences in plant morphology and yield-contributing traits were observed. The performed microanatomical analysis of leaves and flowers in double- and normal-flowered plants revealed micro-diagnostic features (including tissue topography, types of stomata, laticifers, structure of leaf mesophyll, hairs, sepals and petals) important for identification of C. majus raw materials. The analysis of chromosome morphology, DAPI-banding patterns, FISH mapping of 45S and 5S rDNA and also chromosome behavior in meiosis allowed us to identify for the first time all chromosomes in karyotypes and confirm relative genotype stability of the studied plants. Our findings indicate that the examined C. majus populations can be used in further breeding programs.

Phenotypic and molecular cytogenetic variability in calendula (Calendula officinalis L.) cultivars and mutant lines obtained via chemical mutagenesis.

The morphological, meiotic and chromosomal variability were studied in two cultivars of Calendula officinalis L. and their mutant lines obtained though chemical mutagenesis using diethyl sulphate (DES) (0.04%, 0.08%) and dimethyl sulphate (DMS) (0.025%, 0.05%). The studied cultivars displayed different sensitivity to DMS and DES mutagens. More M1 plants with morphological changes were observed in C. officinalis cv. 'Zolotoe more' than in cv. 'Rajskij sad'. DMS and DES at low concentrations had positive effects on main agro-metrical traits in both cultivars including plant height, inflorescence diameter and number of inflorescences per plant. Dose-dependent increase in number of various meiotic abnormalities was revealed in both mutant lines. Comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotype stability in M1 and M2 plants. Seed treatments with DMS and DES at certain concentrations resulted in higher yields of inflorescences in M1 plants compared to the control. In M2 generation, dose-dependent reduction in the yields of inflorescences was observed. Our findings demonstrate that DMS and DES at low concentrations have great potential in calendula mutation breeding.