[Genotype-Specific Features of Cold-Induced Sweetening Process Regulation in Potato Varieties Nikulinsky, Symfonia, and Nevsky].

In this study, we performed expression analysis of genes associated with cold-induced sweetening in potato tubers: vacuolar invertase (Pain-1), sucrose synthase (SUS4), and invertase inhibitor (InvInh2). Potato varieties Nikulinsky, Symfonia, and Nevsky were used. All three varieties were found to accumulate sugars at low temperatures; the maximum accumulation of reducing sugars was observed at 4°C. It was found that the expression pattern of genes associated with cold-induced sweetening differs depending on the variety and storage duration. The increased expression of vacuolar invertase and its inhibitor is more pronounced at the beginning of storage period, whereas the increased expression of sucrose synthase is more pronounced after 3 months of storage. At early storage periods, high expression of invertase and low expression of inhibitor is observed in the Dutch variety Symfonia, and vice versa in the Russian varieties Nikulinsky and Nevsky. The involvement of the studied genes in the process of cold-induced sweetening is discussed.

Diversification of the Homoeologous Lr34 Sequences in Polyploid Wheat Species and Their Diploid Progenitors.

Allopolyploidization induces a multiple processes of genomic reorganization, including the structurally functional diversification of the homoeologous genes. An example of such diversification is the appearance of the Lr34 gene on chromosome 7D of bread wheat T. aestivum (BAD), the gene conferring durable, race non-specific protection against three fungal pathogens. In this study, we focused on the variability of a functionally critical region between exons 10-12 of Lr34 among diploid progenitors of wheat genomes and their respective polyploids. In the diploid A-genome species, two basic forms of the studied region have been revealed: (1) non-functional forms containing stop codons, or/and frameshifts (T. monococcum/T. urartu) and (2) forms with no such a mutations (T. boeoticum). The Lr34 sequence of T. urartu containing a TGA stop codon was inherited by the first tetraploid T. dicoccoides (BA), and then reorganized in some accessions of this species due to the insertion of an LTR retroelement in exon 10. Besides T. boeoticum, the second form of the Lr34 sequence is also characteristic of A. speltoides, which presumably donated this form to all polyploid descendants bearing B-genome. No differences were found between the D-genome-specific Lr34 sequences studied here and downloaded from databases, implying the highest level of conservation of the Lr34 predecessor throughout evolution. The sequence data were later used to construct phylograms, and apparent peculiarities in the evolution of the studied region of Lr34 genes discussed.

The effect of two differentially expressed wheat VRN-B1 alleles on the heading time is associated with structural variation in the first intron.

We measured the level of VRN-B1 transcripts within near-isogenic lines of Triticum aestivum cultivar 'Bezostaya 1' carrying two VRN-B1 alleles from cultivars 'Saratovskaya 29' and 'Diamant 2' (VRN-B1c and VRN-B1a, respectively). Both lines have similar kinetics of VRN-B1 transcript accumulation at the third to fifth leaf stages. However, quantitative PCR analysis at the third leaf stage showed that the VRN-B1c allele was transcribed 10 times faster than the VRN-B1a allele. The F1 hybrid between the lines is more similar to the line carrying the VRN-B1a allele with regard to the level of VRN-B1 transcription. The difference in the transcript levels of the VRN-B1 alleles appears to be due to the previously identified structural changes in the first intron of VRN-B1c (deletion of 0.8 kb and duplication of 0.4 kb) as compared with VRN-B1a, as no other differences were revealed in the present study. We suggested that the first intron structural changes in the VRN-B1c allele as compared with VRN-B1a result in a higher level of VRN-B1 transcripts and an earlier heading time. We confirmed that the expression of the dominant VRN-1 gene induces transcription of the recessive homoeoalleles, coupled with the reduction of the transcript level of a flowering repressor VRN-2.

[Epigenetic regulation of expression of vernalization genes].

We reviewed the mechanisms of epigenetic regulation of vernalization genes of plants which control the transition to the generative stage, depending on the low temperatures. Based on the example of FLC-gene of Arabidopsis, the involvement of the PR2 repression complex and non-coding RNAs in these mechanisms has been shown. On the basis of our own and other data, the authors suggest a similar mechanism of regulation of Vrn-1 gene of cereals.

Chitosan and its derivatives as promising plant protection tools.

In modern conditions, the increase in the yield of agricultural crops is provided not by expanding the areas of their cultivation, but mainly by introducing advanced technologies. The most effective strategy for this purpose is the development of genetically resistant and productive cultivars in combination with the use of a variety of plant protection products (PPPs). However, traditional, chemical PPPs, despite their effectiveness, have significant drawbacks, namely, pollution of environment, ecological damage, toxicity to humans. Recently, biological PPPs based on natural compounds have attracted more attention, since they do not have these disadvantages, but at the same time they can be no less effective. One of such agents is chitosan, a deacetylation product of chitin, one of the most common polysaccharides in nature. The high biological activity, biocompatibility, and safety of chitosan determine the breadth and effectiveness of its use in medicine, industry, and agrobiology. The review considers various mechanisms of action of chitosan as a biopesticide, including both a direct inhibitory effect on pathogens and the induction of plant internal defense systems as a result of chitosan binding to cell surface receptors. The effect of chitosan on the formation of resistance to the main classes of pathogens: fungi, bacteria, and viruses has been shown on a variety of plant objects. The review also discusses various ways of using chitosan: for the treatment of seeds, leaves, fruits, soil, as well as its specific biological effects corresponding to these ways. A separate chapter is devoted to protection products based on chitosan, obtained by its chemical modifications, or by means of combining of a certain molecular forms of chitosan with various substances that enhance its antipathogenic effect. The data presented in the review generally give an idea of chitosan and its derivatives as very effective and promising plant protection products and biostimulants.

[The impact of Ty3-gypsy group retrotransposon Lila on D-genome specificity of wheat Triticum aestivum L].

An analysis of the primary structure of BAC clone 112D20 T. aestivum, that contains D-genome specific Ty3-gypsy-retrotransposon Lila is presented. PCR analysis of nulli-tetrasomic and deletion lines of T. aestivum allowed to localize this BAC clone in the distal region of the long arm of chromosome 5D. Characteristic feature of BAC clone 112D20 is a high concentration of Ty3-gypsy-retrotransposons (61.7%), and low content of the genes (1.2%). Only a single open reading frame was revealed homologous to an unknown gene of Ae. tauschii. Specific to the D-genome Ty3-gypsy-retrotransposon Lila in the BAC clone 112D20 is 14 kb in length and contains unequal in size long terminal repeats. The data of in situ hybridization and PCR analysis of different Triticeae species suggest that this retroelement was amplified within the ancestral species of Ae. tauschii, the donor D-genome. The suggested time of amplification based on estimation of insertion time of Lila 112D20 is 1.7 million years, which corresponds to the formation of the first allopolyploid forms of wheat. Based on comparison with the previously obtained data, it is concluded that the amplification ofretroelements specific to each genome of wheat took place during formation of the diploid progenitors of these genomes.

Development of a marker panel for genotyping of domestic soybean cultivars for genes controlling the duration of vegetation and response to photoperiod.

Soybean, Glycine max L., is one of the most important agricultural crops grown in a wide range of latitude. In this regard, in soybean breeding, it is necessary to pay attention to the set of genes that control the transition to the f lowering stage, which will make it possible to adapt genotypes to local growing conditions as accurately as possible. The possibilities of soybean breeding for this trait have now signif icantly expanded due to identif ication of the main genes (E1-E4, GmFT2a, GmFT5a) that control the processes of f lowering and maturation in soybean, depending on the day length. The aim of this work was to develop a panel of markers for these genes, which could be used for a rapid and eff icient genotyping of domestic soybean cultivars and selection of plant material based on sensitivity to photoperiod and the duration of vegetation. Combinations of 10 primers, both previously developed and our own, were tested to identify different alleles of the E1-E4, GmFT2a, and GmFT5a genes using 10 soybean cultivars from different maturity groups. As a result, 5 combinations of dominant and recessive alleles for the E1-E4 genes were identif ied: (1) e1-nl(e1-as)/ e2-ns/e3-tr(e3-fs)/e4; (2) e1-as/e2-ns/e3-tr/E4; (3) e1-as/e2-ns/E3-Ha/e4; (4) E1/e2-ns/e3-tr/E4; (5) e1-nl/e2-ns/E3-Ha/E4. The studied cultivars contained the most common alleles of the GmFT2a and GmFT5a genes, with the exception of the 'Cassidi' cultivar having a rare dominant allele GmFT5a-H4. The degree of earliness of cultivars positively correlated with the number of recessive genes E1-E4, which is consistent with the data of foreign authors on different sets of cultivars from Japan and North China. Thus, the developed panel of markers can be successfully used in the selection of soybean for earliness and sensitivity to photoperiod.

[Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops].

By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization.

[Genomic changes at early stages of formation of allopolyploid Aegilops longissima x Triticum urartu].

Using the model of synthetic allopolyploid Aegilops longissima TL05 x Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing of PCR spectra obtained while amplifying genome DNA of allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for subtelomere DNA sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some PCR fragments typical for parental genome in RAPD specters of allopolyploid TL05 x TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomere primers such changes in PSR specters were shown only for one of 66 pays of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in specter of synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.

[Intraspecific divergence in wheats of the Timopheevi group as revealed by in situ hybridization with tandem repeats of the Spelt1 and Spelt52 families].

Fluorescent in situ hybridization (FISH) was used to study the distribution of the Spelt1 and Spelt52 repetitive DNA sequences on chromosomes of ten accessions representing three polyploid wheat species of the Timopheevi group: Triticum araraticum (7), T. timopheevii (2), and T. kiharae (1). Sequences of both families were found mostly in the subtelomeric chromosome regions of the G genome. The total number of Spelt1 sites varied from 8 to 14 in the karyotypes of the species under study; their number, location, and size differed among the seven T. araraticum accessions and were the same in the two T. timopheevii accessions and T. kiharae, an amphidiploid T. timopheevii-Aegilops tauschii hybrid. The Spelt52 tandem repeat was detected in the subtelomeric regions of chromosomes 1-4; its sites did not coincide with the Spelt1 sites. The chromosome distribution and signal intensity of the Spelt52 repeats varied in T. araraticum and were the same in T. timopheevii and T. kiharae. The chromosome distributions of the Spelt1 and Spelt52 repeats were compared for the polyploid wheats of the Timopheevi group and diploid Ae. speltoides, a putative donor of the G genome. The comparison revealed a decrease in hybridization level: both the number of sites per genome and the size of sites were lower. The decrease was assumed to result from repeat elimination during polyploidization and subsequent evolution of wheat and from the founder effect, since the origin of Timopheevi wheats might involve the genotype of Ae. speltoides, which is highly polymorphic for the distribution of Spelt1 and Spelt52 sequences and is similar in the chromosome location of the repeats to modern wheat.

[Structural features of the modified BARE-retroelement in the barley (Hordeum vulgare L.) genome]. / Osobennosti struktury modifitsirovannogo BARE-retroélementa genoma iachmenia (Hordeum vulgare L.).

The primary structure of the 4.2-kb BamHI-fragment occurring abundantly in the genome of barley Hordeum vulgare was determined. By means of computer analysis, considerable homology was found between this fragment and the copia-like BARE-l retrotransposon studied earlier. A unique distinction of the BamHI fragment is its symmetrical structure caused by the presence of two mutually inverted parts, each of which is homologous to a long region of BARE-l including a 5'-LTR (long terminal repeat) and the adjacent leader sequence. No sequences homologous to the coding domains of BARE-l were revealed in the fragment. However, potentially functional signals were found: TATA boxes and primer-binding sites (PBS) exhibiting statistically significant homology to the corresponding regulatory signals of known retroelements. Thus, we have revealed and characterized a repeated element of the H. vulgare genome that is a markedly modified derivative of the BARE family of retrotransposons of this genome.

[Analysis of DNA markers specific for G-genome of wheat]. / Analiz DNK-markera, spestifichnogo dlia G-genoma pshenitsy.

A RAPD marker specific for the G genome of wheat was identified. The corresponding 1171-bp DNA sequence was cloned and analyzed. Screening of the database did not reveal any homologies with the known plant DNA sequences. Using the primers specific to the flanking regions of the marker sequence, PCR analysis of the polyploid wheat species and the diploid species of the section Sitopsis was carried out. In addition, using the cloned sequence as a molecular hybridization probe, RFLP analysis of the genomic DNA of these species was performed.

Genetic and epigenetic changes of rDNA in a synthetic allotetraploid, Aegilops sharonensis x Ae. umbellulata.

The synthetic allotetraploid Aegilops sharonensis x Ae. umbellulata (genomic formula S(sh)U) was used to study inheritance and expression of 45S rDNA during early stages of allopolyploid formation. Using silver staining, we revealed suppression of the NORs (nucleolar organizing regions) from the S(sh) genome in response to polyploidization. Most allopolyploid plants of the S(2)-S(4) generations retained the chromosomal location of 45S rDNA typical for the parental species, except for two S(3) plants in which a deletion of the rDNA locus on one of the homologous 6S(sh) chromosomes was revealed. In addition, we found a decrease in NOR signal intensity on both 6S(sh) chromosomes in a portion of the S(3) and S(4) allopolyploid plants. As Southern hybridization showed, the allopolyploid plants demonstrated additive inheritance of parental rDNA units together with contraction of copy number of some rDNA families inherited from Ae. sharonensis. Also, we identified a new variant of amplified rDNA unit with MspAI1 restriction sites characteristic of Ae. umbellulata. These genetic alterations in the allopolyploid were associated with comparative hypomethylation of the promoter region within the Ae. umbellulata-derived rDNA units. The fast uniparental elimination of rDNA observed in the synthetic allopolyploid agrees well with patterns observed previously in natural wheat allotetraploids.

BARE-ID, a representative of a family of BARE-like elements of the barley genome.

In our search for transposable elements in barley, Hordeum vulgare, we have isolated and cloned two BamHI-fragments of 4.7 and 4.2 kb in length containing very abundant DNA sequences. The 4.7 kb fragment is homologous to the extended region, including more than half of the 5'-LTR and some part of the coding domain of BARE-1, a member of copia-like retrotransposon family of barley. The 4.2 kb fragment, bearing homology to BARE-1 and the WIS-2 family isolated from wheat, is unique among studied retroelements of cereals because it consists of two inverted parts, each containing homology to the LTR and UTL of BARE-1. Functional motifs for reverse transcription, two TATA-boxes and two primer-binding sites, were found within the LTRs. The element contained within this fragment was generated by significant rearrangement of a BARE-like retrotransposon, which included inversion of the extended 5'-terminal region and deletion of the internal domain. Therefore this element is named BARE-ID (BARE-inverted, deleted). A family of BARE-like elements is amplified in the H. vulgare genome compared with wild barley species. The terminal inverted repeat of BARE-ID was used as a probe for examination of evolutionary diversity within genus Hordeum. Our data are basically in agreement with the modern classification system. However, they do not support the combination of H. vulgare and H. bulbosum into one group with the same type of genome. New data concerning the possible origin of the polyploid species, H. secalinum, confirm that retrotransposons are a useful tool for phylogenetic studies.

Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis complex.

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 x 10(4) in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex.

Diversity in the integrase coding domain of a gypsy-like retrotransposon among wild relatives of rice in the Oryza officinalis complex.

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75-90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex.