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Abstract Background The APETALA2-like genes form a large multi-gene family of transcription factors which play an important role during the plant life cycle, being key regulators of many developmental processes. Many studies in Arabidopsis have revealed that the APETALA2 (AP2) gene is implicated in the establishment of floral meristem and floral organ identity as well as temporal and spatial regulation of flower homeotic gene expression. Results In this work, we have cloned and characterised the AP2-like gene from accessions of Hordeum chilense and Hordeum vulgare, wild and domesticated barley, respectively, and compared with other AP2 homoeologous genes, including the Q gene in wheat. The Hordeum AP2-like genes contain two plant-specific DNA binding motifs called AP2 domains, as does the Q gene of wheat. We confirm that the H. chilense AP2-like gene is located on chromosome 5Hch. Patterns of expression of the AP2-like genes were examined in floral organs and other tissues in barley, wheat and in tritordeum amphiploids (barley × wheat hybrids). In tritordeum amphiploids, the level of transcription of the barley AP2-like gene was lower than in its barley parental and the chromosome substitutions 1D/1Hch and 2D/2Hch were seen to modify AP2 gene expression levels. Conclusion The results are of interest in order to understand the role of the AP2-like gene in the spike morphology of barley and wheat, and to understand the regulation of this gene in the amphiploids obtained from barley-wheat crossing. This information may have application in cereal breeding programs to up- or down-regulate the expression of AP2-like genes in order to modify spike characteristics and to obtain free-threshing plants.

Key Message: An original RNA-seq mapping strategy, validated with chromosome engineering and physical mapping, identifies candidate genes for fertility restoration in the 6H ch S chromosome of Hordeum chilense in the wheat msH1 system. Cytoplasmic male sterility (CMS) is a valuable trait for hybrid seed production. The msH1 CMS system in common wheat results from the incompatibility between the nuclear genome of wheat and the cytoplasm of the wild barley Hordeum chilense. This work aims to identify H. chilense candidate genes for fertility restoration in the msH1 system with a multidisciplinary strategy based on chromosome engineering, differential expression analysis and genome mapping. Alloplasmic isogenic wheat lines differing for fertility, associated with the presence of an acrocentric chromosome H ch ac resulting from the rearrangement of the short arms of H. chilense chromosomes 1H ch and 6H ch , were used for transcriptome sequencing. Two novel RNA-seq mapping approaches were designed and compared to identify differentially expressed genes of H. chilense associated with male fertility restoration. Minichromosomes (H ch mi), new smaller reorganizations of the H ch ac also restoring fertility, were obtained and used to validate the candidate genes. This strategy was successful identifying a putative restorer-of-fertility region on 6H ch S, with six candidate genes, including the ortholog of the barley restorer gene Rfm1. Additionally, transcriptomics gave preliminary insights on sterility and restoration networks showing the importance of energy supply, stress, protein metabolism and RNA processing.

Bioethanol production obtained from cereal straw has aroused great interest in recent years, which has led to the development of breeding programs to improve the quality of lignocellulosic material in terms of the biomass and sugar content. This process requires the analysis of genotype-phenotype relationships, and although genotyping tools are very advanced, phenotypic tools are not usually capable of satisfying the massive evaluation that is required to identify potential characters for bioethanol production in field trials. However, unmanned aerial vehicle (UAV) platforms have demonstrated their capacity for efficient and non-destructive acquisition of crop data with an application in high-throughput phenotyping. This work shows the first evaluation of UAV-based multi-spectral images for estimating bioethanol-related variables (total biomass dry weight, sugar release, and theoretical ethanol yield) of several accessions of wheat, barley, and triticale (234 cereal plots). The full procedure involved several stages: (1) the acquisition of multi-temporal UAV images by a six-band camera along different crop phenology stages (94, 104, 119, 130, 143, 161, and 175 days after sowing), (2) the generation of ortho-mosaicked images of the full field experiment, (3) the image analysis with an object-based (OBIA) algorithm and the calculation of vegetation indices (VIs), (4) the statistical analysis of spectral data and bioethanol-related variables to predict a UAV-based ranking of cereal accessions in terms of theoretical ethanol yield. The UAV-based system captured the high variability observed in the field trials over time. Three VIs created with visible wavebands and four VIs that incorporated the near-infrared (NIR) waveband were studied, obtaining that the NIR-based VIs were the best at estimating the crop biomass, while the visible-based VIs were suitable for estimating crop sugar release. The temporal factor was very helpful in achieving better estimations. The results that were obtained from single dates [i.e., temporal scenario 1 (TS-1)] were always less accurate for estimating the sugar release than those obtained in TS-2 (i.e., averaging the values of each VI obtained during plant anthesis) and less accurate for estimating the crop biomass and theoretical ethanol yield than those obtained in TS-3 (i.e., averaging the values of each VI obtained during full crop development). The highest correlation to theoretical ethanol yield was obtained with the normalized difference vegetation index ( R 2 = 0.66), which allowed to rank the cereal accessions in terms of potential for bioethanol production.

The global production of cereal straw as an agricultural by-product presents a significant source of biomass, which could be used as feedstock for the production of second generation biofuels by fermentation. The production of sugars for fermentation is an important measure of straw quality and in its suitability for biofuel production. In this paper, we present a characterization of straw digestibility from a wide range of cereal. Our main objective is to evaluate the variability of fermentable sugars released from different species including wheat (Triticum durum L., Triticum aestivum L.), barley (Hordeum vulgare L.) and triticale (X Triticosecale Wittmack). To this end, we adapted a saccharification method (IAS Method) capable of detecting significant differences of released sugars between cultivars and species, while using separately another method that would serve as a control and with which we could contrast our results (CNAP method). ANOVA analyses revealed that barley has a higher saccharification potential than wheat and triticale and shows more variation between genotypes. Thus, populations derived from crosses among them such as Steptoe × Morex and OWB Dominant × OWB Recessive hold potential for the identification of genetic basis for saccharification-related traits. The correlation of glucose released between the two methods was moderate (R2 = 0.57). An evaluation of the inter- and intra- specific correlation between a number of chemical and agronomical parameters and saccharification suggests that the cell wall thickness and lignin content in straw could be used in breeding programs for the improvement of the saccharification potential. Finally, the lack of correlation between grain yield and saccharification suggests that it would be possible to make a selection of genotypes for dual purpose, low recalcitrance and grain yield., Competing Interests: The authors have declared that no competing interests exist.

Exploiting hybrid wheat heterosis has been long pursued to increase crop yield, stability and uniformity. Cytoplasmic male sterility (CMS) systems based in the nuclear-cytoplasmic incompatible interactions are a classic way for hybrid seed production, but to date, no definitive system is available in wheat. The msH1 CMS system results from the incompatibility between the nuclear genome of wheat and the cytoplasmic genome of the wild barley Hordeum chilense. Fertility restoration of the CMS phenotype was first associated with the disomic addition of the short arm of chromosome 6H from H. chilense. In further studies it was observed that chromosome arm 1HchS was also implicated, and the combination of genes in both chromosome arms restored fertility more efficiently. In this work we aim to dissect the effect of each chromosome in fertility restoration when combined in different genomic backgrounds and under different environmental conditions. We propose a model to explain how restoration behaves in the msH1 system and generate valuable information necessary to develop an efficient system for hybrid wheat production.

Celiac disease is a food-sensitive enteropathy triggered by the ingestion of wheat gluten proteins and related proteins from barley, rye, and some varieties of oat. There are no interventional therapies and the only solution is a lifelong gluten-free diet. The down-regulation of gliadins by RNAi provides wheat lines with all the gliadin fractions strongly down-regulated (low-gliadin). The technological properties of doughs prepared from the low-gliadin lines indicated a general weakening effect, although some of the lines displayed similar properties to that of the wild-type lines. In contrast, the stability was increased significantly in some of the transgenic lines, indicating better tolerance to over-mixing. Results reported here are the first analyses of the mixing and bread-making quality of the wheat lines with all gliadin fractions strongly down-regulated. Flour from these lines may be an important breakthrough in the development of new products for the celiac community. These lines might be used directly or blended with other non-toxic cereals, as raw material for developing food products that can be safely tolerated by CD patients and others with gluten intolerance or gluten sensitivity, incrementing the range of available food products and enhancing their diet.

Wheat flour cannot be tolerated by those who suffer allergies to gluten. Human pathologies associated with grain proteins have increased worldwide in recent years, and the only effective treatment available is a lifelong gluten-free diet, which is complicated to follow and detrimental to gut health. This manuscript describes the development of wheat bread potentially suitable for celiac patients and other gluten-intolerant individuals. We have made bread using wheat flour with very low content of the specific gluten proteins (near gliadin-free) that are the causal agents for pathologies such as celiac disease. Loaves were compared with normal wheat breads and rice bread. Organoleptic, nutritional, and immunotoxic properties were studied. The reduced-gliadin breads showed baking and sensory properties, and overall acceptance, similar to those of normal flour, but with up to 97% lower gliadin content. Moreover, the low-gliadin flour has improved nutritional properties since its lysine content is significantly higher than that of normal flour. Conservative estimates indicate that celiac patients could safely consume 67 grams of bread per day that is made with low-gliadin flour. However, additional studies, such as feeding trials with gluten-intolerant patients, are still needed in order to determine whether or not the product can be consumed by the general celiac population, as well as the actual tolerated amount that can be safely ingested. The results presented here offer a major opportunity to improve the quality of life for millions of sufferers of gluten intolerance throughout the world.

For humans, wheat is the most important source of calories, but it is also a source of antioxidant compounds that are involved in the prevention of chronic disease. Among the antioxidant compounds, phenolic acids have great potential to improve human health. In this paper we evaluate the effect of environmental and genetic factors on the phenolics content in the grain of a collection of tritordeums with different cytoplasm and chromosome substitutions. To this purpose, tritordeum flour was used for extraction of the free, conjugates and bound phenolic compounds. These phenolic compounds were identified and quantified by RP-HPLC and the results were analyzed by univariate and multivariate methods. This is the first study that describes the composition of phenolic acids of the amphiploid tritordeum. As in wheat, the predominant phenolic compound is ferulic acid. In tritordeum there is great variability for the content of phenolic compounds and the main factor which determines its content is the genotype followed by the environment, in this case included in the year factor. Phenolic acid content is associated with the substitution of chromosome DS1D(1H(ch)) and DS2D(2H(ch)), and the translocation 1RS/1BL in tritordeum. The results show that there is high potential for further improving the quality and quantity of phenolics in tritordeum because this amphiploid shows high variability for the content of phenolic compounds.

Background: Wheat gluten has unique nutritional and technological characteristics, but is also a major trigger of allergies and intolerances. One of the most severe diseases caused by gluten is coeliac disease. The peptides produced in the digestive tract by the incomplete digestion of gluten proteins trigger the disease. The majority of the epitopes responsible reside in the gliadin fraction of gluten. The location of the multiple gliadin genes in blocks has to date complicated their elimination by classical breeding techniques or by the use of biotechnological tools.As an approach to silence multiple gliadin genes we have produced 38 transgenic lines of bread wheat containing combinations of two endosperm-specific promoters and three different inverted repeat sequences to silence three fractions of gliadins by RNA interference., Results: The effects of the RNA interference constructs on the content of the gluten proteins, total protein and starch, thousand seed weights and SDSS quality tests of flour were analyzed in these transgenic lines in two consecutive years. The characteristics of the inverted repeat sequences were the main factor that determined the efficiency of silencing. The promoter used had less influence on silencing, although a synergy in silencing efficiency was observed when the two promoters were used simultaneously. Genotype and the environment also influenced silencing efficiency., Conclusions: We conclude that to obtain wheat lines with an optimum reduction of toxic gluten epitopes one needs to take into account the factors of inverted repeat sequences design, promoter choice and also the wheat background used.

Scope: The only treatment available for coeliac disease (CD) is a strict diet in which the intake of wheat, barley, rye, or oats is avoided. Barley is a major cereal crop, grown mainly for its use in brewing, and it has high nutritional value. The identification of varieties with a reduced toxicity profile may contribute to improve the diet, the quality of life and health of CD patients., Methods and Results: Searching for harmless barleys, we investigated accessions of malting and wild barley, used for developing new cultivated cereals. The CD toxicity profile of barleys was screened using G12 antibody and cell proliferation and IFN-γ release from peripheral blood mononuclear cells and intestinal biopsies from CD patients. We found a direct correlation between the reactivity with G12 and the immunogenicity of the different barleys., Conclusion: The malting barleys were less immunogenic, with reduced levels of toxic gluten, and were possibly less harmful to CD patients. Our findings could raise the prospect of breeding barley species with low levels of harmful gluten, and the attractive goal of developing nontoxic barley cultivars, always taking into account the Codex standard for foods for special dietary use for persons intolerant to gluten., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

In the present work the effects on dough quality by the down-regulation of γ-gliadins in different genetic backgrounds of bread wheat were investigated. RNAi-mediated silencing of γ-gliadins was introgressed by conventional crossing into three commercial bread wheat lines (namely 'Gazul', 'Podenco' and 'Arpain'), and along with the transgenic line A1152 (cv. Bobwhite) compared with their respective wild types. The protein fractions were quantified by RP-HPLC, whereas the technological and mixing properties were assessed by SDSS test and by the Mixograph instrument. Principal component analysis (PCA) was carried out for both the wild types and the transgenic lines, showing differences in the factors affecting the technological and mixing properties of the dough as a consequence of the reduction of the γ-gliadins. In transgenic lines, the α- and ω-gliadins, and total gliadins negatively affected the dough strength and tolerance to over-mixing, whereas the L/H ratio showed the opposite effect, positively influencing the dough quality. The increase of the SDSS volume in the transgenic lines of 'Gazul', 'Podenco' and 'Arpain' indicates increased gluten strength and quality respect to the wild types. SDSS volume was found to be positively influenced by the amount of glutenins, which were also increased in the transgenic lines. In addition, a positive effect was observed in the MT, PR1 and RBD in some of the transgenic lines of 'Podenco' and 'Arpain'. In conclusion, the down-regulation of γ-gliadins resulted in stronger doughs and a better tolerance to over-mixing in some transgenic lines. Although the reduction of γ-gliadins seems not to have a direct effect on the mixing and bread-making properties, the compensatory effect on the synthesis of the other prolamins may result in stronger doughs with improved over-mixing resistance.

Wheat gluten proteins, gliadins and glutenins, are of great importance in determining the unique biomechanical properties of wheat. Studies have therefore been carried out to determine their pathways and mechanisms of synthesis, folding, and deposition in protein bodies. In the present work, a set of transgenic wheat lines has been studied with strongly suppressed levels of γ-gliadins and/or all groups of gliadins, using light and fluorescence microscopy combined with immunodetection using specific antibodies for γ-gliadins and HMW glutenin subunits. These lines represent a unique material to study the formation and fusion of protein bodies in developing seeds of wheat. Higher amounts of HMW subunits were present in most of the transgenic lines but only the lines with suppression of all gliadins showed differences in the formation and fusion of the protein bodies. Large rounded protein bodies were found in the wild-type lines and the transgenic lines with reduced levels of γ-gliadins, while the lines with all gliadins down-regulated had protein bodies of irregular shape and irregular formation. The size and number of inclusions, which have been reported to contain triticins, were also higher in the protein bodies in the lines with all the gliadins down-regulated. Changes in the protein composition and PB morphology reported in the transgenic lines with all gliadins down-regulated did not result in marked changes in the total protein content or instability of the different fractions.

Background: The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species., Methodology/principal Findings: We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5' UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system., Conclusions/significance: The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.

Comparative transcriptomics are useful to determine the role of orthologous genes among Triticeae species. Thus they constitute an interesting tool to improve the use of wild relatives for crop breeding. Reverse transcription quantitative real-time PCR (qPCR) is the most accurate measure of gene expression but efficient normalization is required. The choice and optimal number of reference genes must be experimentally determined and the primers optimized for cross-species amplification. Our goal was to test the utility of wheat-reference genes for qPCR normalization when species carrying the following genomes (A, B, D, R, H ( v ) and H ( ch )) are compared either simultaneously or in smaller subsets of samples. Wheat/barley/rye consensus primers outperformed wheat-specific ones which indicate that consensus primers should be considered for data normalization in comparative transcriptomics. All genes tested were stable but their ranking in terms of stability differed among subsets of samples. CDC (cell division control protein, AAA-superfamily of ATPases, Ta54227) and RLI (68 kDa protein HP68 similar to Arabidopsis thaliana RNase L inhibitor protein, Ta2776) were always among the three most stable genes. The optimal number of reference genes varied between 2 and 3 depending on the subset of samples and the method used (geNorm vs. coefficient of determination between sequential normalization factors). In any case a maximum number of three reference genes would provide adequate normalization independent of the subset of samples considered. This work constitutes a substantial advance towards comparative transcriptomics using qPCR since it provides useful primers/reference genes.

Background: Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction., Methodology/principal Findings: Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins., Conclusions/significance: Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins.

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins from wheat and similar proteins from barley and rye. The inflammatory reaction is controlled by T cells that recognize gluten peptides in the context of human leukocyte antigen (HLA) DQ2 or HLA-DQ8 molecules. The only available treatment for the disease is a lifelong gluten-exclusion diet. We have used RNAi to down-regulate the expression of gliadins in bread wheat. A set of hairpin constructs were designed and expressed in the endosperm of bread wheat. The expression of gliadins was strongly down-regulated in the transgenic lines. Total gluten protein was extracted from transgenic lines and tested for ability to stimulate four different T-cell clones derived from the intestinal lesion of CD patients and specific for the DQ2-α-II, DQ2-γ-VII, DQ8-α-I, and DQ8-γ-I epitopes. For five of the transgenic lines, there was a 1.5-2 log reduction in the amount of the DQ2-α-II and DQ2-γ-VII epitopes and at least 1 log reduction in the amount of the DQ8-α-I and DQ8-γ-I epitopes. Furthermore, transgenic lines were also tested with two T-cell lines that are reactive with ω-gliadin epitopes. The total gluten extracts were unable to elicit T-cell responses for three of the transgenic wheat lines, and there were reduced responses for six of the transgenic lines. This work shows that the down-regulation of gliadins by RNAi can be used to obtain wheat lines with very low levels of toxicity for CD patients.

The sequence characterized amplified region (SCAR) marker SCK13(603), associated with ascochyta blight resistance in a chickpea recombinant inbred line (RIL) population, was used as anchored sequence for genome walking. The PCRs performed in the walking steps to walk in the same direction produced eight bands in 5' direction and five bands in 3' direction with a length ranking from 530 to 2,871 bp. The assembly of the bands sequences along with the sequence of SCK13(603) resulted in 7,815 bp contig. Blastn analyses showed stretches of DNA sequence mainly distributed from the nucleotides 1,500 to 4,500 significantly similar to Medicago truncatula genomic DNA. Three open reading frames (ORFs) were identified and blastp analysis of predicted amino acids sequences revealed that ORF1, ORF2 and ORF3 had significant similarity to a CCHC zinc finger protein, to an integrase, and to a precursor of the glucoamylase s1/s2, respectively, from M. truncatula. The high homology of the putative proteins derived from ORF1 and ORF2 with retrotransposon proteins and the prediction of the existence of conserved domains usually present in retrotransposon proteins indicate that the marker SCK13(603) is located in a region of a putative retrotransposon. The information generated in this study has contributed to increase the knowledge of this important region for blight resistance in chickpea.

The high molecular weight subunits of wheat (Triticum aestivum L.) glutenin (HMW-GS) are important in determining the bread-making quality of flour and dough. There is therefore interest in transferring orthologous HMW-GS present in other grass species into wheat by wide crossing in order to extend the range of end use properties. In this work, we have isolated and characterized two genes encoding D hordeins from Hordeum chilense (Roem. et Schult.) lines H1 and H7, representing two ecotypes. The fragments were 4,305 bp for line H1 and 4,227 for line H7 and contained the promoter, coding and terminator regions. Both sequences differ in the presence of single base changes (SNPs) and insertions/deletions in the open reading frame (ORF). The encoded proteins comprise 870 and 896 amino acids for lines H1 and H7, respectively. The primary structure is similar to those of D hordeins of cultivated barley (H. vulgare L.) and HMW-GS from wheat. However, the D hordeins from H. chilense are significantly larger than those from cultivated barley due to the presence of longer repetitive regions. The H. chilense D hordeins also differ from those of cultivated barley in the distribution of the cysteine residues: whereas the D hordeins of cultivated barley contain ten cysteines with four in the repetitive domain, only nine are present in the H. chilense proteins with two in the repetitive domain. As in the HMW-GS, the central part of the D hordein proteins comprises repeated sequences based on short peptide motifs. The repetitive domain is divided in three regions named as R1 (N-terminal repeats), R2 (central degenerate repeats) and R3 (C-terminal repeats). Hexapeptide motifs are present throughout the repetitive domains of D hordeins with a consensus motif of PFQGQQ in R1 and R2 and PHQGQQ in R3. In addition, the tetrapeptide motif TTVS, which is characteristic of D hordeins of cultivated barley is present in the repetitive domain close to the protein C-terminus.