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Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.

Russian wheat aphid (RWA) ( Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat ( L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and long-read nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the region facilitated the identification of new markers and precise annotation of the interval, revealing six high-confidence genes. Identification of as the most likely candidate opens an avenue for its validation through functional genomics approaches.

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

Abstract Background Grasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum. Results The DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins. Conclusion The resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.

Abstract Background The wheat tribe Triticeae (Poaceae) is a diverse group of grasses representing a textbook example of reticulate evolution. Apart from globally important grain crops, there are also wild grasses which are of great practical value. Allohexaploid intermediate wheatgrass, Thinopyrum intermedium (2n = 6x = 42), possesses many desirable agronomic traits that make it an invaluable source of genetic material useful in wheat improvement. Although the identification of its genomic components has been the object of considerable investigation, the complete genomic constitution and its potential variability are still being unravelled. To identify the genomic constitution of this allohexaploid, four accessions of intermediate wheatgrass from its native area were analysed by sequencing of chloroplast trnL-F and partial nuclear GBSSI, and genomic in situ hybridization. Results The results confirmed the allopolyploid origin of Thinopyrum intermedium and revealed new aspects in its genomic composition. Genomic heterogeneity suggests a more complex origin of the species than would be expected if it originated through allohexaploidy alone. While Pseudoroegneria is the most probable maternal parent of the accessions analysed, nuclear GBSSI sequences suggested the contribution of distinct lineages corresponding to the following present-day genera: Pseudoroegneria, Dasypyrum, Taeniatherum, Aegilops and Thinopyrum. Two subgenomes of the hexaploid have most probably been contributed by Pseudoroegneria and Dasypyrum, but the identity of the third subgenome remains unresolved satisfactorily. Possibly it is of hybridogenous origin, with contributions from Thinopyrum and Aegilops. Surprising diversity of GBSSI copies corresponding to a Dasypyrum-like progenitor indicates either multiple contributions from different sources close to Dasypyrum and maintenance of divergent copies or the presence of divergent paralogs, or a combination of both. Taeniatherum-like GBSSI copies are most probably pseudogenic, and the mode of their acquisition by Th. intermedium remains unclear. Conclusions Hybridization has played a key role in the evolution of the Triticeae. Transfer of genetic material via extensive interspecific hybridization and/or introgression could have enriched the species' gene pools significantly. We have shown that the genomic heterogeneity of intermediate wheatgrass is higher than has been previously assumed, which is of particular concern to wheat breeders, who frequently use it as a source of desirable traits in wheat improvement.

Abstract Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries. Conclusions The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.