Meiotic instability and irregular chromosome pairing underpin heat-induced infertility in bread wheat carrying the Rht-B1b or Rht-D1b Green Revolution genes.

Mutations in the Rht-B1a and Rht-D1a genes of wheat (Triticum aestivum; resulting in Rht-B1b and Rht-D1b alleles) cause gibberellin-insensitive dwarfism and are one of the most important elements of increased yield introduced during the 'Green Revolution'. We measured the effects of a short period of heat imposed during the early reproductive stage on near-isogenic lines carrying Rht-B1b or Rht-D1b alleles, with respect to the wild-type (WT). The temperature shift caused a significant fertility loss within the ears of Rht-B1b and Rht-D1b wheats, greater than that observed for the WT. Defects in chromosome synapsis, reduced homologous recombination and a high frequency of chromosome mis-segregation were associated with reduced fertility. The transcription of TaGA3ox gene involved in the final stage of gibberellic acid (GA) biosynthesis was activated and ultra-performance liquid chromatography-tandem mass spectrometry identified GA1 as the dominant bioactive GA in developing ears, but levels were unaffected by the elevated temperature. Rht-B1b and Rht-D1b mutants were inclined to meiotic errors under optimal temperatures and showed a higher susceptibility to heat than their tall counterparts. Identification and introduction of new dwarfing alleles into modern breeding programmes is invaluable in the development of climate-resilient wheat varieties.

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH.

BACKGROUND: Though multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity. The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH). RESULTS: The mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations. CONCLUSION: A simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.

A Multigraph-Based Representation of Hi-C Data.

Chromatin-chromatin interactions and three-dimensional (3D) spatial structures are involved in transcriptional regulation and have a decisive role in DNA replication and repair. To understand how individual genes and their regulatory elements function within the larger genomic context, and how the genome reacts to environmental stimuli, the linear sequence information needs to be interpreted in three-dimensional space, which is still a challenging task. Here, we propose a novel, heuristic approach to represent Hi-C datasets by a whole-genomic pseudo-structure in 3D space. The baseline of our approach is the construction of a multigraph from genomic-sequence data and Hi-C interaction data, then applying a modified force-directed layout algorithm. The resulting layout is a pseudo-structure. While pseudo-structures are not based on direct observation and their details are inherent to settings, surprisingly, they demonstrate interesting, overall similarities of known genome structures of both barley and rice, namely, the Rabl and Rosette-like conformation. It has an exciting potential to be extended by additional omics data (RNA-seq, Chip-seq, etc.), allowing to visualize the dynamics of the pseudo-structures across various tissues or developmental stages. Furthermore, this novel method would make it possible to revisit most Hi-C data accumulated in the public domain in the last decade.

Effect of additional water supply during grain filling on protein composition and epitope characteristics of winter oats.

Pure oats in gluten-free diets (GFD) represent important nutritional benefits for people suffering from celiac disease (CD). However, oat cultivars do not contain the typical CD-related wheat gliadin analog polypeptides. Emerging evidence suggests that oat cultivars containing gluten-like epitopes in avenin sequences may pose potential health risks for celiac patients in rare cases, depending on the individual's susceptibility. Consequently, it is necessary to screen oats in terms of protein and epitope composition, to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variation of oat protein composition directly related to health-related and techno-functional properties and to examine how the protein compositional parameters change due to irrigation during the grain-filling period as compared to the natural rain-fed grown, in a large winter oat population of different geographic origin. Elements of an oat sample population representing 164 winter oat varieties from 8 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by SE-HPLC, while the 70% ethanol extracted proteins were analyzed by RP-HPLC. Protein extracts are separated into 3 main groups of fractions on the SE-HPLC column; polymeric, avenin, and non-avenin monomeric protein groups, representing 59.17-80.87%, 12.89-31.03%, and 3.40-9.41% of total protein content, respectively. The ratio of polymeric to monomeric proteins varied between 1.71 and 6.07. 91 RP-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. The various parameters identified a lot of variation, confirming the significance of genotypic variation. In addition, it was also established that the additional water supply during grain filling significantly affected the various quantitative parameters of protein content, but not its qualitative structure. This environmental effect, however, was strongly genotype-dependent. Winter oat genotypes with low levels of epitope content were identified and it was proven that these characteristics were independent of the environmental factor of water availability. These genotypes are appropriate for initiating a specific breeding program to yield oat cultivars suitable for CD patients.

Estimation of Nuclear DNA Content in Some Aegilops Species: Best Analyzed Using Flow Cytometry.

The genera Triticum and Aegilops have been considered as the main gene pool of wheat due to their features, such as tolerance of all types of abiotic and biotic stresses. This study was conducted to evaluate the cytogenetic analyses in 115 native and wild populations from eleven Aegilops species using their nuclear DNA quantification. Mean 2C nuclear DNA contents of different ploidy levels in the wild wheat of Turkey and Iran were measured using the flow cytometry technique. The obtained results showed that the mean nuclear DNA content in diploid species varied from 10.09 pg/2C (Ae. umbellulata) to 10.95 pg/2C (Ae. speltoides var. ligustica) in Turkey. In Iranian diploids, the mean nuclear DNA content varied from 10.20 pg/2C (Ae. taushii) to 11.56 pg/2C (Ae. speltoides var. ligustica). This index in the tetraploid species of Turkey varied from 18.09 pg/2C (Ae. cylindrica) to 21.65 pg/2C (Ae. triaristata), and in Iranian species, it was from 18.61 pg/2C (Ae. cylindrica) to 21.75 pg/2C (Ae. columnaris). On the other hand, in the hexaploid species of Turkey, this index varied from 31.59 pg/2C (Ae. crassa) to 31.81 pg/2C (Ae. cylindrica); in the Iranian species, it varied from 32.58 pg/2C (Ae. cylindrica) to 33.97 pg/2C (Ae. crassa). There was a significant difference in the DNA content of Turkey and Iran diploid as well as tetraploid species; however, in hexaploid species, the difference was not significant. It was concluded that the variation in intraspecific genome size was very low in diploid and tetraploid populations; this means that the low variation is not dependent on geographic and climatic parameters. On the other hand, the interspecific variation is significant at the diploid and tetraploid populations. It is generally very difficult to distinguish Aegilops species from each other in natural conditions; meanwhile, in this study, all species could be, easily, quickly and unambiguously, distinguished and separated using the FCM technique.

Effects of genetic components of plant development on yield-related traits in wheat (Triticum aestivum L.) under stress-free conditions.

The dynamics of plant development not only has an impact on ecological adaptation but also contributes to the realization of genetically determined yield potentials in various environments. Dissecting the genetic determinants of plant development becomes urgent due to the global climate change, which can seriously affect and even disrupt the locally adapted developmental patterns. In order to determine the role plant developmental loci played in local adaptation and yield formation, a panel of 188 winter and facultative wheat cultivars from diverse geographic locations were characterized with the 15K Illumina Single Nucleotide Polymorphism (SNP) chip and functional markers of several plant developmental genes and included into a multiseason field experiment. Genome-wide association analyses were conducted on five consecutive developmental phases spanning from the first node appearance to full heading together with various grain yield-related parameters. The panel was balanced for the PPD-D1 photoperiod response gene, which facilitated the analyses in the two subsets of photoperiod-insensitive and -sensitive genotypes in addition to the complete panel. PPD-D1 was the single highest source, explaining 12.1%-19.0% of the phenotypic variation in the successive developmental phases. In addition, 21 minor developmental loci were identified, each one explaining only small portions of the variance, but, together, their effects amounted to 16.6%-50.6% of phenotypic variance. Eight loci (2A_27, 2A_727, 4A_570, 5B_315, 5B_520, 6A_26, 7A_1-(VRN-A3), and 7B_732) were independent of PPD-D1. Seven loci were only detectable in the PPD-D1-insensitive genetic background (1A_539, 1B_487, 2D_649, 4A_9, 5A_584-(VRN-A1), 5B_571-(VRN-B1), and 7B_3-(VRN-B3)), and six loci were only detectable in the sensitive background, specifically 2A_740, 2D_25, 3A_579, 3B_414, 7A_218, 7A_689, and 7B_538. The combination of PPD-D1 insensitivity and sensitivity with the extremities of early or late alleles in the corresponding minor developmental loci resulted in significantly altered and distinct plant developmental patterns with detectable outcomes on some yield-related traits. This study examines the possible significance of the above results in ecological adaptation.

Exploring the legacy of Central European historical winter wheat landraces.

Historical wheat landraces are rich sources of genetic diversity offering untapped reservoirs for broadening the genetic base of modern varieties. Using a 20K SNP array, we investigated the accessible genetic diversity in a Central European bread wheat landrace collection with great drought, heat stress tolerance and higher tillering capacity. We discovered distinct differences in the number of average polymorphisms between landraces and modern wheat cultivars, and identified a set of novel rare alleles present at low frequencies in the landrace collection. The detected polymorphisms were unevenly distributed along the wheat genome, and polymorphic markers co-localized with genes of great agronomic importance. The geographical distribution of the inferred Bayesian clustering revealed six genetically homogenous ancestral groups among the collection, where the Central European core bared an admixed background originating from four ancestral groups. We evaluated the effective population sizes (Ne) of the Central European collection and assessed changes in diversity over time, which revealed a dramatic ~ 97% genetic erosion between 1955 and 2015.

Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals.

The reciprocal exchange of genetic information between homologous chromosomes during meiotic recombination is essential to secure balanced chromosome segregation and to promote genetic diversity. The chromosomal position and frequency of reciprocal genetic exchange shapes the efficiency of breeding programmes and influences crop improvement under a changing climate. In large genome cereals, such as wheat and barley, crossovers are consistently restricted to subtelomeric chromosomal regions, thus preventing favourable allele combinations being formed within a considerable proportion of the genome, including interstitial and pericentromeric chromatin. Understanding the key elements driving crossover designation is therefore essential to broaden the regions available for crossovers. Here, we followed early meiotic chromatin dynamism in cereals through the visualisation of a homologous barley chromosome arm pair stably transferred into the wheat genetic background. By capturing the dynamics of a single chromosome arm at the same time as detecting the undergoing events of meiotic recombination and synapsis, we showed that subtelomeric chromatin of homologues synchronously transitions to an open chromatin structure during recombination initiation. By contrast, pericentromeric and interstitial regions preserved their closed chromatin organisation and become unpackaged only later, concomitant with initiation of recombinatorial repair and the initial assembly of the synaptonemal complex. Our results raise the possibility that the closed pericentromeric chromatin structure in cereals may influence the fate decision during recombination initiation, as well as the spatial development of synapsis, and may also explain the suppression of crossover events in the proximity of the centromeres.

Single versus repeated heat stress in wheat: What are the consequences in different developmental phases?

With a possible reference to heat priming and to characterize the extent and variation in the heat stress responses in wheat, the effects of single vs. repeated heat stresses were examined by measuring the changes in morphological and grain yield-related traits and photosynthetic parameters. To achieve these objectives, 51 winter wheat cultivars of various geographic origins were included in two independent experiments covering different phenological stages. In Experiment I, a single heat stress event was applied at stem elongation (SE) and booting (B), and the repeated heat stress was applied at both of these stages (SE+B). In Experiment II, the single heat stress was applied at stem elongation (SE) and full heading (CH), while the repeated heat stress was applied at both stages (SE+CH). While genotype was a more important factor for determining the morphological and yield-related traits, it was the treatment effect that mostly influenced the photosynthetic parameters, with the exception of the chlorophyll content. The heading stage was more sensitive to heat stress than the booting stage, which was primarily due to the larger decrease in the average seed number. The importance of biomass in contributing to grain yield intensified with the heat stress treatments. There was a large variation between the wheat cultivars not only in yielding abilities under control conditions but also in sensitivities to the various heat stresses, based on which 7 distinct groups with specific response profiles could be identified at a highly significant level. The 7 wheat groups were also characterized by their reaction patterns of different magnitudes and directions in their responses to single vs. repeated heat stresses, which depended on the phenological phases during the second cycle of heat stress. The possible association between these findings and heat priming is discussed.

Development and validation of an exome-based SNP marker set for identification of the St, Jr and Jvs genomes of Thinopyrym intermedium in a wheat background.

KEY MESSAGE: Cytogenetic analysis and array-based SNP genotyping of wheat- Th. intermedium introgression lines allowed identification of 634 chromosome-specific SNP markers across all twenty-one chromosomes of Th. intermedium (StJ r J vs , 2 n = 6 x = 42). Thinopyrum intermedium (2n = 6x = 42, StJrJvs) is one of the most promising reservoirs of useful genes including tolerance to abiotic stresses, perenniality and disease resistance not available in the cultivated bread wheat. The transfer of genetic diversity from wild species to wheat offers valuable responses to the effects of climate change. The new array-based single-nucleotide polymorphism (SNP) marker technology provides cheap and easy-to-use molecular markers for marker-assisted selection (MAS) in wheat breeding programmes. Here, we focus on the generation of a new chromosome-specific SNP marker set that can be used to characterize and identify the Th. intermedium chromosomes or chromosome segments transferred into wheat. A progressive investigation of marker development was conducted using 187 various newly developed wheat-Th. intermedium introgression lines and the Axiom® Wheat-Relative Genotyping array. We employed molecular cytogenetic techniques to clarify the genome constitution of the Th. intermedium parental lines and validated 634 chromosome-specific SNPs. Our data confirmed the allohexaploid nature of Th. intermedium and demonstrated that the St genome-specific GISH signal and markers are present at the centromeric regions of chromosomes 1Jvs, 2Jvs, 3Jvs and 7Jvs. The SNP markers presented here will be introduced into current wheat improvement programmes, offering a significant speed-up in wheat breeding and making it possible to deal with the transfer of the full genetic potential of Th. intermedium into wheat.

Development of a New Am -Genome-Specific Single Nucleotide Polymorphism Marker Set for the Molecular Characterization of Wheat-Triticum monococcum Introgression Lines.

CORE IDEAS: We identified 1247 polymorphic single nucleotide polymorphisms between Triticum monococcum and wheat. We identified 191 markers validated across all seven chromosomes of T. monococcum. Detected a T. monococcum introgression in leaf-rust-resistant lines. Cultivated einkorn wheat (Triticum monococcum L. subsp. monococcum, 2n = 2x = 14, Am Am ) and its wild relative T. monococcum subsp. aegilopoides are important sources of economically useful genes that can be exploited for wheat (Triticum aestivum L.) breeding. Einkorn has excellent resistance to fungal diseases and gene transfer is relatively simple via standard breeding methods. To fulfill the growing demand by modern prebreeding programs for a cost-effective high-throughput procedure for accurately detecting introgressed chromosomes or chromosome segments from T. monococcum into wheat, we used the Axiom Wheat-Relative Genotyping Array and developed a set of Am genome-specific exome-based single nucleotide polymorphism (SNP) markers suitable for rapid identification of T. monococcum chromatin in a wheat background. We identified 1247 polymorphic SNPs between T. monococcum and wheat. We identified 191 markers across all seven chromosomes of T. monococcum that are also present on an existing Triticum urartu Thum. ex Gandil. genetic map and potentially ordered them on the basis of the high macrocollinearity and conservation of marker order between T. monococcum and T. urartu. The marker set has been tested on leaf-rust-resistant BC3 F4 progenies of wheat-T. monococcum hybrids. Two markers (AX-94492165, AX-95073542) placed on the distal end of the chromosome arm 7AL detected a T. monococcum introgression into wheat. The SNP marker set thus proved highly effective in the identification of T. monococcum chromatin in a wheat background, offering a reliable method for screening and selecting wheat-T. monococcum introgression lines, a procedure that could significantly speed up prebreeding programs.

Development of a new 7BS.7HL winter wheat-winter barley Robertsonian translocation line conferring increased salt tolerance and (1,3;1,4)-ß-D-glucan content.

Interspecific hybridization between bread wheat (Triticum aestivum, 2n = 42) and related species allows the transfer of agronomic and quality traits, whereby subsequent generations comprise an improved genetic background and can be directly applied in wheat breeding programmes. While wild relatives are frequently used as sources of agronomically favourable traits, cultivated species can also improve wheat quality and stress resistance. A salt-tolerant 'Asakaze'/'Manas' 7H disomic addition line (2n = 44) with elevated ß-glucan content, but with low fertility and an unstable genetic background was developed in an earlier wheat-barley prebreeding programme. The aim of the present study was to take this hybridization programme further and transfer the favourable barley traits into a more stable genetic background. Taking advantage of the breakage-fusion mechanism of univalent chromosomes, the 'Rannaya' winter wheat 7B monosomic line was used as female partner to the 7H addition line male, leading to the development of a compensating wheat/barley Robertsonian translocation line (7BS.7HL centric fusion, 2n = 42) exhibiting higher salt tolerance and elevated grain ß-glucan content. Throughout the crossing programme, comprising the F1-F4 generations, genomic in situ hybridization, fluorescence in situ hybridization and chromosome-specific molecular markers were used to trace and identify the wheat and barley chromatin. Investigations on salt tolerance during germination and on the (1,3;1,4)-ß-D-glucan (mixed-linkage glucan [MLG]) content of the seeds confirmed the salt tolerance and elevated grain MLG content of the translocation line, which can be directly applied in current wheat breeding programmes.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Addition of Manas barley chromosome arms to the hexaploid wheat genome.

BACKGROUND: Cultivated barley belongs to the tertiary genepool of hexaploid wheat. Genes of interest can be transferred from barley into wheat through wide hybridization. The application of wheat-barley introgression lines could provide an excellent tool for the transfer of earliness, favourable amino acid composition, biotic stress resistance, abiotic stress tolerance, or good tillering ability into wheat. RESULTS: A set of 10 wheat-barley ditelosomic addition lines (2HS, 2HL, 3HS, 3HL, 4HS, 4HL, 6HS, 6HL, 7HS and 7HL) was developed from the progenies of an Asakaze/Manas wheat-barley hybrid produced in Martonvásár, Hungary. The addition lines were selected from self-fertilized plants of the BC2F2-BC2F4 generations using genomic in situ hybridization (GISH) and were identified by fluorescence in situ hybridization (FISH) with repetitive DNA probes [HvT01, (GAA)7 and centromere-specific (AGGGAG)4 probes]. The cytogenetic identification was confirmed using barley arm-specific SSR and STS markers. The ditelosomic additions were propagated in the phytotron and in the field, and morphological parameters (plant height, tillering, length of the main spike, number of seeds/spike and seeds/plant, and spike characteristics) were described. In addition, the salt stress response of the ditelosomic additions was determined. CONCLUSIONS: The six-rowed winter barley cultivar Manas is much better adapted to Central European environmental conditions than the two-rowed spring barley Betzes previously used in wheat-barley crosses. The production of wheat-barley ditelosomic addition lines has a wide range of applications both for breeding (transfer of useful genes to the recipient species) and for basic research (mapping of barley genes, genetic and evolutionary studies and heterologous expression of barley genes in the wheat background).

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.

BACKGROUND AND AIMS: Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. METHODS: The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. KEY RESULTS: FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. CONCLUSIONS: The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.

Increased micronutrient content (Zn, Mn) in the 3M(b)(4B) wheat - Aegilops biuncialis substitution and 3M(b).4BS translocation identified by GISH and FISH.

3M(b) Triticum aestivum L. (Mv9kr1) - Aegilops biuncialis Vis. (MvGB642) addition lines were crossed with the Chinese Spring ph1b mutant genotype (CSph1b) to produce 3M(b)-wheat chromosome rearrangements. In the F3 generation, 3M(b)(4B) substitution lines and 3M(b).4BS centric fusions were identified with in situ hybridization using repetitive and genomic DNA probes, and with SSR markers. Grain micronutrient analysis showed that the investigated Ae. biuncialis accession MvGB382 and the parental line MvGB642 are suitable gene sources for improving the grain micronutrient content of wheat, as they have higher K, Zn, Fe, and Mn contents. The results suggested that the Ae. biuncialis chromosome 3M(b) carries genes determining the grain micronutrient content, as the 3M(b).4BS centric fusion had significantly higher Zn and Mn contents compared with the recipient wheat cultivar. As yield-related traits, such as the number of tillers, the length of main spike, and spikelets per main spike, were similar in the 3M(b).4BS centric fusion and the parental wheat genotype, it can be concluded that this line could be used in pre-breeding programs aimed at enriching elite wheat cultivars with essential micronutrients.

High-frequency generation and characterization of intergeneric hybrids and haploids from new wheat-barley crosses.

KEY MESSAGE: Hybrid plants and a high frequency of maternal haploids were obtained using an efficient wheat-barley hybridization system (with new genotype combinations) and confirmed by several cytological and molecular tools. An efficient hybridization system between wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is presented on the basis of three new genotype combinations. A particularly high, 14% frequency of plant regeneration per florets was achieved in the wheat-barley genotype combination of 'Sichuan' × 'Morex'. The genome composition in 42 of the 95 plants regenerated by embryo rescue was determined using ploidy analysis, genomic in situ hybridization and the application of chromosome arm-specific molecular markers (SSR and STS). A high overall frequency (76%) of maternal (wheat) haploids was observed in all the tests for all three cross combinations. A major implication of this observation is that this new hybridization system represents a useful tool to study the mechanism of uniparental chromosome elimination in cereals.

Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii.

KEY MESSAGE: Chromosomes 5A (u) , 5S and 5D can be isolated from wild progenitors, providing a chromosome-based approach to develop tools for breeding and to study the genome evolution of wheat. The three subgenomes of hexaploid bread wheat originated from Triticum urartu (A(u)A(u)), from a species similar to Aegilops speltoides (SS) (progenitor of the B genome), and from Ae. tauschii (DD). Earlier studies indicated the potential of chromosome genomics to assist gene transfer from wild relatives of wheat and discover novel genes for wheat improvement. This study evaluates the potential of flow cytometric chromosome sorting in the diploid progenitors of bread wheat. Flow karyotypes obtained by analysing DAPI-stained chromosomes were characterized and the contents of the chromosome peaks were determined. FISH analysis with repetitive DNA probes proved that chromosomes 5A(u), 5S and 5D could be sorted with purities of 78-90 %, while the remaining chromosomes could be sorted in groups of three. Twenty-five conserved orthologous set (COS) markers covering wheat homoeologous chromosome groups 1-7 were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. These assays validated the cytomolecular results as follows: peak I on flow karyotypes contained chromosome groups 1, 4 and 6, peak II represented homoeologous group 5, while peak III consisted of groups 2, 3 and 7. The isolation of individual chromosomes of wild progenitors provides an attractive opportunity to investigate the structure and evolution of the polyploid genome and to deliver tools for wheat improvement.