SSR Variability of Stem Rust Pathogen on Spring Bread Wheat in Russia.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, which used to be a harmful disease of winter wheat in the southern part of Russia, has been largely affecting the yield of spring bread wheat in the territories of the temperate climate zone since 2009. In total, 222 P. graminis f. sp. tritici isolates were obtained from samples of susceptible cultivars of spring bread wheat in Central and Volga regions and Omsk and Novosibirsk provinces in 2019. Genotyping of the isolates was carried out at 16 simple-sequence repeat (SSR) loci. Number of alleles, proportion of heterozygotes, and deviation from Hardy-Weinberg equilibrium were determined at each SSR locus. Based on genetic variability of SSR genotypes, it was shown that the P. graminis f. sp. tritici population is subdivided into two large clusters in the territory of the Russian temperate climate zone: the "European" population (the Central region) and the "Asian" one (the Volga region and two main wheat provinces of Western Siberia). Both of the P. graminis f. sp. tritici populations are characterized by a mixed mode of reproduction (sexual and clonal) but different sources of inoculum seem to shape a genotype structure within them. A group of P. graminis f. sp. tritici genotypes with high variability, the inbreeding coefficient closed to zero, and low observed heterozygosity was revealed among samples from Omsk. Moreover, two singular SSR genotypes identified among the Asian samples of P. graminis f. sp. tritici isolates should attract special attention in the monitoring of stem rust in order to disclose unexpected rapid changes of the pathogen in the corresponding regions and to prevent disease outbreak.

Features of the organization of bread wheat chromosome 5BS based on physical mapping.

BACKGROUND: The IWGSC strategy for construction of the reference sequence of the bread wheat genome is based on first obtaining physical maps of the individual chromosomes. Our aim is to develop and use the physical map for analysis of the organization of the short arm of wheat chromosome 5B (5BS) which bears a number of agronomically important genes, including genes conferring resistance to fungal diseases. RESULTS: A physical map of the 5BS arm (290 Mbp) was constructed using restriction fingerprinting and LTC software for contig assembly of 43,776 BAC clones. The resulting physical map covered ~ 99% of the 5BS chromosome arm (111 scaffolds, N50 = 3.078 Mb). SSR, ISBP and zipper markers were employed for anchoring the BAC clones, and from these 722 novel markers were developed based on previously obtained data from partial sequencing of 5BS. The markers were mapped using a set of Chinese Spring (CS) deletion lines, and F2 and RICL populations from a cross of CS and CS-5B dicoccoides. Three approaches have been used for anchoring BAC contigs on the 5BS chromosome, including clone-by-clone screening of BACs, GenomeZipper analysis, and comparison of BAC-fingerprints with in silico fingerprinting of 5B pseudomolecules of T. dicoccoides. These approaches allowed us to reach a high level of BAC contig anchoring: 96% of 5BS BAC contigs were located on 5BS. An interesting pattern was revealed in the distribution of contigs along the chromosome. Short contigs (200-999 kb) containing markers for the regions interrupted by tandem repeats, were mainly localized to the 5BS subtelomeric block; whereas the distribution of larger 1000-3500 kb contigs along the chromosome better correlated with the distribution of the regions syntenic to rice, Brachypodium, and sorghum, as detected by the Zipper approach. CONCLUSION: The high fingerprinting quality, LTC software and large number of BAC clones selected by the informative markers in screening of the 43,776 clones allowed us to significantly increase the BAC scaffold length when compared with the published physical maps for other wheat chromosomes. The genetic and bioinformatics resources developed in this study provide new possibilities for exploring chromosome organization and for breeding applications.

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.

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

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 6A(t) chromosome. (GAA)n site observed in Triticum monococcum are undetectable in the A(b) 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.

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.

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.

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.