Dissection of figured wood trait in curly birch (Betula pendula Roth var. carelica (Mercklin) Hämet-Ahti) using high-throughput genotyping.

Curly (Karelian) birch is a special variety of Betula pendula Roth distributed in the northwestern part of Europe. Karelian birch is well-known for its valuable figured curly wood also known as "wooden marble". The genetic basis underlying curly wood formation has been debated since last century, however, there was no data about loci responsible for the curly wood trait. In the present study, we analyzed two full-sibs populations derived from experimental crosses of curly birches and segregating for the trait. RADseq genotyping was applied to reveal how many loci are involved in 'curliness' formation and to search for genetic variants associated with this trait. One single interval on chromosome 10 was detected containing possible candidate genes. InDel marker BpCW1 was suggested for the first time for marker-assisted selection of trees with curly wood at their earliest stages of development.

Development of a High-Density Genetic Map for Muscadine Grape Using a Mapping Population from Selfing of the Perfect-Flowered Vine 'Dixie'.

Intraspecific diversity of the immune grape Muscadinia rotundifolia Michaux. can serve as a rich source of valuable resistance loci to the most widespread pathogens and pests of grapevine. While only one Run1/Rpg1 resistance locus has been introgressed from M. rotundifolia to the Vitis vinifera gene pool, a number of other genes conferring resistance to powdery mildew and downy mildew have been identified in various Muscadinia cultivars. A larger introduction of Muscadinia varieties to the European continent would greatly facilitate experiments of interspecific crosses as well as stimulate biotechnological efforts to overcome the main barrier to F1 fertility caused by the differences in chromosome number. For the successful introduction of Muscadinia into the new European environment, it is necessary to overcome the difficulties associated with the physiological characteristics of the species, such as insufficient cold tolerance and very late fruit ripening. To facilitate the further discovery of valuable loci in Muscadinia and their transfer to grapevine breeding programs, we constructed a high-density linkage map using an S1 mapping population obtained from the self-pollination of M. rotundifolia cv. Dixie maintained on the southern coast of Crimea. Using ddRADseq, 3730 SNPs were ordered across 20 linkage groups spanning 2753.6 cM of the total map length. No segregation in resistance to diseases and pests was observed among the 'Dixie' S1 population, suggesting the presence of homozygous non-segregating resistant loci in the genetic background of 'Dixie'. Markers with high segregation distortion showed a bias towards chromosomal intervals on linkage groups 10 and 20, where loci affecting the survival of 'Dixie' S1 progeny may be localized. QTLs with significant additive and dominance effects were discovered on LG14 and LG18, affecting the morphological traits associated with the vigor of growth and adaptability of young Muscadinia vines in the conditions of Crimea.

Development of SNP Set for the Marker-Assisted Selection of Guar (Cyamopsis tetragonoloba (L.) Taub.) Based on a Custom Reference Genome Assembly.

Guar gum, a polysaccharide derived from guar seeds, is widely used in a variety of industrial applications, including oil and gas production. Although guar is mostly propagated in India, interest in guar as a new industrial legume crop is increasing worldwide, demanding the development of effective tools for marker-assisted selection. In this paper, we report a wide-ranging set of 4907 common SNPs and 327 InDels generated from RADseq genotyping data of 166 guar plants of different geographical origin. A custom guar reference genome was assembled and used for variant calling. A consensus set of variants was built using three bioinformatic pipelines for short variant discovery. The developed molecular markers were used for genome-wide association study, resulting in the discovery of six markers linked to the variation of an important agronomic trait-percentage of pods matured to the harvest date under long light day conditions. One of the associated variants was found inside the putative transcript sequence homologous to an ABC transporter in Arabidopsis, which has been shown to play an important role in D-myo-inositol phosphates metabolism. Earlier, we suggested that genes involved in myo-inositol phosphate metabolism have significant impact on the early flowering of guar plants. Hence, we believe that the developed SNP set allows for the identification of confident molecular markers of important agrobiological traits.

Identification of Key Metabolic Pathways and Biomarkers Underlying Flowering Time of Guar (Cyamopsis tetragonoloba (L.) Taub.) via Integrated Transcriptome-Metabolome Analysis.

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an annual legume crop native to India and Pakistan. Seeds of the plant serve as a source of galactomannan polysaccharide (guar gum) used in the food industry as a stabilizer (E412) and as a gelling agent in oil and gas fracturing fluids. There were several attempts to introduce this crop to countries of more northern latitudes. However, guar is a plant of a short photoperiod, therefore, its introduction, for example, to Russia is complicated by a long day length during the growing season. Breeding of new guar varieties insensitive to photoperiod slowed down due to the lack of information on functional molecular markers, which, in turn, requires information on guar genome. Modern breeding strategies, e.g., genomic predictions, benefit from integration of multi-omics approaches such as transcriptome, proteome and metabolome assays. Here we present an attempt to use transcriptome-metabolome integration to understand the genetic determination of flowering time variation among guar plants that differ in their photoperiod sensitivity. This study was performed on nine early- and six delayed-flowering guar varieties with the goal to find a connection between 63 metabolites and 1,067 differentially expressed transcripts using Shiny GAM approach. For the key biomarker of flowering in guar myo-inositol we also evaluated the KEGG biochemical pathway maps available for Arabidopsis thaliana. We found that the phosphatidylinositol signaling pathway is initiated in guar plants that are ready for flowering through the activation of the phospholipase C (PLC) gene, resulting in an exponential increase in the amount of myo-inositol in its free form observed on GC-MS chromatograms. The signaling pathway is performed by suppression of myo-inositol phosphate kinases (phosphorylation) and alternative overexpression of phosphatases (dephosphorylation). Our study suggests that metabolome and transcriptome information taken together, provide valuable information about biomarkers that can be used as a tool for marker-assisted breeding, metabolomics and functional genomics of this important legume crop.

The Assessment of Agrobiological and Disease Resistance Traits of Grapevine Hybrid Populations (Vitis vinifera L. × Muscadinia rotundifolia Michx.) in the Climatic Conditions of Crimea.

The Crimean autochthonous grape varieties are unique by their origin and serve as a valuable source for breeding new cultivars with increased salt and frost resistance, as well as high-quality berries. However, they suffer from fungal pathogens, as the dry and hot summer months contribute to the epiphytotic course of diseases. An increase in the resistance of Crimean grape varieties is currently achieved through interspecific hybridization. In this study, we describe the genetic and agrobiological diversity of three hybrid populations obtained using the Vitis interspecific hybrid 'Magarach 31-77-10' as a female parent and Muscadinia rotundifolia × Vitis vinifera BC5 hybrid plants as male parents. The hybrid nature of the populations was assessed using RADseq high-throughput genotyping. We discovered 12,734 SNPs, which were common to all three hybrid populations. We also proved with the SSR markers that the strong powdery and downy mildew resistance of the paternal genotypes is determined by the dominant Run1/Rpv1 locus inherited from M. rotundifolia. As a result, the disease development score (R, %) for both mildew diseases in the female parent 'Magarach 31-77-10' was three times higher than in male parents 2000-305-143 and 2000-305-163 over two years of phytopathological assessment. The highest values of yield-contributing traits (average bunch weight ~197 g and 1.3 kg as yield per plant) were detected in the population 4-11 (♀M. No. 31-77-10 × 2000-305-163). Despite the epiphytotic development of PM, the spread of oidium to the vegetative organs of hybrids 4-11 did not exceed 20%. Some hybrid genotypes with high productivity and resistance to pathogens were selected for further assessment as promising candidates for new varieties.

Key metabolites associated with the onset of flowering of guar genotypes (Cyamopsis tetragonoloba (L.) Taub).

BACKGROUND: Guar (Cyamopsis tetragonoloba (L.) Taub.), a short-day plant, is an economically valuable legume crop. Seeds of guar serve as a source of galactomannan polysaccharide, known as guar gum, which is in demand in the gas and oil industries. The rapid and complete maturation of guar seeds depends on the flowering time of a particular genotype. It is known that flowering in guar is controlled by several gene systems. However, no information about the process and mechanisms that trigger flowering in guar on the molecular and biochemical levels was previously reported. The aim of the study was to investigate the metabolic landscape underlying transition to the flowering in guar using GC-MS-metabolomic analysis. RESULTS: 82 diverse guar genotypes (each in 8 replicates) from the VIR collection were grown under experimental conditions of high humidity and long photoperiod. In the stress environment some guar genotypes turned to flowering early (41 ± 1,8 days from the first true leaf appearance) while for others the serious delay of flowering (up to 95 ± 1,7 days) was observed. A total of 244 metabolites were detected by GC-MS analysis on the third true leaves stage of 82 guar genotypes. Among them some molecules were associated with the transition of the guar plants to flowering. Clear discrimination was observed in metabolomic profiles of two groups of «early flowering¼ and «delayed flowering¼ plants, with 65 metabolites having a significantly higher abundance in early flowering genotypes. Among them 7 key molecules were identified by S-plot, as potential biomarkers discriminating of «early flowering¼ and «delayed flowering¼ guar genotypes. CONCLUSIONS: The metabolomic landscape accompanying transition to flowering in guar was firstly described. The results obtained can be used in subsequent genomic research for identifying metabolite-gene associations and revealing genes responsible for the onset of flowering and photoperiod sensitivity of guar. In addition, the detected key metabolites associated with flowering of guar can be employed as biomarkers allowing rapid screening of breeding material for the potentially early flowering genotypes.

The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae.

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.

Impact of the 7-bp deletion in HvGA20ox2 gene on agronomic important traits in barley (Hordeum vulgare L.).

BACKGROUND: Alike to Reduced height-1 (Rht-1) genes in wheat and the semi dwarfing (sd-1) gene in rice, the sdw1/denso locus involved in the metabolism of the GA, was designated as the 'Green Revolution' gene in barley. The recent molecular characterization of the candidate gene HvGA20ox2 for sdw1/denso locus allows to estimate the impact of the functional polymorphism of this gene on the variation of agronomically important traits in barley. RESULTS: We investigated the effect of the 7-bp deletion in exon 1 of HvGA20ox2 gene (sdw1.d mutation) on the variation of yield-related and malting quality traits in the population of DHLs derived from cross of medium tall barley Morex and semi-dwarf barley Barke. Segregation of plant height, flowering time, thousand grain weight, grain protein content and grain starch was evaluated in two diverse environments separated from one another by 15° of latitude. The 7-bp deletion in HvGA20ox2 gene reduced plant height by approximately 13 cm and delayed flowering time by 3-5 days in the barley segregating DHLs population independently on environmental cue. On other hand, the sdw1.d mutation did not affect significantly either grain quality traits (protein and starch content) or thousand grain weight. CONCLUSIONS: The beneficial effect of the sdw1.d allele could be associated in barley with lodging resistance and extended period of vegetative growth allowing to accumulate additional biomass that supports higher yield in certain environments. However, no direct effect of the sdw1.d mutation on thousand grain weight or grain quality traits in barley was detected.

Features of Ppd-B1 expression regulation and their impact on the flowering time of wheat near-isogenic lines.

BACKGROUND: Photoperiod insensitive Ppd-1a alleles determine early flowering of wheat. Increased expression of homoeologous Ppd-D1a and Ppd-A1a result from deletions in the promoter region, and elevated expression of Ppd-B1a is determined by an increased copy number. RESULTS: In this study, using bread wheat cultivars Sonora and PSL2, which contrast in flowering time, and near-isogenic lines resulting from their cross, "Ppd-m" and "Ppd-w" with Ppd-B1a introgressed from Sonora, we investigated the putative factors that influence Ppd-B1a expression. By analyzing the Ppd-B1a three distinct copies, we identified an indel and the two SNPs, which distinguished the investigated allele from other alleles with a copy number variation. We studied the expression of the Ppd-A1, Ppd-B1a, and Ppd-D1 genes along with genes that are involved in light perception (PhyA, PhyB, PhyC) and the flowering initiation (Vrn-1, TaFT1) and discussed their interactions. Expression of Ppd-B1a in the "Ppd-m" line, which flowered four days earlier than "Ppd-w", was significantly higher. We found PhyC to be up-regulated in lines with Ppd-B1a alleles. Expression of PhyC was higher in "Ppd-m". Microsatellite genotyping demonstrated that in the line "Ppd-m", there is an introgression in the pericentromeric region of chromosome 5B from the early flowering parental Sonora, while the "Ppd-w" does not have this introgression. FHY3/FAR1 is known to be located in this region. Expression of the transcription factor FHY3/FAR1 was higher in the "Ppd-m" line than in "Ppd-w", suggesting that FHY3/FAR1 is important for the wheat flowering time and may cause earlier flowering of "Ppd-m" as compared to "Ppd-w". CONCLUSIONS: We propose that there is a positive bidirectional regulation of Ppd-B1a and PhyC with an FHY3/FAR1 contribution. The bidirectional regulation can be proposed for Ppd-A1a and Ppd-D1a. Using in silico analysis, we demonstrated that the specificity of the Ppd-B1 regulation compared to that of homoeologous genes involves not only a copy number variation but also distinct regulatory elements.

Development of F1 hybrid population and the high-density linkage map for European aspen (Populus tremula L.) using RADseq technology.

BACKGROUND: Restriction-site associated DNA sequencing (RADseq) technology was recently employed to identify a large number of single nucleotide polymorphisms (SNP) for linkage mapping of a North American and Eastern Asian Populus species. However, there is also the need for high-density genetic linkage maps for the European aspen (P. tremula) as a tool for further mapping of quantitative trait loci (QTLs) and marker-assisted selection of the Populus species native to Europe. RESULTS: We established a hybrid F1 population from the cross of two aspen parental genotypes diverged in their phenological and morphological traits. We performed RADseq of 122 F1 progenies and two parents yielding 15,732 high-quality SNPs that were successfully identified using the reference genome of P. trichocarpa. 2055 SNPs were employed for the construction of maternal and paternal linkage maps. The maternal linkage map was assembled with 1000 SNPs, containing 19 linkage groups and spanning 3054.9 cM of the genome, with an average distance of 3.05 cM between adjacent markers. The paternal map consisted of 1055 SNPs and the same number of linkage groups with a total length of 3090.56 cM and average interval distance of 2.93 cM. The linkage maps were employed for QTL mapping of one-year-old seedlings height variation. The most significant QTL (LOD = 5.73) was localized to LG5 (96.94 cM) of the male linkage map, explaining 18% of the phenotypic variation. CONCLUSIONS: The set of 15,732 SNPs polymorphic in aspen and high-density genetic linkage maps constructed for the P. tremula intra-specific cross will provide a valuable source for QTL mapping and identification of candidate genes facilitating marker-assisted selection in European aspen.

Expression quantitative trait loci analysis in plants.

An expression Quantitative Trait Locus or eQTL is a chromosomal region that accounts for a proportion of the variation in abundance of a mRNA transcript observed between individuals in a genetic mapping population. A single gene can have one or multiple eQTLs. Large scale mRNA profiling technologies advanced genome-wide eQTL mapping in a diverse range of organisms allowing thousands of eQTLs to be detected in a single experiment. When combined with classical or trait QTLs, correlation analyses can directly suggest candidates for genes underlying these traits. Furthermore, eQTL mapping data enables genetic regulatory networks to be modelled and potentially provide a better understanding of the underlying phenotypic variation. The mRNA profiling data sets can also be used to infer the chromosomal positions of thousands of genes, an outcome that is particularly valuable for species with unsequenced genomes where the chromosomal location of the majority of genes remains unknown. In this review we focus on eQTL studies in plants, addressing conceptual and technical aspects that include experimental design, genetic polymorphism prediction and candidate gene identification.

Transcript profiling and expression level mapping.

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.

Robust detection and genotyping of single feature polymorphisms from gene expression data.

It is well known that Affymetrix microarrays are widely used to predict genome-wide gene expression and genome-wide genetic polymorphisms from RNA and genomic DNA hybridization experiments, respectively. It has recently been proposed to integrate the two predictions by use of RNA microarray data only. Although the ability to detect single feature polymorphisms (SFPs) from RNA microarray data has many practical implications for genome study in both sequenced and unsequenced species, it raises enormous challenges for statistical modelling and analysis of microarray gene expression data for this objective. Several methods are proposed to predict SFPs from the gene expression profile. However, their performance is highly vulnerable to differential expression of genes. The SFPs thus predicted are eventually a reflection of differentially expressed genes rather than genuine sequence polymorphisms. To address the problem, we developed a novel statistical method to separate the binding affinity between a transcript and its targeting probe and the parameter measuring transcript abundance from perfect-match hybridization values of Affymetrix gene expression data. We implemented a Bayesian approach to detect SFPs and to genotype a segregating population at the detected SFPs. Based on analysis of three Affymetrix microarray datasets, we demonstrated that the present method confers a significantly improved robustness and accuracy in detecting the SFPs that carry genuine sequence polymorphisms when compared to its rivals in the literature. The method developed in this paper will provide experimental genomicists with advanced analytical tools for appropriate and efficient analysis of their microarray experiments and biostatisticians with insightful interpretation of Affymetrix microarray data.

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

BACKGROUND: 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. DESCRIPTION: Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. CONCLUSION: By integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets.

Tissue-dependent limited pleiotropy affects gene expression in barley.

Non-synonymous coding mutations in a gene change the resulting protein, no matter where it is expressed, but the effects of cis-regulatory mutations could be spatially or temporally limited - a phenomenon termed limited pleiotropy. Here, we report the genome-wide occurrence of limited pleiotropy of cis-regulatory mutations in barley (Hordeum vulgare L.) using Affymetrix analysis of 22,840 genes in a population of 139 doubled haploid lines derived from a cross between the cultivars Steptoe (St) and Morex (Mx). We identified robust cis-acting expression regulators that segregate as major genes in two successive ontogenetic stages: germinating embryo tissues and seedling leaves from the embryonic axis. We show that these polymorphisms may be consistent in both tissues or may cause a dramatic change in transcript abundance in one tissue but not in another. We also show that the parental allele that increases expression can vary with the tissue, suggesting nucleotide polymorphism in enhancer sequences. Because of the limited pleiotropy of cis-regulating mutations, the number of cis expression quantitative trait loci (cis-eQTLs) discovered by 'genetical genomics' is strongly affected by the particular tissue or developmental stage studied. Given that limited pleiotropy is a common feature of cis-regulatory mutations in barley, we predict that the phenomenon would be relevant to developmental and/or tissue-specific interactions across wide taxonomic boundaries in both plants and animals.

Methods for evaluating gene expression from Affymetrix microarray datasets.

BACKGROUND: Affymetrix high density oligonucleotide expression arrays are widely used across all fields of biological research for measuring genome-wide gene expression. An important step in processing oligonucleotide microarray data is to produce a single value for the gene expression level of an RNA transcript using one of a growing number of statistical methods. The challenge for the researcher is to decide on the most appropriate method to use to address a specific biological question with a given dataset. Although several research efforts have focused on assessing performance of a few methods in evaluating gene expression from RNA hybridization experiments with different datasets, the relative merits of the methods currently available in the literature for evaluating genome-wide gene expression from Affymetrix microarray data collected from real biological experiments remain actively debated. RESULTS: The present study reports a comprehensive survey of the performance of all seven commonly used methods in evaluating genome-wide gene expression from a well-designed experiment using Affymetrix microarrays. The experiment profiled eight genetically divergent barley cultivars each with three biological replicates. The dataset so obtained confers a balanced and idealized structure for the present analysis. The methods were evaluated on their sensitivity for detecting differentially expressed genes, reproducibility of expression values across replicates, and consistency in calling differentially expressed genes. The number of genes detected as differentially expressed among methods differed by a factor of two or more at a given false discovery rate (FDR) level. Moreover, we propose the use of genes containing single feature polymorphisms (SFPs) as an empirical test for comparison among methods for the ability to detect true differential gene expression on the basis that SFPs largely correspond to cis-acting expression regulators. The PDNN method demonstrated superiority over all other methods in every comparison, whilst the default Affymetrix MAS5.0 method was clearly inferior. CONCLUSION: A comprehensive assessment of seven commonly used data extraction methods based on an extensive barley Affymetrix gene expression dataset has shown that the PDNN method has superior performance for the detection of differentially expressed genes.

Exploiting regulatory variation to identify genes underlying quantitative resistance to the wheat stem rust pathogen Puccinia graminis f. sp. tritici in barley.

We previously mapped mRNA transcript abundance traits (expression-QTL or eQTL) using the Barley1 Affymetrix array and 'whole plant' tissue from 139 progeny of the Steptoe x Morex (St/Mx) reference barley mapping population. Of the 22,840 probesets (genes) on the array, 15,987 reported transcript abundance signals that were suitable for eQTL analysis, and this revealed a genome-wide distribution of 23,738 significant eQTLs. Here we have explored the potential of using these mRNA abundance eQTL traits as surrogates for the identification of candidate genes underlying the interaction between barley and the wheat stem rust fungus Puccinia graminis f. sp. tritici. We re-analysed quantitative 'resistance phenotype' data collected on this population in 1990/1991 and identified six loci associated with barley's reaction to stem rust. One of these coincided with the major stem rust resistance locus Rpg1, that we had previously positionally cloned using this population. Correlation analysis between phenotype values for rust infection and mRNA abundance values reported by the 22,840 GeneChip probe sets placed Rpg1, which is on the Barley1 GeneChip, in the top five candidate genes for the major QTL on chromosome 7H corresponding to the location of Rpg1. A second co-located with the rpg4/Rpg5 stem rust resistance locus that has been mapped in a different population and the remaining four were novel. Correlation analyses identified candidate genes for the rpg4/Rpg5 locus on chromosome 5H. By combining our data with additional published mRNA profiling data sets, we identify a putative sensory transduction histidine kinase as a strong candidate for a novel resistance locus on chromosome 2H and compile candidate gene lists for the other three loci.

Gene expression quantitative trait locus analysis of 16 000 barley genes reveals a complex pattern of genome-wide transcriptional regulation.

Transcript abundance from cRNA hybridizations to Affymetrix microarrays can be used for simultaneous marker development and genome-wide gene expression quantitative trait locus (eQTL) analysis of crops. We have previously shown that it is easily possible to use Affymetrix expression arrays to profile individuals from a segregating population to accurately identify robust polymorphic molecular genetic markers. We applied the method to identify more than 2000 genetic polymorphisms (transcript derived markers, TDMs) from an experiment involving two commercial varieties of barley (Hordeum vulgare; Steptoe and Morex) and their doubled-haploid progeny. With this set of TDMs, we constructed a genetic map and used it for the genome-wide eQTL analysis of about 16 000 genes in a relatively large population (n = 139). We identified 23 738 significant eQTLs at a genome-wide significance (P

Large-scale analysis of the barley transcriptome based on expressed sequence tags.

To provide resources for barley genomics, 110,981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one-third of the 380,000 publicly available barley ESTs. Clustering and assembly resulted in 14,151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category 'Energy' was further analysed to reveal tissue- and stage-specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination-specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence-related and antioxidant genes during germination might be controlled by the ethylene-signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.