Fat element-a new marker for chromosome and genome analysis in the Triticeae.

Chromosomal distribution of the Fat element that was isolated from bacterial artificial chromosome (BAC) end sequences of wheat chromosome 3B was studied in 45 species representing eight genera of Poaceae (Aegilops, Triticum, Agropyron, Elymus, Secale, Hordeum, Avena and Triticale) using fluorescence in situ hybridisation (FISH). The Fat sequence was not present in oats and in two barley species, Hordeum vulgare and Hordeum spontaneum, that we investigated. Only very low amounts of the Fat element were detected on the chromosomes of two other barley species, Hordeum geniculatum and Hordeum chilense, with different genome compositions. The chromosomes of other cereal species exhibited distinct hybridisation patterns with the Fat probe, and labelling intensity varied significantly depending on the species or genome. The highest amount of hybridisation was detected on chromosomes of the D genome of Aegilops and Triticum and on chromosomes of the S genome of Agropyron. Despite the bioinformatics analysis of several BAC clones that revealed the tandem organisation of the Fat element, hybridisation with the Fat probe produces uneven, diffuse signals in the proximal regions of chromosomes. In some of the genomes we investigated, however, it also forms distinct, sharp clusters in chromosome-specific positions, and the brightest fluorescence was always observed on group 4 chromosomes. Thus, the Fat element represents a new family of Triticeae-specific, highly repeated DNA elements with a clustered-dispersed distribution pattern. These elements may have first emerged in cereal genomes at the time of divergence of the genus Hordeum from the last common ancestor. During subsequent evolution, the amount and chromosomal distribution of the Fat element changed due to amplification, elimination and re-distribution of this sequence. Because the labelling patterns that we detected were highly specific, the Fat element can be used as an accessory probe in FISH analysis for chromosome identification and investigation of evolutionary processes at the chromosomal level.

Insertion site-based polymorphism markers open new perspectives for genome saturation and marker-assisted selection in wheat.

In wheat, the deployment of marker-assisted selection has long been hampered by the lack of markers compatible with high-throughput cost-effective genotyping techniques. Recently, insertion site-based polymorphism (ISBP) markers have appeared as very powerful new tools for genomics and genetic studies in hexaploid wheat. To demonstrate their possible use in wheat breeding programmes, we assessed their potential to meet the five main requirements for utilization in MAS: flexible and high-throughput detection methods, low quantity and quality of DNA required, low cost per assay, tight link to target loci and high level of polymorphism in breeding material. Toward this aim, we developed a programme, IsbpFinder, for the automated design of ISBP markers and adapted three detection methods (melting curve analysis, SNaPshot Multiplex System and Illumina BeadArray technology) for high throughput and flexible detection of ISBP or ISBP-derived SNP markers. We demonstrate that the high level of polymorphism of the ISBPs combined with cost-effective genotyping methods can be used to efficiently saturate genetic maps, discriminate between elite cultivars, and design tightly linked diagnostic markers for virtually all target loci in the wheat genome. All together, our results suggest that ISBP markers have the potential to lead to a breakthrough in wheat marker-assisted selection.

Reactivity profiling: covalent modification of single nucleophile peptides for skin sensitization risk assessment.

The molecular basis of chemical allergy is rooted in the ability of an allergen (hapten) to modify endogenous proteins. This mechanistic understanding aided development of screening assays which generate reproducible quantitative and qualitative reactivity data. Such assays use model peptides with a limited number and type of protein nucleophiles, and the data does not reflect the specificity, variety, and complexity of hapten interactions with multiple nucleophiles. Building on these developments, we extended the standardized approach to maximize the type and the amount of information that can be derived from an in chemico assay. We used a panel of six single nucleophile peptides and individually optimized the incubation conditions to favor chemical modification. Employing liquid chromatography tandem mass spectrometry (LC-MS/MS) technique, we simultaneously obtained multiple quantitative and qualitative measurements (% peptide depletion, adducts formation, and peptide dimerization for Cys-containing peptide). Using these methods, we obtained reactivity data for 36 chemicals of known skin sensitizing potency. By optimizing incubation conditions, we ensured detection of all reactive chemicals. We explored the LC-MS/MS approach to generate kinetic data for 10 chemicals allowing further characterization of reactivity and a potentially more robust quantitative reactivity descriptor. Our ultimate aim is to integrate this dataset with available physicochemical data and outputs from other predictive assays, all addressing different key steps in the induction of sensitization, to help us make decisions about the safe use of chemicals without using animal tests. The epidermal protein target sites, modification of which may be immunogenic and lead to induction of skin sensitization, are currently unknown. Increasing the understanding of this process may help further refine in chemico reactivity assays as well as aid the interpretation of the reactivity data.

Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B.

Bread wheat (Triticum aestivum) is one of the most important crops worldwide. However, because of its large, hexaploid, highly repetitive genome it is a challenge to develop efficient means for molecular analysis and genetic improvement in wheat. To better understand the composition and molecular evolution of the hexaploid wheat homoeologous genomes and to evaluate the potential of BAC-end sequences (BES) for marker development, we have followed a chromosome-specific strategy and generated 11 Mb of random BES from chromosome 3B, the largest chromosome of bread wheat. The sequence consisted of about 86% of repetitive elements, 1.2% of coding regions, and 13% remained unknown. With 1.2% of the sequence length corresponding to coding sequences, 6000 genes were estimated for chromosome 3B. New repetitive sequences were identified, including a Triticineae-specific tandem repeat (Fat) that represents 0.6% of the B-genome and has been differentially amplified in the homoeologous genomes before polyploidization. About 10% of the BES contained junctions between nested transposable elements that were used to develop chromosome-specific markers for physical and genetic mapping. Finally, sequence comparison with 2.9 Mb of random sequences from the D-genome of Aegilops tauschii suggested that the larger size of the B-genome is due to a higher content in repetitive elements. It also indicated which families of transposable elements are mostly responsible for differential expansion of the homoeologous wheat genomes during evolution. Our data demonstrate that BAC-end sequencing from flow-sorted chromosomes is a powerful tool for analysing the structure and evolution of polyploid and highly repetitive genomes.