András Cseh

Advances in plant gene-targeted and functional markers: a review

Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the potential to generate phenotypically linked functional markers, especially when fingerprints are generated from the transcribed or expressed region of the genome. It is to be expected that these recently developed techniques will generate larger datasets, but their shortcomings should also be acknowledged and carefully investigated.

Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata.

This study evaluates the potential of flow cytometry for chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes were characterized and content of chromosome peaks was determined. Peaks of chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the chromosome could be sorted with purities exceeding 95%. The remaining chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops chromosomes using sorted chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.

Syntenic Relationships between the U and M Genomes of Aegilops, Wheat and the Model Species Brachypodium and Rice as Revealed by COS Markers

Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat–Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.

Development and identification of a 4HL.5DL wheat/barley centric fusion using GISH, FISH and SSR markers

The 4H(4D) wheat/barley substitution line was crossed with the ‘Chinese Spring’ ph1b mutant genotype in order to induce wheat-barley homoeologous recombinations. F3 and F4 seeds of the 4H(4D) × ‘Chinese Spring’ ph1b mutant cross were analysed using genomic in situ hybridization, and a Robertsonian translocation was detected in monosomic form. Disomic centric fusions were selected among the self-fertilized progenies. The presence of the long arm of 4H was confirmed with SSR markers. The long arm of the 5D wheat chromosome in the Robertsonian translocation was identified using fluorescent in situ hybridization with the help of three DNA probes: pSc119.2, Afa family and pTa71. The wheat/barley centric fusion was identified as a 4HL.5DL translocation. This line exhibited supernumerary spikelet character, but the number of seeds/plant did not increase. The 4HL.5DL centric fusion line is suitable genetic material to study the expression of genes located on 4HL in a wheat genetic background.

Addition of Manas barley chromosome arms to the hexaploid wheat genome

BackgroundCultivated 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.ResultsA 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.ConclusionsThe 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).