Genome Variability in Artificial Allopolyploid Hybrids of Avena sativa L. and Avena macrostachya Balansa ex Coss. et Durieu Based on Marker Sequences of Satellite DNA and the ITS1-5.8S rDNA Region.

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.

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.

Integration of Repeatomic and Cytogenetic Data on Satellite DNA for the Genome Analysis in the Genus Salvia (Lamiaceae).

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.

A Pipeline NanoTRF as a New Tool for De Novo Satellite DNA Identification in the Raw Nanopore Sequencing Reads of Plant Genomes.

High-copy tandemly organized repeats (TRs), or satellite DNA, is an important but still enigmatic component of eukaryotic genomes. TRs comprise arrays of multi-copy and highly similar tandem repeats, which makes the elucidation of TRs a very challenging task. Oxford Nanopore sequencing data provide a valuable source of information on TR organization at the single molecule level. However, bioinformatics tools for de novo identification of TRs in raw Nanopore data have not been reported so far. We developed NanoTRF, a new python pipeline for TR repeat identification, characterization and consensus monomer sequence assembly. This new pipeline requires only a raw Nanopore read file from low-depth (<1×) genome sequencing. The program generates an informative html report and figures on TR genome abundance, monomer sequence and monomer length. In addition, NanoTRF performs annotation of transposable elements (TEs) sequences within or near satDNA arrays, and the information can be used to elucidate how TR−TE co-evolve in the genome. Moreover, we validated by FISH that the NanoTRF report is useful for the evaluation of TR chromosome organization­clustered or dispersed. Our findings showed that NanoTRF is a robust method for the de novo identification of satellite repeats in raw Nanopore data without prior read assembly. The obtained sequences can be used in many downstream analyses including genome assembly assistance and gap estimation, chromosome mapping and cytogenetic marker development.

Repeatome Analyses and Satellite DNA Chromosome Patterns in Deschampsia sukatschewii, D. cespitosa, and D. antarctica (Poaceae).

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.

Integration of Genomic and Cytogenetic Data on Tandem DNAs for Analyzing the Genome Diversity Within the Genus Hedysarum L. (Fabaceae).

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.

Clarification of the Position of Linum stelleroides Planch. within the Phylogeny of the Genus Linum L.

The phylogeny of members of the family Linaceae DC. ex Perleb has not been adequately studied. In particular, data on the phylogenetic relationship between Linum stelleroides Planch. and other representatives of the blue-flowered flax are very controversial. In the present work, to clarify this issue, we obtained DNA sequences of three nuclear loci (IGS and ITS1 + 5.8S rDNA + ITS2 of the 35S rRNA gene and the 5S rRNA gene) and eight chloroplast loci (rbcL, the trnL-trnF intergenic spacer, matK, the 3' trnK intron, ndhF, trnG, the psbA-trnH intergenic spacer, and rpl16) of 10 Linum L. species (L. stelleroides, L. hirsutum, L. perenne, L. leonii, L. lewisii, L. narbonense, L. decumbens, L. grandiflorum, L. bienne (syn. L. angustifolium), and L. usitatissimum) using high-throughput sequencing data. The phylogenetic analysis showed that L. stelleroides forms a basal branch in the blue-flowered flax clade. Previously found inconsistencies in the position of L. stelleroides and some other species in the Linaceae phylogenetic tree resulted from the erroneous species identification of some of the studied plant samples.

Cytogenomics of Deschampsia P. Beauv. (Poaceae) Species Based on Sequence Analyses and FISH Mapping of CON/COM Satellite DNA Families.

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.

Molecular Cytogenetics of Eurasian Species of the Genus Hedysarum L. (Fabaceae).

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.

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.

Molecular cytogenetics of valuable Arctic and sub-Arctic pasture grass species from the Aveneae/Poeae tribe complex (Poaceae).

BACKGROUND: Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships, and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of insufficiently studied pasture grass species (Poaceae) from related genera representing two neighboring clades: 1) Deschampsia and Holcus; 2) Alopecurus, Arctagrostis and Beckmannia, which are the primary fodder resources in the Arctic tundra. RESULTS: We constructed the integrated schematic maps of distribution of these species in the northern, central and eastern parts of Eurasia based on the currently available data as only scattered data on their occurrence is currently available. The species karyotypes were examined with the use of DAPI-banding, multicolour FISH with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid multocolour GISH with genomic DNAs of Deschampsia sukatschewii, Deschampsia flexuosa and Holcus lanatus belonging to one of the studied clades. Cytogenomic structures of the species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus. CONCLUSIONS: The peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.

Phenotypic, biochemical and genomic variability in generations of the rapeseed (Brassica napus L.) mutant lines obtained via chemical mutagenesis.

The phenotypic, biochemical and genetic variability was studied in M2-M5 generations of ethyl methansulfonat (EMS, 0.2%) mutagenized rapeseed lines generated from canola, '00', B. napus cv. Vikros. EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny resulted in selection of EMS populations of B. napus- and B. rapa-morphotypes. The seeds of the obtained mutant lines were high-protein, low in oil and stabilized in contents of main fatty acids which make them useful for feed production. Despite the increased level of various meiotic abnormalities revealed in EMS populations, comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotypic stability in M2-M5 plants, and therefore, the obtained mutant lines could be useful in further rapeseed improvement. The revealed structural chromosomal reorganizations in karyotypes of several plants of B. rapa-type indicate that rapeseed breeding by chemical mutagenesis can result in cytogenetic instability in the mutant progeny, and therefore, it should include the karyotype examination. Our findings demonstrate that EMS at low concentrations has great potential in rapeseed improvement.

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.

Molecular Cytogenetics of Pisum sativum L. Grown under Spaceflight-Related Stress.

The ontogenesis and reproduction of plants cultivated aboard a spacecraft occur inside the unique closed ecological system wherein plants are subjected to serious abiotic stresses. For the first time, a comparative molecular cytogenetic analysis of Pisum sativum L. (Fabaceae) grown on board the RS ISS during the Expedition-14 and Expedition-16 and also plants of their succeeding (F1 and F2) generations cultivated on Earth was performed in order to reveal possible structural chromosome changes in the pea genome. The karyotypes of these plants were studied by multicolour fluorescence in situ hybridization (FISH) with five different repeated DNA sequences (45S rDNA, 5S rDNA, PisTR-B/1, microsatellite motifs (AG)12, and (GAA)9) as probes. A chromosome aberration was revealed in one F1 plant. Significant changes in distribution of the examined repeated DNAs in karyotypes of the "space grown" pea plants as well as in F1 and F2 plants cultivated on Earth were not observed if compared with control plants. Additional oligo-(GAA)9 sites were detected on chromosomes 6 and 7 in karyotypes of F1 and F2 plants. The detected changes might be related to intraspecific genomic polymorphism or plant cell adaptive responses to spaceflight-related stress factors. Our findings suggest that, despite gradual total trace contamination of the atmosphere on board the ISS associated with the extension of the space station operating life, exposure to the space environment did not induce serious chromosome reorganizations in genomes of the "space grown" pea plants and generations of these plants cultivated on Earth.

Integration of Physical, Genetic, and Cytogenetic Mapping Data for Cellulose Synthase (CesA) Genes in Flax (Linum usitatissimum L.).

Flax, Linum usitatissimum L., is a valuable multi-purpose plant, and currently, its genome is being extensively investigated. Nevertheless, mapping of genes in flax genome is still remaining a challenging task. The cellulose synthase (CesA) multigene family involving in the process of cellulose synthesis is especially important for metabolism of this fiber crop. For the first time, fluorescent in situ hybridization (FISH)-based chromosomal localization of the CesA conserved fragment (KF011584.1), 5S, and 26S rRNA genes was performed in landrace, oilseed, and fiber varieties of L. usitatissimum. Intraspecific polymorphism in chromosomal distribution of KF011584.1 and 5S DNA loci was revealed, and the generalized chromosome ideogram was constructed. Using BLAST analysis, available data on physical/genetic mapping and also whole-genome sequencing of flax, localization of KF011584.1, 45S, and 5S rRNA sequences on genomic scaffolds, and their anchoring to the genetic map were conducted. The alignment of the results of FISH and BLAST analyses indicated that KF011584.1 fragment revealed on chromosome 3 could be anchored to linkage group (LG) 11. The common LG for 45S and 5S rDNA was not found probably due to the polymorphic localization of 5S rDNA on chromosome 1. Our findings indicate the complexity of integration of physical, genetic, and cytogenetic mapping data for multicopy gene families in plants. Nevertheless, the obtained results can be useful for future progress in constructing of integrated physical/genetic/cytological maps in L. usitatissimum which are essential for flax breeding.

Comparative molecular cytogenetic characterization of seven Deschampsia (Poaceae) species.

The genus Deschampsia P. Beauv (Poaceae) involves a group of widespread polymorphic species. Some of them are highly tolerant to stressful and variable environmental conditions, and D. antarctica is one of the only two vascular plants growing in Antarctic. This species is a source of useful for selection traits and a valuable model for studying an environmental stress tolerance in plants. Genome diversity and comparative chromosomal phylogeny within the genus have not been studied yet as karyotypes of most Deschampsia species are poorly investigated. We firstly conducted a comparative molecular cytogenetic analysis of D. antarctica (Antarctic Peninsula) and related species from various localities (D. cespitosa, D. danthonioides, D. elongata, D. flexuosa (= Avenella flexuosa), D. parvula and D. sukatschewii by fluorescence in situ hybridization with 45S and 5S rDNA, DAPI-banding and sequential rapid in situ hybridization with genomic DNA of D. antarctica, D. cespitosa, and D. flexuosa. Based on patterns of distribution of the examined markers, chromosomes of the studied species were identified. Within these species, common features as well as species peculiarities in their karyotypic structure and chromosomal distribution of molecular cytogenetic markers were characterized. Different chromosomal rearrangements were detected in D. antarctica, D. flexuosa, D. elongata and D. sukatschewii. In karyotypes of D. antarctica, D. cespitosa, D. elongata and D. sukatschewii, 0-3 B chromosomes possessed distinct DAPI-bands were observed. Our findings suggest that the genome evolution of the genus Deschampsia involved polyploidy and also different chromosomal rearrangements. The obtained results will help clarify the relationships within the genus Deschampsia, and can be a basis for the further genetic and biotechnological studies as well as for selection of plants tolerant to extreme habitats.

Identification, Expression Analysis, and Target Prediction of Flax Genotroph MicroRNAs Under Normal and Nutrient Stress Conditions.

Cultivated flax (Linum usitatissimum L.) is an important plant valuable for industry. Some flax lines can undergo heritable phenotypic and genotypic changes (LIS-1 insertion being the most common) in response to nutrient stress and are called plastic lines. Offspring of plastic lines, which stably inherit the changes, are called genotrophs. MicroRNAs (miRNAs) are involved in a crucial regulatory mechanism of gene expression. They have previously been assumed to take part in nutrient stress response and can, therefore, participate in genotroph formation. In the present study, we performed high-throughput sequencing of small RNAs (sRNAs) extracted from flax plants grown under normal, phosphate deficient and nutrient excess conditions to identify miRNAs and evaluate their expression. Our analysis revealed expression of 96 conserved miRNAs from 21 families in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of the identified miRNAs were transcribed from LIS-1. Expression of seven miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as most suitable for this purpose. Down-regulation of novel potential lus-miR-N1 and up-regulation of conserved miR399 were revealed under the phosphate deficient conditions. In addition, the negative correlation of expression of lus-miR-N1 and its predicted target, ubiquitin-activating enzyme E1 gene, as well as, miR399 and its predicted target, ubiquitin-conjugating enzyme E2 gene, was observed. Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide new insights into nutrient stress response regulation in plastic flax cultivars.

Molecular Cytogenetic Analysis of Deschampsia antarctica Desv. (Poaceae), Maritime Antarctic.

Deschampsia antarctica Desv. (Poaceae) (2n = 26) is one of the two vascular plants adapted to the harshest environment of the Antarctic. Although the species is a valuable model for study of environmental stress tolerance in plants, its karyotype is still poorly investigated. We firstly conducted a comprehensive molecular cytogenetic analysis of D. antarctica collected on four islands of the Maritime Antarctic. D. antarctica karyotypes were studied by Giemsa C- and DAPI/C-banding, Ag-NOR staining, multicolour fluorescence in situ hybridization with repeated DNA probes (pTa71, pTa794, telomere repeats, pSc119.2, pAs1) and the GAA simple sequence repeat probe. We also performed sequential rapid in situ hybridization with genomic DNA of D. caespitosa. Two chromosome pairs bearing transcriptionally active 45S rDNA loci and five pairs with 5S rDNA sites were detected. A weak intercalary site of telomere repeats was revealed on the largest chromosome in addition to telomere hybridization signals at terminal positions. This fact confirms indirectly the hypothesis that chromosome fusion might have been the cause of the unusual for cereals chromosome number in this species. Based on patterns of distribution of the examined molecular cytogenetic markers, all chromosomes in karyotypes were identified, and chromosome idiograms of D. antarctica were constructed. B chromosomes were found in most karyotypes of plants from Darboux Island. A mixoploid plant with mainly triploid cells bearing a Robertsonian rearrangement was detected among typical diploid specimens from Great Jalour Island. The karyotype variability found in D. antarctica is probably an expression of genome instability induced by environmental stress factors. The differences in C-banding patterns and in chromosome distribution of rDNA loci as well as homologous highly repeated DNA sequences detected between genomes of D. antarctica and its related species D. caespitosa indicate that genome reorganization involving coding and noncoding repeated DNA sequences had occurred during the divergence of these species.

A Set of Cytogenetic Markers Allows the Precise Identification of All A-Genome Chromosomes in Diploid and Polyploid Wheat.

Karyotypes of 3 diploid wheat species containing different variants of the A-genome, Triticum boeoticum (A(b)), T. monococcum (A(b)), and T. urartu (A(u)), were examined using C-banding and FISH with DNA probes representing 5S and 45S rDNA families, the microsatellite sequences GAAn and GTTn, the already known satellite sequences pSc119.2, Spelt52, Fat, pAs1, and pTa535, and a newly identified repeat called Aesp_SAT86. The C-banding patterns of the 3 species in general were similar; differences were observed in chromosomes 4A and 6A. Besides 2 major 45S rDNA loci on chromosomes 1A and 5A, 2 minor polymorphic NORs were observed in the terminal part of 5AL and in the distal part of 6AS in all species. An additional minor locus was found in the distal part of 7A(b)L of T. boeoticum and T. monococcum, but not in T. urartu. Two 5S rDNA loci were observed in 1AS and 5AS. The pTa535 probe displayed species- and chromosome-specific hybridization patterns, allowing complete chromosome identification and species discrimination. The distribution of pTa535 on the A(u)-genome chromosomes was more similar to that on the A-genome chromosomes of T. dicoccoides and T. araraticum, thus confirming the origin of these genomes from T. urartu. The probe pAs1 allowed the identification of 4 chromosomes of T. urartu and 2 of T. boeoticum or T. monococcum. The Aesp_SAT86-derived patterns were polymorphic; main clusters were observed on chromosomes 1A(u )and 3A(u) of T. urartu and chromosomes 3A(b) and 6A(b) of T. boeoticum. Thus, a set of probes, pTa535, pAs1, GAAn and GTTn, pTa71, pTa794, and Aesp_SAT86, proved to be most informative for the analysis of A-genomes in diploid and polyploid wheat species.