Alexandra V. Amosova

Genome differentiation in Aegilops. 3. Evolution of the D-genome cluster

Abstract. Six polyploid Aegilops species containing the D genome were studied by C-banding and fluorescence in situ hybridization (FISH) using clones pTa71 (18S-5.8S-26S rDNA), pTa794 (5S rDNA), and pAs1 (non-coding repetitive DNA sequence) as probes. The C-banding and pAs1-FISH patterns of Ae. cylindrica chromosomes were identical to those of the parental species. However, inactivation of the NOR on chromosome 5D with a simultaneous decrease in the size of the pTa71-FISH site was observed. The Nv and Dv genomes of Ae. ventricosa were somewhat modified as compared with the N genome of Ae. uniaristata and the D genome of Ae. tauschii. Modifications included minor changes in the C-banding and pAs1-FISH patterns, complete deletion of the NOR on chromosome 5Dv, and the loss of several minor 18S-5.8S-26S rDNA loci on Nv genome chromosomes. According to C-banding and FISH analyses, the Dcr1 genome of Ae. crassa is more similar to the Dv genome of Ae. ventricosa than to the D genome of Ae. tauschii. Mapping of the 18S-5.8S-26S rDNA and 5S rDNA loci by multicolor FISH suggests that the second (Xcr) genome of tetraploid Ae. crassa is a derivative of the S genome (section Emarginata of the Sitopsis group). Both genomes of Ae. crassa were significantly modified as the result of chromosomal rearrangements and redistribution of highly repetitive DNA sequences. Hexaploid Ae. crassa and Ae. vavilovii arose from the hybridization of chromosomal type N of tetraploid Ae. crassa with Ae. tauschii and Ae. searsii, respectively. Chromosomal type T1 of tetraploid Ae. crassa and Ae. umbellulata were the ancestral forms of Ae. juvenalis. The high level of genome modification in Ae. juvenalis indicates that it is the oldest hexaploid species in this group. The occurrence of hexaploid Ae. crassa was accompanied by a species-specific translocation between chromosomes 4Dcr1 and 7Xcr. No chromosome changes relative to the parental species were detected in Ae. vavilovii, however, its intraspecific diversity was accompanied by a translocation between chromosomes 3Xcr and 3Dcr1.

9-Aminoacridine: An efficient reagent to improve human and plant chromosome banding patterns and to standardize chromosome image analysis

Successful automated chromosome analysis requires the development of new techniques to increase and standardize chromosome length and improve banding patterns.

Genetic and epigenetic changes of rDNA in a synthetic allotetraploid, Aegilops sharonensis × 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.

Genome Comparisons with Chromosomal and Molecular Markers for Three Closely Related Flax Species and Their Hybrids

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimumL., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienneMill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1–3 μm), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. InL. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed thatL. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifoliumbeing closest to it.

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the Section Linum (Syn. Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18S–5.8S–26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA were colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum, L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.

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.

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 (Ab), T. monococcum (Ab), and T. urartu (Au), 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 7AbL 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 Au-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 1Au and 3Au of T. urartu and chromosomes 3Ab and 6Ab 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.

Retrotransposon-based molecular markers for analysis of genetic diversity within the Genus Linum.

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.

The diversity of karyotypes and genomes within section Syllinum of the Genus Linum (Linaceae) revealed by molecular cytogenetic markers and RAPD analysis.

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.

Excess fertilizer responsive miRNAs revealed in Linum usitatissimum L.

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.