Amaranthus cruentus L. and Amaranthus hypochondriacus L. are valuable and promising food crops for multi-purpose use that are distributed worldwide in temperate, subtropical, and tropical zones. However, their karyotypes and genomic relationships still remain insufficiently studied. For the first time, a comparative repeatome analysis of A. cruentus and A. hypochondriacus was performed based on the available NGS data; bioinformatic analyses using RepeatExplorer/TAREAN pipelines; and chromosome FISH mapping of 45S rDNA, 5S rDNA, and the most abundant satellite DNAs. In the repeatomes of these species, interspecific variations in the amount of Ty3/Gypsy and Ty1/Copia retroelements, DNA transposons, ribosomal, and satellite DNA were detected. In the repeatomes of both species, shared satDNAs with high sequence similarity were identified. The chromosome distribution patterns of four effective molecular markers, 45S rDNA, 5S rDNA, AmC4, and AmC9, allowed us to identify all chromosome pairs in the species karyotypes, construct unique karyograms of A. cruentus and A. hypochondriacus, and confirm the close relationship between their genomes. These results are important for comparative karyotypic studies within the genus Amaranthus. Our findings demonstrated that cytogenomic analyses might provide important data on genomic relationships within Amaranthus and increase knowledge on genome organization in these valuable crops.

The genus Hedysarum L. (Fabaceae) includes about 200 species of annual and perennial herbs distributed in Asia, Europe, North Africa, and North America. Many species of this genus are valuable medicinal, melliferous, and forage resources. In this review, we consider the taxonomic history of the genus Hedysarum, the chromosomal organization of the species from the sections Hedysarum and Multicaulia, as well as phylogenetic relationships between these sections. According to morphological, genetic, and phylogenetic data, the genus Hedysarum is divided into three main sections: Hedysarum (= syn. Gamotion), Multicaulia, and Stracheya. In species of this genus, two basic chromosome numbers, x = 7 (section Hedysarum) and x = 8 (sections Multicaulia and Stracheya), were determined. The systematic positions of some species within the sections are still uncertain due to their morphological similarities. The patterns of distribution of molecular chromosomal markers (45S rDNA, 5S rDNA, and different satellite DNAs) in karyotypes of various Hedysarum species made it possible to determine their ploidy status and also specify genomic relationships within the sections Hedysarum and Multicaulia. Recent molecular phylogenetic studies clarified significantly the taxonomy and evolutionary development of the genus Hedysarum.

The cosmopolitan genus Hedysarum L. (Fabaceae) is divided into sections Hedysarum, Stracheya, and Multicaulia. This genus includes many valuable medicinal, melliferous, and forage species. The species taxonomy and genome relationships within the sections are still unclear. We examined intra- and interspecific diversity in the section (sect.) Hedysarum based on repeatome analyses using NGS data, bioinformatic technologies, and chromosome FISH mapping of 35S rDNA, 5S rDNA, and the identified satellite DNA families (satDNAs). A comparison of repeatomes of H. alpinum, H. theinum, and H. flavescens revealed differences in their composition. However, similarity in sequences of most satDNAs indicated a close relationship between genomes within sect. Hedysarum. New effective satDNA chromosomal markers were detected, which is important for karyotype analyses within Hedysarum. Intra- and interspecific variability in the chromosomal distribution patterns of the studied markers were revealed, and species karyograms were constructed. These results provided new insight into the karyotype structures and genomic diversity within sect. Hedysarum, clarified the systematic position of H. sachalinense and H. arcticum, and confirmed the distant genomic relationships between species from sections Hedysarum and Multicaulia. Our findings are important for further comparative genome studies within the genus Hedysarum.

Artificial hybrids between cultivated Avena species and wild Avena macrostachya that possess genes for resistance to biotic and abiotic stresses can be important for oat breeding. For the first time, a comprehensive study of genomes of artificial fertile hybrids Avena sativa × Avena macrostachya and their parental species was carried out based on the chromosome FISH mapping of satellite DNA sequences (satDNAs) and also analysis of intragenomic polymorphism in the 18S–ITS1–5.8S rDNA region, using NGS data. Chromosome distribution patterns of marker satDNAs allowed us to identify all chromosomes in the studied karyotypes, determine their subgenomic affiliation, and detect several chromosome rearrangements. Based on the obtained cytogenomic data, we revealed differences between two A. macrostachya subgenomes and demonstrated that only one of them was inherited in the studied octoploid hybrids. Ribotype analyses showed that the second major ribotype of A. macrostachya was species-specific and was not represented in rDNA pools of the octoploids, which could be related to the allopolyploid origin of this species. Our results indicate that the use of marker satDNAs in cytogenomic studies can provide important data on genomic relationships within Avena allopolyploid species and hybrids, and also expand the potential for interspecific crosses for breeding.

Background. Currently, it is quite relevant in breeding to create varieties with high yields of raw materials and the content of biologically active compounds. This work aim is to identify promising forms of P. caeruleum L. is for further inclusion in the breeding process based on biomorphological, anatomical, cytogenetic studies and the bioproductivity of colchicine-induced and wild-growing forms in comparison with the Lazur variety. Materials and methods. To obtain polyploids in 2018, the seeds of the P. caeruleum L. the Lazur variety were germinated in Petri dishes on filter paper at room temperature. When the roots reached the length of the seed, they were placed in different concentrations of colchicine solution 0.05 %, 0.1 %, 0.2 % and 0.52 % for 12 and 24 hours, then washed in running water for three hours. Results. The article for the first time presents data on a comparative study of three forms of Polemonium caeruleum L. in terms of morphological characteristics, microscopy of the leaf epidermis and cytogenetic analysis. It was established that the raw materials productivity of the underground part of the plants tetraploid forms in the C1 generation significantly exceeded the diploid and wild forms of plants by 32 %, as well as the yield of seeds by 22 %. In the C2 generation, the yield of raw materials (rhizomes with roots) in tetraploid plants increases in relation to the control by 56 %, seeds by 34 %. Quantitative determination of the amount of triterpene saponins in terms of β-escin in the plant material of three forms of P. caeruleum L. showed an excess of this indicator in the colchicine form by 24 % in the grass and by 36 % in rhizomes with roots, in the wild-growing form - by 15 % in the aerial part and decrease in the underground part by 9 % in relation to the control. It was found that plants of colchicine and wild forms differ in the presence of larger stomata compared to the control. Based on the pattern of DAPI-bandin and the distribution of 45S rDNA and 5S rDNA genes, a generalized species idiogram of blue cyanosis was constructed, taking into account polymorphic variants of DAPI-bandin patterns and indicating the location of the sites of 45S rDNA and 5S rDNA genes. The cytogenetic stability of the resulting tetraploid form was revealed. Conclusion. The article presents comparative studies on morphological characteristics, bioproductivity, microscopy of the leaf epidermis and cytogenetic analysis of the tetraploid form (colchicine) of P. caeruleum L. in comparison with the variety Lazur (diploid form) and the wild form of P. caeruleum L. plants.

Aim. Comparative study of the source material of four populations of Sedum roseum (L.) Scop. belonging to different ecological and geographical zones of origin and assessment of their suitability for cultivation in the conditions of the non‐chernozem zone of the Russian Federation.Materials and methods. The following populations were studied: the cultivated population of VILAR (Altai, 1980), the cultivated population of the Botanical Garden of Pitirim Sorokin Syktyvkar State University (Syktyvkar, Komi Republic), an introduced population from Kirovsk, N. A. Avrorin Polar‐Alpine Botanical Garden‐Institute and a cultivated population of Innsbruck Botanical Garden, Austria.Results. Morphological description of the samples was carried out and the productivity of raw materials and seeds and the main biological and karyological features of the populations studied were determined. The results of made it possible to identify Rhodiola populations characterized by high winter hardiness (A and D) and low winter hardiness (B and C). A high productivity of roots and rhizomes in the fourth year of life in populations C and A (30–31 g) was established, and in terms of the content of active substances (phenylpropanoids) in population B (Komi) – 5.4%. The numbers of chromosomes (2n=22) were determined and their morphometric analysis was carried out and formulas of karyotypes of the Rhodiola populations studied were compiled. Chromosomal analysis using C/DAPI‐differential staining showed that no chromosomal abnormalities were detected in the karyotypes, which indicates their stability.Conclusion. Morphological variability indicates the ecological plasticity of the populations studied, which indicates the prospects for their field cultivation.

A comparative analysis of genomes of six Hedysarum species was carried out using the rapid genomic in situ hybridization (rapidGISH) technique, which makes it possible to identify common highly repetitive DNA sequences, and also shows the patterns of their distribution on chromosomes of the studied species. Dispersed localization of genomic DNA of H. flavescens (sect. Hedysarum) was found on chromosomes of H. alpinum and H. theinum (both belonged to sect. Hedysarum). On chromosomes of H. neglectum from the section Hedysarum, clustered hybridization signals of genomic DNA of H. flavescens were observed. The localization of H. alpinum genomic DNA was found along the all chromosomes of H. flavescens and H. theinum. On chromosomes of H. grandiflorum and H. dahuricum (both from sect. Multicaulia), small clustered and dispersed hybridization signals of genomic DNA of H. flavescens and H. alpinum were found. Thus, in the studied species, common highly repetitive DNA sequences of different types of their chromosome organization were revealed. Our findings indicate the presence of common origin of the species from the sections Hedysarum and Multicaulia with different degree of their relationship.

Salvia officinalis L. (Lamiaceae) is a widespread economically valuable species. Due to the high content of essential oil, it is actively used in pharmaceutical, food and perfume industries. However, the chromosome structure and organization of its genome is still not well understood. For the first time, an analysis of intraspecific variability of S. officinalis was carried out based on the patterns of FISH localization of various DNA repeats in karyotypes of four species samples and two varieties from different habitats. Patterns of chromosome localization on of classical markers, 45S rDNA and 5S rDNA, confirmed the stability of genomes of the studied species samples and varieties. However, the intraspecific variability in localization of SO202 in karyotypes of the studied samples was found. In addition to the main set of chromosomes, B chromosomes were found in some karyotypes, including those containing 45S rDNA and SO202 clusters. The results of this study allowed us to identify all chromosome pairs in karyotypes and construct karyograms of the studied samples of S. officinalis. Our findings confirmed the stability of karyotypes of these samples according to the classical chromosomal markers but also demonstrated the presence of intraspecific variability in localization of the SO202 repeat, which can be used in breeding new varieties of S. officinalis.

For the first time, a comparative molecular cytogenetic study of four species of Calendula L. (Asteraceae): C. officinalis, C. stellata, C. tripterocarpa, and C. arvensis was carried out. In each species, chromosome numbers were determined: C. stellata (2n = 2х = 14), C. officinalis (2n = 4x = 32), C. tripterocarpa (2n = 2x = 30), and C. arvensis (2n = 4x = 44), and also specific chromosome localization of 45S and 5S rDNA clusters was revealed with the use of fluorescence in situ hybridization. An additional polymorphic minor 45S rDNA hybridization signal was found in the C. officinalis karyotype, which was located median in the short arm of one chromosome. The patterns of chromosome distribution of the major sites of 45S and 5S rDNA in karyotypes of the studied species confirmed the hybrid origin of C. arvensis (2n = 4x = 44) which could be a result of the introgressive hybridization of two other species: C. stellata (2n = 2x = 14) and C. tripterocarpa (2n = 2x = 30) during speciation.

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.

Within the complicated and controversial taxonomy of cosmopolitan genus Salvia L. (Lamiaceae) are valuable species Salvia officinalis L. and Salvia sclarea L., which are important for the pharmaceutical, ornamental horticulture, food, and perfume industries. Genome organization and chromosome structure of these essential oil species remain insufficiently studied. For the first time, the comparative repeatome analysis of S. officinalis and S. sclarea was performed using the obtained NGS data, RepeatExplorer/TAREAN pipelines and FISH-based chromosome mapping of the revealed satellite DNA families (satDNAs). In repeatomes of these species, LTR retrotransposons made up the majority of their repetitive DNA. Interspecific variations in genome abundance of Class I and Class II transposable elements, ribosomal DNA, and satellite DNA were revealed. Four (S. sclarea) and twelve (S. officinalis) putative satDNAs were identified. Based on patterns of chromosomal distribution of 45S rDNA; 5S rDNA and the revealed satDNAs, karyograms of S. officinalis and S. sclarea were constructed. Promising satDNAs which can be further used as chromosome markers to assess inter- and intraspecific chromosome variability in Salvia karyotypes were determined. The specific localization of homologous satDNA and 45S rDNA on chromosomes of the studied Salvia species confirmed their common origin, which is consistent with previously reported molecular phylogenetic data.

Subpolar and polar ecotypes of Deschampsia sukatschewii (Popl.) Roshev, D. cespitosa (L.) P. Beauv, and D. antarctica E. Desv. are well adapted to stressful environmental conditions, which make them useful model plants for genetic research and breeding. For the first time, the comparative repeatome analyses of subpolar and polar D. sukatschewii, D. cespitosa, and D. antarctica was performed using RepeatExplorer/TAREAN pipelines and FISH-based chromosomal mapping of the identified satellite DNA families (satDNAs). In the studied species, mobile genetic elements of class 1 made up the majority of their repetitive DNA; interspecific variations in the total amount of Ty3/Gypsy and Ty1/Copia retroelements, DNA transposons, ribosomal, and satellite DNA were revealed; 12–18 high confident and 7–9 low confident putative satDNAs were identified. According to BLAST, most D. sukatschewii satDNAs demonstrated sequence similarity with satDNAs of D. antarctica and D. cespitosa indicating their common origin. Chromosomal mapping of 45S rDNA, 5S rDNA, and satDNAs of D. sukatschewii allowed us to construct the species karyograms and detect new molecular chromosome markers important for Deschampsia species. Our findings confirmed that genomes of D. sukatschewii and D. cespitosa were more closely related compared to D. antarctica according to repeatome composition and patterns of satDNA chromosomal distribution.

GenBio2022: III Международная научно-практическая конференция «Геномика и современные биотехнологии в размножении

The section Multicaulia is the largest clade in the genus Hedysarum L. (Fabaceae). Representatives of the sect. Multicaulia are valuable plants used for medicinal and fodder purposes. The taxonomy and phylogeny of the sect. Multicaulia are still ambiguous. To clarify the species relationships within sect. Multicaulia, we, for the first time, explored repeatomes of H. grandiflorum Pall., H. zundukii Peschkova, and H. dahuricum Turcz. using next-generation sequencing technologies and a subsequent bioinformatic analysis by RepeatExplorer/TAREAN pipelines. The comparative repeatome analysis showed that mobile elements made up 20-24% (Class I) and about 2-2.5% (Class II) of their repetitive DNAs. The amount of ribosomal DNA varied from 1 to 2.6%, and the content of satellite DNA ranged from 2.7 to 5.1%. For each species, five high confident putative tandem DNA repeats and 5-10 low confident putative DNA repeats were identified. According to BLAST, these repeats demonstrated high sequence similarity within the studied species. FISH-based mapping of 35S rDNA, 5S rDNA, and satDNAs made it possible to detect new effective molecular chromosome markers for Hedysarum species and construct the species karyograms. Comparison of the patterns of satDNA localization on chromosomes of the studied species allowed us to assess genome diversity within the sect. Multicaulia. In all studied species, we revealed intra- and interspecific variabilities in patterns of the chromosomal distribution of molecular chromosome markers. In H. gmelinii Ledeb. and H. setigerum Turcz. ex Fisch. et Meyer, similar subgenomes were detected, which confirmed the polyploid status of their genomes. Our findings demonstrated a close genomic relationship among six studied species indicating their common origin and confirmed the taxonomic status of H. setigerum as a subspecies of H. gmelinii as well as the validity of combining the sect. Multicaulia and Subacaulia into one sect. Multicaulia.

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.

The genus Deschampsia P. Beauv. (Poaceae) involves a group of widespread polymorphic species, and many of them are highly tolerant to stressful environmental conditions. Genome diversity and chromosomal phylogeny within the genus are still insufficiently studied. Satellite DNAs, including CON/COM families, are the main components of the plant repeatome, which contribute to chromosome organization. For the first time, using PCR-based (Polymerase Chain Reaction) techniques and sequential BLAST (Basic Local Alignment Search Tool) and MSA (Multiple Sequence Alignment) analyses, we identified and classified CON/COM repeats in genomes of eleven Deschampsia accessions and three accessions from related genera. High homology of CON/COM sequences were revealed in the studied species though differences in single-nucleotide alteration profiles detected in homologous CON/COM regions indicated that they tended to diverge independently. The performed chromosome mapping of 45S rDNA, 5S rDNA, and CON/COM repeats in six Deschampsia species demonstrated interspecific variability in localization of these cytogenetic markers and facilitated the identification of different chromosomal rearrangements. Based on the obtained data, the studied Deschampsia species were distinguished into karyological groups, and MSA-based schematic trees were built, which could clarify the relationships within the genus. Our findings can be useful for further genetic and phylogenetic studies.

The systematic knowledge on the genus Hedysarum L. (Fabaceae: Hedysareae) is still incomplete. The species from the section Hedysarum are valuable forage and medicinal resources. For eight Hedysarum species, we constructed the integrated schematic map of their distribution within Eurasia based on currently available scattered data. For the first time, we performed cytogenomic characterization of twenty accessions covering eight species for evaluating genomic diversity and relationships within the section Hedysarum. Based on the intra- and interspecific variability of chromosomes bearing 45S and 5S rDNA clusters, four main karyotype groups were detected in the studied accessions: (1) H. arcticum, H. austrosibiricum, H. flavescens, H. hedysaroides, and H. theinum (one chromosome pair with 45S rDNA and one pair bearing 5S rDNA); (2) H. alpinum and one accession of H. hedysaroides (one chromosome pair with 45S rDNA and two pairs bearing 5S rDNA); (3) H. caucasicum (one chromosome pair with 45S rDNA and one chromosome pair bearing 5S rDNA and 45S rDNA); (4) H. neglectum (two pairs with 45S rDNA and one pair bearing 5S rDNA). The species-specific chromosomal markers detected in karyotypes of H. alpinum, H. caucasicum, and H. neglectum can be useful in taxonomic studies of this section.

For the first time, a comparative karyotype analysis of closely related species Hedysarum gmelinii andH. setigerum (Hedysarum section Multicaulia) grown in Southern Siberia, has been performed by molecular cytogeneticmarkers. Chromosome numbers in karyotypes of these species were specified – 2n = 4х = 32. In some accessions, additionalB chromosomes were revealed. FISH analyses indicated high similarities in chromosome morphology and also patternsof chromosomal distributions of 45S and 5S rDNA clusters in karyotypes of H. gmelinii and H. setigerum, which confirmsthe close relationship between their genomes.

The influence of chemical mutagens DMS (dimethyl sulphate) and DES (diethyl sulphate) in different concentrations on two cultivarsof Calendula officinalis L. (cv. 'Zolotoe more' and Rayskiy sad') was studied. The morphological, meiotic and cytological variability in their mutant lines (M1 and M2 generations) was estimated. More families (in percentage terms) with morphological changes were observed among MI plants of cv. 'Zolotoe more' compared to cv. `Rayskiy sad'. Between Calendula cultivars, significant differences in plant height, ramification degree and number of leaves were revealed after the DMS and DES treatment. In cv. 'Rayskiy sad', mutagenesis resulted in increase of number of inflorescences in MI and M2 generations. The analysis of meiosis indicated dose-dependent increase of polyvalent frequency in microsporocytes of mutant plants. The karyotype of the studied cultivars and mutant forms consists of 16 pairs of small-sized chromosomes (3.5-5.0 tim). In the studied C. officina/is karyotypes, any chromosomal rearrangements were not detected. The AgNOR staining revealed transcriptionally active (Ag-positive) regions in the secondary constriction areas of the satellite chromosomes.