E. M. Sergeeva

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.

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.

Common wheat chromosome 5B composition analysis using low-coverage 454 sequencing

The sequencing of individual chromosomes of common wheat is in progress. The molecular size of wheat chromosome 5B is nearly 870 Mb (5BL = 580 Mb and 5BS = 290 Mb). We produced the first low coverage 454‐sequencing of the long and short arms of wheat chromosome 5B (110,793 and 39,695 reads, which compose 8 and 6% of total 5BL and 5BS length, respectively) and calculated the ratios of the different families of repetitive sequences, including transposable elements (TEs), satellite repeats (Afa, pSc119.2, 5S rDNA and 45S rDNA), and microsatellites, as well as direct and inverted repeat motifs. The TEs accounted for 70% of the total analyzed nucleotide sequences. The content of the Cereba retrotransposon family differed between the two arms of chromosome 5B. Comparing the reads of chromosome 5B with the data from chromosome 5A, we found the retrotransposons Fatima and Sakura and DNA transposon Jorge were prevalent in 5B. The hypothetical coding sequences accounted for 2.0% of the short arm and 2.07% of the long arm. Using in silico mapping, we identified the regions of synteny with rice and Brachypodium chromosomes (1,073,526 and 1,767,298 bp aligned, respectively), and the result was consistent with the data from the expressed sequence tag (EST) mapping of wheat 5B chromosome to the genomes of these grasses. Thus, these results show that low coverage survey sequencing can provide useful information about the composition and evolution of wheat chromosome 5B.

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.

Analysis of 5S rDNA changes in synthetic allopolyploids Triticum × Aegilops

Changes of 5S rDNA at the early stage of allopolyploidization were investigated in three synthetic allopolyploids: Aegilops sharonensis × Ae. umbellulata (2n = 28), Triticum urartu × Ae. tauschii (2n = 28), and T. dicoccoides × Ae. tauschii (2n = 42). Fluorescent in situ hybridization (FISH) revealed quantitative changes affecting separate loci of one of the parental genomes in S3 plants of each hybrid combination. Southern hybridization with genomic DNA of the allopolyploid T. urartu × Ae. tauschii (TMU38 × TQ27) revealed a lower intensity of signals from Ae. tauschii fragments compared with those derived from T. urartu. This confirmed the signal reduction revealed for chromosome 1D of this hybrid by FISH. Neither Southern hybridization nor PCR testing of 5–15 plants of the S2-S3 generations revealed an appearance of new 5S rDNA fragments or a complete disappearance of parental fragments from the allopolyploids under study. No changes were found by aligning nine 5S rDNA sequences of the allopolyploid TMU38 × TQ27 with corresponding sequences of the parental species. The similarity between one of the synthetic allopolyploids examined and a natural allopolyploid with the same genome composition points to an early formation of the 5S rDNA organization unique for each allopolyploid.

A comparative genetic and cytogenetic mapping of wheat chromosome 5B using introgression lines

The genetic map of chromosome 5B has been constructed by using microsatellite (SSR) analysis of 381 plants from the F2 population produced by cross of the Chinese Spring (CS) and Renan cultivars. Initially, 180 SSR markers for the common wheat 5B chromosome have been used for analysis of these cultivars. The 32 markers able to detect polymorphism between these cultivars have been located on the genetic map of chromosome 5B. Cytogenetic mapping has involved a set of CS 5B chromosome deletion lines. Totally, 51 SSR markers have been located in ten regions (deletion bins) of this chromosome by SSR analysis of these deletion lines. Five genes—TaCBFIIIc-B10, Vrn-B1, Chi-B1, Skr, and Ph1—have been integrated into the cytogenetic map of chromosome 5B using the markers either specific of or tightly linked to the genes in question. Comparison of the genetic and cytogenetic maps suggests that recombination is suppressed in the pericentromeric region of chromosome 5B, especially in the short arm segment. The 18 markers localized to deletion bins 5BL16-0.79-1.00 and 5BL18-0.66-0.79 have been used to analyze common wheat introgression lines L842, L5366-180, L73/00i, and L21-4, carrying fragments of alien genomes in the terminal region of 5B long arm. L5366-180 and L842 lines carry a fragment of the Triticum timopheevii 5GL chromosome, while L73/00i and L21-4 lines, a fragment of the Aegilops speltoides 5SL chromosome. As has been shown, the translocated fragments in these four lines are of different lengths, allowing bin 5BL18-0.66-0.79 to be divided into three shorter regions. The utility of wheat introgression lines carrying alien translocations for increasing the resolution of cytogenetic mapping is discussed.

GAA)n microsatellite as an indicator of the A genome reorganization during wheat evolution and domestication

Abstract Although the wheat A genomes have been intensively studied over past decades, many questions concerning the mechanisms of their divergence and evolution still remain unsolved. In the present study we performed comparative analysis of the A genome chromosomes in diploid (Triticum urartu Tumanian ex Gandilyan, 1972, Triticum boeoticum Boissier, 1874 and Triticum monococcum Linnaeus, 1753) and polyploid wheat species representing two evolutionary lineages, Timopheevi (Triticum timopheevii (Zhukovsky) Zhukovsky, 1934 and Triticum zhukovskyi Menabde & Ericzjan, 1960) and Emmer (Triticum dicoccoides (Körnicke ex Ascherson & Graebner) Schweinfurth, 1908, Triticum durum Desfontaines, 1798, and Triticum aestivum Linnaeus, 1753) using a new cytogenetic marker – the pTm30 probe cloned from Triticum monococcum genome and containing (GAA)56 microsatellite sequence. Up to four pTm30 sites located on 1AS, 5AS, 2AS, and 4AL chromosomes have been revealed in the wild diploid species, although most accessions contained one–two (GAA)n sites. The domesticated diploid species Triticum monococcum differs from the wild diploid species by almost complete lack of polymorphism in the distribution of (GAA)n site. Only one (GAA)n site in the 4AL chromosome has been found in Triticum monococcum. Among three wild emmer (Triticum dicoccoides) accessions we detected 4 conserved and 9 polymorphic (GAA)n sites in the A genome. The (GAA)n loci on chromosomes 2AS, 4AL, and 5AL found in of Triticum dicoccoides were retained in Triticum durum and Triticum aestivum. In species of the Timopheevi lineage, the only one, large (GAA)n site has been detected in the short arm of 6At chromosome. (GAA)n site observed in Triticum monococcum are undetectable in the Ab genome of Triticum zhukovskyi, this site could be eliminated over the course of amphiploidization, while the species was established. We also demonstrated that changes in the distribution of (GAA)n sequence on the A-genome chromosomes of diploid and polyploid wheats are associated with chromosomal rearrangements/ modifications, involving mainly the NOR (nucleolus organizer region)-bearing chromosomes, that took place during the evolution of wild and domesticated species.

Identification of microsatellite loci based on BAC sequencing data and their physical mapping into the soft wheat 5B chromosome

The shortage of polymorphic markers for the regions of the wheat chromosomes that encode commercially valuable traits determines the need for studying the wheat microsatellite SSR loci. In this work, SSR markers for individual regions of the short arm of soft wheat chromosome 5B (5BS) were designed based on the sequence data obtained from BAC clones, and regions of the corresponding chromosome were saturated with these markers. Totally, 130 randomly selected BAC clones from 5BS library were sequenced using the IonTorrent platform and assembled in contigs using MIRA software. The assembly characteristics (N50 = 4136 bp) are comparable to the recently obtained data for wheat and related species and are acceptable for the identification of the microsatellite loci. The algorithm utilizing the properties of complex decompositions in the sliding-window mode was used to detect DNA sequences with a repeat unit of 2–4 bp. Analysis of 17770 contigs with a total length of 25879921 bp allowed for the design of 113, 79, and 67 microsatellite SSR loci with a repeat unit of 2, 3, and 4 bp, respectively. SSR markers with a motif of 3 bp were tested using nullitetrasomic lines of Chinese Spring wheat homoeologous group 5. In total, 21 markers specific for chromosome 5B were identified. Eight of these markers were mapped into the distal region of this chromosome (bin 5BS6) using a set of Chinese Spring deletion lines for 5BS. Eight and four markers were mapped to the interstitial region (bins 5BS5 and 5BS4, respectively). One marker was mapped to a pericentromeric bin. Comparative analysis of the distribution of trinucleotide microsatellites over wheat chromosome 5B, and in different cereal species, suggests that the (AAG)n repeat proliferates and is conserved during the evolution of cereals.

Genomic changes at early stages of formation of allopolyploid Aegilops longissima × Triticum urartu

Using the model of synthetic allopolyploid Aegilops longissima TL05 × 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 PCR fragments of the allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for these chromosomal regions 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 RAPD fragments typical for parental genome in allopolyploid TL05 × TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomeric primers the changes in PCR fragments were shown only for one of 66 pairs of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in the 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.