Molecular Mapping of the rps1.a Recessive Gene for Resistance to Stripe Rust in BBA 2890 Barley.

Phytopathology. 2007 Jun;97(6):668-73

Authors: Yan G, Chen X

ABSTRACT Stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important diseases of barley in the south-central and western United States. Growing resistant cultivars is the best approach for controlling the disease. The barley genotype BBA 2890 has all-stage resistance against all races of P. striiformis f. sp. hordei (PSH) identified thus far in the United States. The resistance in BBA 2890 is controlled by a single recessive gene, rps1.a. The objectives of this study were to identify resistance gene analog polymorphism (RGAP) markers for the all-stage resistance gene rps1.a, to map the gene on a barley chromosome using chromosome-specific simple sequence repeat (SSR) markers, and to determine the presence or absence of the flanking RGAP markers for the gene in 24 barley genotypes. Seedlings of the parents and 200 F(8) recombinant inbred lines (RILs) were tested for resistance to pathogen races PSH-14, PSH-48, and PSH-54 in the greenhouse in 2005. Genomic DNA was extracted from the parents and 150 F(8) RILs. The RGAP technique was used to identify molecular markers for the rps1.a gene. Twelve primer pairs generating repeatable polymorphic bands were selected for genotyping the 150 F(8) RILs. A genetic linkage group was constructed for the resistance gene with 13 RGAP markers and four chromosome-specific SSR markers. The four SSR markers mapped the gene on the long arm of barley chromosome 3H. The closest RGAP marker for the resistant allele was within a genetic distance of 2.1 centimorgans (cM). The closest marker for the susceptible allele was 6.8 cM away from the locus. The two closest RGAP markers for the resistant allele detected polymorphisms in 67 and 71% of the 24 barley genotypes when used individually, and detected polymorphism in 88% of the genotypes when used in combination. This information should be useful in incorporating the resistance gene into barley cultivars and in pyramiding the gene with other resistance genes for superior stripe rust resistance.

PMID: 18943597 [PubMed - in process]

Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork.

BMC Genet. 2008;9:73

Authors: Druka A, Druka I, Centeno AG, Li H, Sun Z, Thomas WT, Bonar N, Steffenson BJ, Ullrich SE, Kleinhofs A, Wise RP, Close TJ, Potokina E, Luo Z, Wagner C, Schweizer GF, Marshall DF, Kearsey MJ, Williams RW, Waugh R

A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community.

PMID: 19017390 [PubMed - indexed for MEDLINE]

Transcript profiling and expression level mapping.

Methods Mol Biol. 2009;513:81-92

Authors: Potokina E, Druka A, Kearsey MJ

Transcript abundance data from cRNA hybridizations to Affymetrix microarrays can potentially be used to identify genetic markers to facilitate high-throughput genotyping. We have shown that it is easily possible to use the information from Affymetrix expression arrays to accurately identify over 4,000 robust polymorphic transcript-derived markers (TDMs). We developed the method to identity TDM polymorphisms from experiments involving two tissues in two commercial varieties of barley and their doubled-haploid progeny. These TDMs represent ~18% of the total barley genes on the chip and can be used to predict the genotypes in an F(1)-derived, doubled-haploid population. According to our estimates, 35% of the TDMs reveal nucleotide polymorphism of the particular gene (single feature polymorphisms, SFPs) while 65% mark polymorphism resulting in extreme variation of gene expression (genetic expression markers, GEMs). These latter are probably mainly cis-acting regulators while a small proportion, approximately 5%, are loosely or un-linked transregulators.

PMID: 19347647 [PubMed - indexed for MEDLINE]

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

Theor Appl Genet. 2009 Sep;119(5):851-65

Authors: Hofinger BJ, Jing HC, Hammond-Kosack KE, Kanyuka K

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

PMID: 19578831 [PubMed - indexed for MEDLINE]