Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

The CC-NB-LRR-type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death.

BACKGROUND: Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance. PRINCIPAL FINDINGS: We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5' regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism. CONCLUSIONS: This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death.

Constitutive differences between steely and mealy barley samples associated with endosperm modification.

BACKGROUND: Structurally different areas may occur in the endosperm of the barley grain, and they can be visually classified as either mealy or steely. Barleys with a high proportion of grains that are mostly steely often show uneven physical-chemical modification of the endosperm during malting. To study the relationship between steeliness and endosperm modification, two samples of barley cv. Scarlett with contrasting malting quality were analysed. RESULTS: The proportions of steely grains were 77% and 46% in the two samples, which were then defined as steely sample and mealy sample, respectively. The steely sample showed slower modification during malting (in terms of beta-glucan degradation, friability increase, and Calcofluor staining), lower hot water extract (HWE) and acrospire growth, and higher extract viscosity. Endosperm permeation to large molecules (tested with the fluorescein isothiocyanate-dextran conjugate, FITC-D) closely followed cell wall modification in the steely sample, but this was not so in the mealy sample. CONCLUSIONS: Higher steeliness was associated with higher levels of C hordeins in the grain of barley cv. Scarlett. It is proposed that such hordeins can increase the permeability to large molecules (FITC-D) but slow modification. Like steeliness and the level of C hordeins, permeability to FITC-D appears to be more linked to environmental rather than genetic effects. Although a more general association of C hordeins with steeliness of malting barley still has to be ascertained, the negative role of C hordeins in malting quality has been confirmed.