I. G. Adonina

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

BackgroundTransposable 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.ResultsFISH 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.ConclusionThe 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.

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.

A comparative analysis of the composition and organization of two subtelomeric repeat families in Aegilops speltoidesTausch. and related species

The structural organization and evolution of two tandemly repeated families, Spelt1 and Spelt52, located in the subtelomeric regions of Aegilops speltoides chromosomes were studied. The Spelt1 family of sequences with a monomer length of 178 bp was characterized by cloning and sequence analysis of polymerase chain reaction (PCR) products. Members of the Spelt1 family revealed sequence similarities exceeding 95\%. This conservation has remained despite divergence of species in Aegilops section Sitopsis and after independent multiple amplification events in the genome of Ae. speltoides. Sequences representing the Spelt52 family were cloned, sequenced and compared with other sequences in databases. The Spelt52 repeat family contains monomers of two types, Spelt52.1 and Spelt52.2. The two monomers share a homologous stretch of 280 bp and have two regions without sequence similarity of 96 bp and 110 bp, respectively. PCR analysis was conducted to 15 lines in Ae. speltoidesTausch., Ae. longissimaSchw.&Mushc.,Ae. sharonensisEig.,Ae. bicornis(Forssk)Jaub.&Sp., andAe. searsii Feld.&Kis. using primers to the homologous and non- homologous regions of Spelt52 family. Intraspecies and interspecies differences in the occurrence and abundance of combinations of Spelt52.1 and Spelt52.2 monomers were detected. The use of primers to telomeric and subtelomeric repeats followed by Southern hybridization, cloning, and sequence analysis demonstrated that Spelt1 and Spelt52 are localized close to each other and to telomeric repeats. The efficiency of a PCR approach for the analysis of telomeric/subtelomeric junction regions of chromosomes is discussed.

Isolation and sequence analysis of the wheat B genome subtelomeric DNA

BackgroundTelomeric 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.ResultsThe 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.ConclusionSatellite 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.

Production and molecular and cytogenetic analyses of euploid (2n = 42) and telocentric addition (2n = 42 + 2t) alloplasmic lines (Hordeum marinum subsp. gussoneanum)-Triticum aestivum

Individual plants from the BC1F6 and BC1F8 backcross progenies of barley-wheat [H. marinum subsp. gussoneanum Hudson (=H. geniculatum All.) (2n = 28) × T. aestivum L. (2n = 42)] and the BC1F6 progeny of their amphiploids were used to obtain alloplasmic euploid (2n = 42) lines L-28, L-29, and L-49 and alloplasmic telocentric addition (2n = 42 + 2t) lines L-37, L-38, and L-50. The lines were examined by genomic in situ hybridization (GISH), microsatellite analysis, chromosome C-banding, and PCR analysis of the mitochondrial 18S/5S repeat. Lines L-29 and L-49 were characterized by substitution of wild barley chromosome 7H1 for common wheat chromosome 7D. In line L-49, common wheat chromosomes 1B, 5D, and 7D were substituted with homeologous barley chromosomes. Lines L-37, L-38, and L-50 each contained a pair of telocentric chromosomes, which corresponded to barley chromosome arm 7H1L. All lines displayed heteroplasmy for the mitochondrial 18S/5S locus; i.e., both barley and wheat sequences were found.

Evolutionary analysis of the CACTA DNA-transposon Caspar across wheat species using sequence comparison and in situ hybridization

Mobile elements constitute a considerable part of the eukaryotic genome. This work is focused on the distribution and evolution of DNA-transposons in the genomes of diploid and allopolyploid Triticeae species and their role in the formation of functionally important chromosomal subtelomeric regions. The Caspar family is among the most abundant of CACTA DNA-transposons in Triticeae. To study the evolution of Caspar-like elements in Triticeae genomes, we analyzed their sequences and distribution in chromosomes by in situ hybridization. In total, 46 Caspar-like elements from the wheat and barley Caspar, Clifford, and Donald families were analyzed after being extracted from databases using the transposase consensus sequence. Sequence alignment and subsequent phylogenetic analyses revealed that the transposase DNA sequences formed three major distinct groups: (1) Clifford, (2) Caspar_Triticinae, and (3) Caspar_Hordeinae. Additionally, in situ hybridization demonstrated that Caspar_Triticinae transposons are predominantly compartmentalized in the subtelomeric chromosomal regions of wheat and its progenitors. Analysis of data suggested that compartmentalization in the subtelomeric chromosomal region was a characteristic feature of all the main groups of Caspar-like elements. Furthermore, a dot plot analysis of the terminal repeats demonstrated that the divergence of these repeats strictly correlated with the divergence of Caspar coding sequences. A clear distinction in the Caspar DNA sequences among the species Triticum/Aegilops (Caspar_Triticinae), Hordeum (Caspar_Hordeinae), and different distributions in individual hexaploid wheat genomes (A/B and D) suggest an independent proliferation of these elements in wheat (or its progenitors) and barley genomes. Thus, Caspar-like transposons can significantly contribute to the formation and differentiation of subtelomeric regions in Triticeae species.

Construction and study of leaf rust-resistant common wheat lines with translocations of Aegilops speltoides Tausch. Genetic material

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum × Aegilops speltoides). Fluorescence in situ hybridization (FISH) with probes Spelt1 and pSc119.2 in combination with microsatellite analysis were used to determine the locations and sizes of the Ae. speltoides genetic fragments integrated into the line genome. Translocations were identified in the long arms of chromosomes 5B and 6B and in the short arm of chromosome 1B. The Spelt1 and pSc119.2 molecular cytological markers made it possible to rapidly establish lines with single translocation in the long arms of chromosomes 5B and 6B. The line carrying the T5BS · 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS · 6BL-6SL translocation displayed moderate resistance. The translocations differed in chromosomal location from known leaf resistance genes transferred into common wheat from Ae. speltoides. Hence, it was assumed that new genes were introduced into the common wheat genome from Ae. speltoides. The locus that determined high resistance to leaf rust and was transferred into the common wheat genome from the long arm of Ae. speltoides chromosome 5S by the T5BS · 5BL-5SL translocation was preliminarily designated as LrAsp5.

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.

Impact of Ty3/Gypsy group retrotransposon Lila on the D-Genome specificity of common 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 of retroelements specific to each genome of wheat took place during formation of the diploid progenitors of these genomes.

The mechanisms of variation of subtelomeric repeats Spelt1 in the progeny of introgressive line Triticum aestivum L. × Aegilops speltoides Tausch.

Quantitative variation of species-specific subtelomeric repeat Spelt1 was studied in the progeny of an individual plant from the introgressive line Triticum aestivum × Aegilops speltoides. In the progeny, no cases of the Spelt1 increased content were observed. On the contrary, in some cases statistically significant decrease of the repeat copy number was detected. It seems likely that the mechanisms of the Spelt1 elimination involve either the selection at the gamete level versus the increase of the satellite DNA content in the telomeres, or intramolecular (within one chromatid) homologous recombination.