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.

The appearance of a new scientific term is a significant event in the human cognitive process and the result of the realization of the separateness of an object or a phenomenon. Our article concentrates on the origins of basic genetic terms, such as genetics, gene, genotype, genome, gene pool, and genomics. We propose using the term karyogenomics for the special direction of genomics related to the study of the organization and evolution of eukaryotic genomes by means of modern chromosome analysis, as well as by full genome sequencing.

The appearance of a new scientific term is a significant event in the human cognitive process and the result of the realization of the separateness of an object or a phenomenon. Our article concentrates on the origins of basic genetic terms, such as genetics, gene, genotype, genome, gene pool, and genomics. We propose using the term karyogenomics for the special direction of genomics related to the study of the organization and evolution of eukaryotic genomes by means of modern chromosome analysis, as well as by full genome sequencing.

The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.

Polyploid wheats are represented by two evolutionary lineages – Emmer and Timopheevi. It was reported that the species of these groups differentiated by species-specific translocations; they showed distinct karyotype structures, i.e., the amount and the distribution of heterochromatin. Analysis of more than 1,500 accessions representing 21 wild and cultivated polyploid wheat species using C-banding revealed that intra- and interspecific divergence within these two groups was accompanied by chromosomal rearrangements. Intraspecific diversity was the highest for wild species, followed by landraces and commercial cultivars. Chromosomal rearrangements were more frequent in T. araraticum than in T. dicoccoides (55.7

The comparative studies of diploid Matricaria chamomilla L (2n=18) (Azulena and Sybirskaya bisabololnaya varieties), Matricaria discoidea L. (2n=18) and Matricaria inodora L. (2n=18) were carried out by DAPI/C-banding, FISH with 26S and 5S rDNA probes, the analysis of meiosis and composition of essential oil. Based on DAPI/C banding and FISH with 26S and 5S rDNA probes, all the chromosomes in karyotypes of the studied diploid chamomile species were identified, and generalized idiograms of chromosomes with account of all possibilities of DAPI/C-banding patterns as well as chromosomal localization of 26S and 5S rDNA sites were constructed. The results of comparative analysis of DAPI/C-banding and localization of ribosomal genes in the studied related chamomile species conformed to the hypothesis on monophyletic origin of their genomes. In most cases, we observed the classical type of meiosis with formation of 9 baculiform bivalents. Insignificant percentage of abnormalities suggested genetic stability of genomes of the studied species. The analysis of essential oil composition confirmed that M. discoidea could be used for medical purposes in common with M. chamomillа. The comparative analysis of interspecific karyotype differences and also the qualitative composition of essential oil allowed us to conclude that M d genome took an intermediate position between M ch and M i genomes, and M. discoidea was considerably closer to M. chamomillа than M. inodora.

SSAP method was used to study the genetic diversity of 22 Linum species from sections Linum, Adenolinum, Dasylinum, Stellerolinum, and 46 flax cultivars. All the studied flax varieties were distinguished using SSAP for retrotransposons FL9 and FL11. Thus, the validity of SSAP method was demonstrated for flax marking, identification of accessions in genebank collections, and control during propagation of flax varieties. Polymorphism of Fl1a, Fl1b, and Cassandra insertions were very low in flax varieties, but these retrotransposons were successfully used for the investigation of Linum species. Species clusterization based on SSAP markers was in concordance with their taxonomic division into sections Dasylinum, Stellerolinum, Adenolinum, and Linum. All species of sect. Adenolinum clustered apart from species of sect. Linum. The data confirmed the accuracy of the separation in these sections. Members of section Linum are not as closely related as members of other sections, so taxonomic revision of this section is desirable. L. usitatissimum accessions genetically distant from modern flax cultivars were revealed in our work. These accessions are of utmost interest for flax breeding and introduction of new useful traits into flax cultivars. The chromosome localization of Cassandra retrotransposon in Linum species was determined.

C помощью DAPI-бэндинга, флуоресцентной гибридизации in situ (FISH) с зондами 26S и 5S рДНК и анализа мейоза проведено сравнительное цитогенетическое изучение автотетраплоидного сорта Подмосковная ромашки аптечной Matricaria chamomilla L. (M. recutita L.) и дикорастущей тетраплоидной ромашки непахучей M. inodora L. В кариотипах обеих форм по рисункам DAPI-бэндинга и распределения 26S и 5S рДНК идентифицированы все хромосомы и построены видовые идиограммы M. chamomilla и M. inodora с учетом полиморфных вариантов рисунков DAPI-бэндинга и указанием расположения сайтов 26S и 5S рДНК.

Effective fertilizer application is necessary to increase crop yields and reduce risk of plant overdosing. It is known that expression level of microRNAs (miRNAs) alters in plants under different nutrient concentrations in soil. The aim of our study was to identify and characterize miRNAs with expression alterations under excessive fertilizer in agriculturally important crop – flax (Linum usitatissimum L.). We have sequenced small RNAs in flax grown under normal and excessive fertilizer using Illumina GAIIx. Over 14 million raw reads was obtained for two small RNA libraries. 84 conserved miRNAs from 20 families were identified. Differential expression was revealed for several flax miRNAs under excessive fertilizer according to high-throughput sequencing data. For 6 miRNA families (miR395, miR169, miR408, miR399, miR398 and miR168) expression level alterations were evaluated on the extended sampling using qPCR. Statistically significant up-regulation was revealed for miR395 under excessive fertilizer. It is known that target genes of miR395 are involved in sulfate uptake and assimilation. However, according to our data alterations of the expression level of miR395 could be associated not only with excess sulfur application, but also with redundancy of other macro- and micronutrients. Furthermore expression level was evaluated for miRNAs and their predicted targets. The negative correlation between miR399 expression and expression of its predicted target ubiquitin-conjugating enzyme E2 gene was shown in flax for the first time. So we suggested miR399 involvement in phosphate regulation in L. usitatissimum. Revealed in our study expression alterations contribute to miRNA role in flax response to excessive fertilizer.

С использованием биобаллистической трансформации гипокотильных эксплантов льна-долгунца сорта Василек получены первичные трансформанты, несущие генетическую конструкцию с химерным геном gfp-tua6. Жизнеспособные модифицированные растения послужили основой для создания самоопыленных линий, у которых подтверждено наследование введенной генетической конструкции при воспроизведении в течение трех генераций. У трансгенных растений изучены особенности фенологических стадий роста, высота растений, число коробочек и мейоз. Сравнение трансформированных линий по годам воспроизводства выявило значительное снижение образования семян у одной из них. Это послужило причиной проведения анализа мейоза этих линий на стадиях метафазы I и анафазы I. Показано, что процент клеток с нарушениями мейоза был самым высоким у трансгенных растений из линии с самой низкой семенной продуктивностью. Таким образом, неспецифическое встраивание генетической конструкции с использованием биобаллистической трансформации в геном трансформантов льна-долгунца в разной степени нарушают течение мейоза, что и является основной причиной неодинаковой воспроизводимости трансгенных линий льна-долгунца. Можно заключить, что для получения стабильно воспроизводящихся линий трасгенных растений целесообразно проводить у них систематический анализ мейоза.

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.

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.

С использованием биобаллистической трансформации гипокотильных эксплантов льна-долгунца сорта Василек получены первичные трансформанты, несущие генетическую конструкцию с химерным геном gfp-tua6. Жизнеспособные модифицированные растения послужили основой для создания самоопыленных линий, у которых подтверждено наследование введенной генетической конструкции при воспроизведении в течение трех генераций. У трансгенных растений изучены особенности фенологических стадий роста, высота растений, число коробочек и мейоз. Сравнение трансформированных линий по годам воспроизводства выявило значительное снижение образования семян у одной из них. Это послужило причиной проведения анализа мейоза этих линий на стадиях метафазы I и анафазы I. Показано, что процент клеток с нарушениями мейоза был самым высоким у трансгенных растений из линии с самой низкой семенной продуктивностью. Таким образом, неспецифическое встраивание генетической конструкции с использованием биобаллистической трансформации в геном трансформантов льна-долгунца в разной степени нарушают течение мейоза, что и является основной причиной неодинаковой воспроизводимости трансгенных линий льна-долгунца. Можно заключить, что для получения стабильно воспроизводящихся линий трасгенных растений целесообразно проводить у них систематический анализ мейоза.

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A(3) (CMA)/4',6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S-5.8S-25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S-5.8S-25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.

MicroRNAs (miRNAs) control a variety of biological processes and are involved in plant response to abiotic stress including phosphate starvation. Phosphate deficiency is one of the major limiting factors for crop yield. In the present work inorganic phosphate deficiency responsive miRNAs were identified in flax for the first time by high-throughput sequencing. Three conserved miRNA families (miR398, miR399 and miR408) were differentially expressed under inorganic phosphate starvation. In other plant species it was demonstrated that these miRNAs are involved in response to a combination of different stresses. In phosphate starvation conditions miR399 and miR408 are up-regulated but miR398 is differentially expressed. We found that in flax under inorganic phosphate starvation stress conditions expression level alterations for conserved miRNAs miR399 and miR408 were close to those in other plant species and for miR398 – to those in tomato but not in Arabidopsis. A better understanding of miRNAs stress regulatory functions would provide new tools for genetic improvement of stress tolerance in flax and other plants.