Short Interrupted Repeat Cassette (SIRC)-Novel Type of Repetitive DNA Element Found in Arabidopsis thaliana.

Short interrupted repeat cassette (SIRC)-a novel DNA element found throughout the A. thaliana nuclear genome. SIRCs are represented by short direct repeats interrupted by diverse DNA sequences. The maxima of SIRC's distribution are located within pericentromeric regions. We suggest that originally SIRC was a special case of the complex internal structure of the miniature inverted repeat transposable element (MITE), and further MITE amplification, transposition, and loss of terminal inverted repeats gave rise to SIRC as an independent DNA element. SIRC sites were significantly enriched with several histone modifications associated with constitutive heterochromatin and mobile genetic elements. The majority of DNA-binding proteins, strongly associated with SIRC, are related to histone modifications for transcription repression. A part of SIRC was found to overlap highly inducible protein-coding genes, suggesting a possible regulatory role for these elements, yet their definitive functions need further investigation.

Targeting the B1 Gene and Analysis of Its Polymorphism Associated with Awned/Awnless Trait in Russian Germplasm Collections of Common Wheat.

The presence of awns on the ear is associated with a number of important plant properties, such as drought resistance, quality of the grain mass during processing, etc. The main manifestations of this trait are controlled by the B1 gene, which has recently been identified and encodes the C2H2 zinc finger transcription factor. Based on the previously identified SNPs in the promoter region of this gene, we constructed markers for dominant and recessive alleles which determine awnless and awned phenotypes, respectively. The markers were successful for use in targeting the respective alleles of the B1 gene in 176 varieties of common wheat, accessions of T. spelta L., as well as on F2/F3 hybrids from crosses between awned and awnless forms of T. aestivum. We first identified a new allele, b1mite, which has both an insert of a miniature Stowaway-like transposon, 261 bp in length, and 33 novel SNPs in the promoter region. Despite these changes, this allele had no effect on the awned phenotype. The possible mechanisms of the influence of the analyzed gene on phenotype are discussed.

Genetic Features of Triticale-Wheat Hybrids with Vaviloid-Type Spike Branching.

Vaviloid spike branching, also called sham ramification, is a typical trait of Triticum vavilovii Jakubz. and is characterized by a lengthening of the spikelet axis. In this article, we present the results of a study of three triticale-wheat hybrid lines with differences in terms of the manifestation of the vaviloid spike branching. Lines were obtained by crossing triticale with hexaploid wheat, T. aestivum var. velutinum. The parental triticale is a hybrid of synthetic wheat (T. durum × Ae. tauschii var. meyrei) with rye, S. cereale ssp. segetale. Line 857 has a karyotype corresponding to hexaploid wheat and has a spike morphology closest to normal, whereas Lines 808/1 and 844/4 are characterized by the greatest manifestation of vaviloid spike branching. In Lines 808/1 and 844/4, we found the substitution 2RL(2DL). The karyotypes of the latter lines differ in that a pair of telocentric chromosomes 2DS is detected in Line 808/1, and these telocentrics are fused into one unpaired chromosome in Line 844/4. Using molecular genetic analysis, we found a deletion of the wheat domestication gene Q located on 5AL in the three studied hybrid lines. The deletion is local since an analysis of the adjacent gene B1 showed the presence of this gene. We assume that the manifestation of vaviloid spike branching in two lines (808/1 and 844/4) is associated with a disturbance in the joint action of genes Q and AP2L2-2D, which is another important gene that determines spike morphology and is located on 2DL.

Drought Stress Tolerance and Photosynthetic Activity of Alloplasmic Lines T. dicoccum x T. aestivum.

Tetraploid species T. dicoccum Shuebl is a potential source of drought tolerance for cultivated wheat, including common wheat. This paper describes the genotyping of nine stable allolines isolated in the offspring from crossing of T. dicoccum x T. aestivum L. using 21 microsatellite (simple sequence repeats-SSR) markers and two cytoplasmic mitochondrial markers to orf256, rps19-p genes; evaluation of drought tolerance of allolines at different stages of ontogenesis (growth parameters, relative water content, quantum efficiency of Photosystem II, electron transport rate, energy dissipated in Photosystem II); and the study of drought tolerance regulator gene Dreb-1 with allele-specific PCR (AS-MARKER) and partial sequence analysis. Most allolines differ in genomic composition and T. dicoccum introgressions. Four allolines-D-b-05, D-d-05, D-d-05b, and D-41-05-revealed signs of drought tolerance of varying degrees. The more drought tolerant D-41-05 line was also characterized by Dreb-B1 allele introgression from T. dicoccum. A number of non-specific patterns and significant differences in allolines in regulation of physiological parameters in drought conditions is identified. Changes in photosynthetic activity in stress-drought are shown to reflect the level of drought tolerance of the forms studied. The contribution of different combinations of nuclear/cytoplasmic genome and alleles of Dreb-1 gene in allolines to the formation of stress tolerance and photosynthetic activity is discussed.

Evolution of VRN-1 homoeologous loci in allopolyploids of Triticum and their diploid precursors.

BACKGROUND: The key gene in genetic system controlling the duration of the vegetative period in cereals is the VRN1 gene, whose product under the influence of low temperature (vernalization) promotes the transition of the apical meristem cells into a competent state for the development of generative tissues of spike. As early genetic studies shown, the dominant alleles of this gene underlie the spring forms of plants that do not require vernalization for this transition. In wheat allopolyploids various combinations of alleles of the VRN1 homoeologous loci (VRN1 homoeoalleles) provide diversity in such important traits as the time to heading, height of plants and yield. Due to genetical mapping of VRN1 loci it became possible to isolate the dominant VRN1 alleles and to study their molecular structure compared with the recessive alleles defining the winter type of plants. Of special interest is the process of divergence of VRN1 loci in the course of evolution from diploid ancestors to wheat allopolyploids of different levels of ploidy. RESULTS: Molecular analysis of VRN1 loci allowed to establish that various dominant alleles of these loci appeared as a result of mutations in two main regulatory regions: the promoter and the first intron. In the diploid ancestors of wheat, especially, in those of A- genome (T. boeoticum, T. urartu), the dominant VRN1 alleles are rare in accordance with a limited distribution of spring forms in these species. In the first allotetraploid wheat species including T. dicoccoides, T. araraticum (T. timopheevii), the spring forms were associated with a new dominant alleles, mainly, within the VRN-A1 locus. The process of accumulation of new dominant alleles at all VRN1 loci was significantly accelerated in cultivated wheat species, especially in common, hexaploid wheat T. aestivum, as a result of artificial selection of spring forms adapted to different climatic conditions and containing various combinations of VRN1 homoeoalleles. CONCLUSIONS: This mini-review summarizes data on the molecular structure and distribution of various VRN1 homoeoalleles in wheat allopolyploids and their diploid predecessors.

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

BACKGROUND: The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. RESULTS: Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. CONCLUSION: A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

Identification of new heading date determinants in wheat 5B chromosome.

BACKGROUND: Variability of heading date may assist in wheat adaptation to local environments. Thereafter, discovery of new heading date determinants is important for cereal improvement. In this study we used common wheat cultivar Chinese Spring (CS) and the substitution line of CS with 5B chromosome from T. dicoccoides (CS-5Bdic), different in their heading date by two weeks, to detect determinants of heading date on 5B chromosome. RESULTS: The possible influence of the VRN-B1 gene, the most powerful regulator of flowering, located on 5B chromosome, to differences in heading time between CS and CS-5Bdic was studied. The sequencing of this gene from CS-5Bdic showed that an insertion of a nucleotide triplet produced an additional amino acid in the corresponding protein. No changes in the transcription levels of each homoeologous VRN-1 loci were found in CS-5Bdic by comparison with CS. To ascertain the loci determining heading date difference, a set of 116 recombinant inbred 5В chromosomal lines as a result of hybridization of CS with CS-5Bdic were developed and their heading dates were estimated. Using the Illumina Infinium 15 k Wheat platform, 379 5B-specific polymorphic markers were detected and a genetic map with 82 skeletal markers was constructed. Phenotype (heading date) - genotype association analysis revealed seventy eight markers in pericentromeric region of 5B chromosome significantly associated with heading date variation. Based on this estimation and synteny with model crop genomes we identified the three best candidate genes: WRKY, ERF/AP2 and FHY3/FAR1. CONCLUSIONS: We supposed that the difference in activity of WRKY, ERF/AP2 and/or FHY3/FAR1 transcription factors between CS and CS-5Bdic to be a probable reason for the observed difference in heading dates. Data obtained in this study provide a good basis for the subsequent investigation of heading time pathways in wheat.

VRN-1 gene- associated prerequisites of spring growth habit in wild tetraploid wheat T. dicoccoides and the diploid A genome species.

BACKGROUND: In order to clarify the origin of spring growth habit in modern domesticated wheat, allelic variability of the VRN-1 gene was investigated in a wide set of accessions of the wild tetraploid species Triticum dicoccoides (BBAA), together with diploid species T. monococcum, T. boeoticum and T. urartu, presumable donors of the A genome to polyploid wheats. RESULTS: No significant variation was found at the VRN-B1 locus of T. dicoccoides, whereas at VRN-A1 a number of previously described alleles were found with small deletions in the promoter (VRN-A1b, VRN-A1d) or a large deletion in the first (1st) intron (VRN-A1L). The diploid A genome species were characterized by their own set of VRN-1 alleles including previously described VRN-A1f and VRN-A1h alleles with deletions in the promoter region and the VRN-A1ins allele containing a 0.5 kb insertion in the 1st intron. Based on the CAPS screening data, alleles VRN-A1f and VRN-A1ins were species-specific for T. monococcum, while allele VRN-A1h was specific for T. boeoticum. Different indels were revealed in both the promoter and 1(st) intron of the recessive VRN-A1u allele providing specific identification of T. urartu, the proposed donor of the A genome to modern wheat. We found that alleles VRN-A1b and VRN-A1h, previously described as dominant, have either no or weak association with spring growth habit, while in some diploid accessions this habit was associated with the recessive VRN-A1 allele. CONCLUSIONS: Spring growth habit in diploid wheats was only partially associated with indels in regulatory regions of the VRN-1 gene. An exception is T. monococcum where dominant mutations in both the promoter region and, especially, the 1st intron were selected during domestication resulting in a greater variety of spring forms. The wild tetraploid T. dicoccoides had a distinct set of VRN-A1 alleles compared to the diploids in this study, indicating an independent origin of spring tetraploid forms that likely occurred after combining of diploid genomes. These alleles were subsequently inherited by cultivated polyploid (tetraploid and hexaploid) descendants.

The impact of Ty3-gypsy group LTR retrotransposons Fatima on B-genome specificity of polyploid wheats.

BACKGROUND: Transposable elements (TEs) are a rapidly evolving fraction of the eukaryotic genomes and the main contributors to genome plasticity and divergence. Recently, occupation of the A- and D-genomes of allopolyploid wheat by specific TE families was demonstrated. Here, we investigated the impact of the well-represented family of gypsy LTR-retrotransposons, Fatima, on B-genome divergence of allopolyploid wheat using the fluorescent in situ hybridisation (FISH) method and phylogenetic analysis. RESULTS: FISH analysis of a BAC clone (BAC_2383A24) initially screened with Spelt1 repeats demonstrated its predominant localisation to chromosomes of the B-genome and its putative diploid progenitor Aegilops speltoides in hexaploid (genomic formula, BBAADD) and tetraploid (genomic formula, BBAA) wheats as well as their diploid progenitors. Analysis of the complete BAC_2383A24 nucleotide sequence (113,605 bp) demonstrated that it contains 55.6% TEs, 0.9% subtelomeric tandem repeats (Spelt1), and five genes. LTR retrotransposons are predominant, representing 50.7% of the total nucleotide sequence. Three elements of the gypsy LTR retrotransposon family Fatima make up 47.2% of all the LTR retrotransposons in this BAC. In situ hybridisation of the Fatima_2383A24-3 subclone suggests that individual representatives of the Fatima family contribute to the majority of the B-genome specific FISH pattern for BAC_2383A24. Phylogenetic analysis of various Fatima elements available from databases in combination with the data on their insertion dates demonstrated that the Fatima elements fall into several groups. One of these groups, containing Fatima_2383A24-3, is more specific to the B-genome and proliferated around 0.5-2.5 MYA, prior to allopolyploid wheat formation. CONCLUSION: The B-genome specificity of the gypsy-like Fatima, as determined by FISH, is explained to a great degree by the appearance of a genome-specific element within this family for Ae. speltoides. Moreover, its proliferation mainly occurred in this diploid species before it entered into allopolyploidy.Most likely, this scenario of emergence and proliferation of the genome-specific variants of retroelements, mainly in the diploid species, is characteristic of the evolution of all three genomes of hexaploid wheat.

Isolation and sequence analysis of the wheat B genome subtelomeric DNA.

BACKGROUND: Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. RESULTS: The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119,737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11,666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. CONCLUSION: Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time that Spelt52 sequences were involved in the evolution of terminal regions of common wheat chromosomes. Our research provides new insights into the microcollinearity in the terminal regions of wheat chromosomes 4BL and rice chromosome 3S.

Phylogenetic reconstruction of Aegilops section Sitopsis and the evolution of tandem repeats in the diploids and derived wheat polyploids.

The evolution of 2 tandemly repeated sequences Spelt1 and Spelt52 was studied in Triticum species representing 2 evolutionary lineages of wheat and in Aegilops sect. Sitopsis, putative donors of their B/G genomes. Using fluorescence in situ hybridization we observed considerable polymorphisms in the hybridization patterns of Spelt1 and Spelt52 repeats between and within Triticum and Aegilops species. Between 2 and 28 subtelomeric sites of Spelt1 probe were detected in Ae. speltoidies, depending on accession. From 8 to 12 Spelt1 subtelomeric sites were observed in species of Timopheevi group (GAt genome), whereas the number of signals in emmer/aestivum accessions was significantly less (from 0 to 6). Hybridization patterns of Spelt52 in Ae. speltoides, Ae. longissima, and Ae. sharonensis were species specific. Subtelomeric sites of Spelt52 repeat were detected only in T. araraticum (T. timopheevii), and their number and chromosomal location varied between accessions. Superimposing copy number data onto our phylogenetic scheme constructed from RAPD data suggests 2 major independent amplifications of Spelt52 and 1 of Spelt1 repeats in Aegilops divergence. It is likely that the Spelt1 amplification took place in the ancient Ae. speltoides before the divergence of polyploid wheats. The Spelt52 repeat was probably amplified in the lineage of Ae. speltoides prior to divergence of the allopolyploid T. timopheevii but after the divergence of T. durum. In a separate amplification event, Spelt52 copy number expanded in the common ancestor of Ae. longissima and Ae. sharonensis.