High-throughput genotyping of wheat-barley amphiploids utilising diversity array technology (DArT).

BMC Plant Biol. 2013 Jun 3;13(1):87

Authors: Castillo A, Ramírez MC, Martín AC, Kilian A, Martín A, Atienza SG

Abstract
BACKGROUND: Hordeum chilense, a native South American diploid wild barley, is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. Hexaploid tritordeum (xTritordeum Ascherson et Graebner, 2n=6x=42, AABBHchHch) is the fertile amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense and durum wheat. Approaches used in the improvement of this crop have included crosses with hexaploid wheat to promote D/Hch chromosome substitutions. While this approach has been successful as was the case with triticale, it has also complicated the genetic composition of the breeding materials. Until now tritordeum lines were analyzed based on molecular cytogenetic techniques and screening with a small set of DNA markers. However, the recent development of DArT markers in H. chilense offers new possibilities to screen large number of accessions more efficiently. RESULTS: Here, we have applied DArT markers to genotype composition in forty-six accessions of hexaploid tritordeum originating from different stages of tritordeum breeding program and to H. chilense-wheat chromosome addition lines to allow their physical mapping. Diversity analyses were conducted including dendrogram construction, principal component analysis and structure inference. Euploid and substituted tritordeums were clearly discriminated independently of the method used. However, dendrogram and Structure analyses allowed the clearest discrimination among substituted tritordeums. The physically mapped markers allowed identifying these groups as substituted tritordeums carrying the following disomic substitutions (DS): DS1D (1Hch), DS2D (2Hch), DS5D (5Hch), DS6D (6Hch) and the double substitution DS2D (2Hch), DS5D (5Hch). These results were validated using chromosome specific EST and SSR markers and GISH analysis. CONCLUSION: In conclusion, DArT markers have proved to be very useful to detect chromosome substitutions in the tritordeum breeding program and thus they are expected to be equally useful to detect translocations both in the tritordeum breeding program and in the transference of H. chilense genetic material in wheat breeding programs.

PMID: 23725040 [PubMed - as supplied by publisher]

The rpg4-mediated resistance to wheat stem rust (Puccinia graminis) in barley (Hordeum vulgare) requires Rpg5, a second NBS-LRR gene and an actin depolymerization factor.

Mol Plant Microbe Interact. 2012 Dec 5;

Authors: Wang X, Richards J, Gross T, Druka A, Kleinhofs A, Steffenson B, Acevedo M, Brueggeman RS

Abstract
The rpg4 gene confers recessive resistance to several races of wheat stem rust (Puccinia graminis f. sp. tritici) and Rpg5 provides dominant resistance against isolates of the rye stem rust (Puccinia graminis f. sp. secalis) in barley. The rpg4 and Rpg5 genes are tightly linked on chromosome 5H and positional cloning using high-resolution populations clearly separated the genes, unambiguously identifying Rpg5 but the identity of rpg4 remained unclear. High-resolution genotyping of critical recombinants at the rpg4/Rpg5 locus, designated here as RMRL (rpg4-mediated resistance locus) delimited two distinct yet tightly linked loci required for resistance, designated as RMRL1 and RMRL2. Utilizing virus-induced gene silencing, each gene at RMRL1, HvRga1 (a nucleotide binding site-leucine rich repeat (NBS-LRR) domain gene), Rpg5 (an NBS-LRR-protein kinase domain gene) and HvAdf3 (an actin depolymerizing factor-like gene), were individually silenced followed by inoculation with Pgt race QCCJ. Silencing each gene changed the reaction type from incompatible to compatible, indicating that all three genes are required for rpg4-mediated resistance. This stem rust resistance mechanism in barley follows the emerging theme of unrelated pairs of genetically linked NBS-LRR genes required for specific pathogen recognition and resistance. It also appears that actin cytoskeleton dynamics may play an important role in determining resistance against several races of stem rust in barley.

PMID: 23216085 [PubMed - as supplied by publisher]

Evaluation of diagnostic molecular markers for DUS phenotypic assessment in the cereal crop, barley (Hordeum vulgare ssp. vulgare L.).

Theor Appl Genet. 2012 Aug 17;

Authors: Cockram J, Jones H, Norris C, O'Sullivan DM

Abstract
The deployment of genetic markers is of interest in crop assessment and breeding programmes, due to the potential savings in cost and time afforded. As part of the internationally recognised framework for the awarding of Plant Breeders' Rights (PBR), new barley variety submissions are evaluated using a suite of morphological traits to ensure they are distinct, uniform and stable (DUS) in comparison to all previous submissions. Increasing knowledge of the genetic control of many of these traits provides the opportunity to assess the potential of deploying diagnostic/perfect genetic markers in place of phenotypic assessment. Here, we identify a suite of 25 genetic markers assaying for 14 DUS traits, and implement them using a single genotyping platform (KASPar). Using a panel of 169 UK barley varieties, we show that phenotypic state at three of these traits can be perfectly predicted by genotype. Predictive values for an additional nine traits ranged from 81 to 99 %. Finally, by comparison of varietal discrimination based on phenotype and genotype resulted in correlation of 0.72, indicating that deployment of molecular markers for varietal discrimination could be feasible in the near future. Due to the flexibility of the genotyping platform used, the genetic markers described here can be used in any number or combination, in-house or by outsourcing, allowing flexible deployment by users. These markers are likely to find application where tracking of specific alleles is required in breeding programmes, or for potential use within national assessment programmes for the awarding of PBRs.

PMID: 22898724 [PubMed - as supplied by publisher]

Application of real-time PCR-based SNP detection for mapping of <i>Net2</i>, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>.

Genes Genet Syst. 2012;87(2):137-43

Authors: Matsuda R, Iehisa JC, Takumi S

Abstract
Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F<inf>2</inf> individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.

PMID: 22820387 [PubMed - in process]