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Brassicaceae are natural sources of bioactive compounds able to promote gut health. Belonging to this plant family, Camelina sativa is an ancient oil crop rich in glucosinolates, polyunsaturated fatty acids, and antioxidants that is attracting renewed attention for its nutraceutical potential. This work aimed at investigating the therapeutic effects of a defatted seed meal (DSM) of Camelina sativa on the colon damage and the persistent visceral hypersensitivity associated with colitis in rats. Inflammation was induced by the intrarectal injection of 2,4-dinitrobenzenesulfonic acid (DNBS). The acute administration of Camelina sativa DSM (0.1-1 g kg -1 ) showed a dose-dependent pain-relieving effect in DNBS-treated rats. The efficacy of the meal was slightly enhanced after bioactivation with myrosinase, which increased isothiocyanate availability, and drastically decreased by pre-treating the animals with the selective peroxisome proliferator-activated receptor alpha (PPAR α) receptor antagonist GW6471. Repeated treatments with Camelina sativa DSM (1 g kg -1 ) meal counteracted the development, as well as the persistence, of visceral hyperalgesia in DNBS-treated animals by reducing the intestinal inflammatory damage and preventing enteric neuron damage. In conclusion, Camelina sativa meal might be employed as a nutraceutical tool to manage persistent abdominal pain in patients and to promote gut healing.

Leaf rust (LR) and stem rust (SR) are diseases increasingly impacting wheat production worldwide. Fungal pathogens producing rust diseases in wheat may cause yield losses of up to 50−60%. One of the most effective methods for preventing such losses is the development of resistant cultivars with high yield potential. This goal can be achieved through complex breeding studies, including the identification of key genetic factors controlling rust disease resistance. The objective of this study was to identify sources of tetraploid wheat resistance to LR and SR races, both at the seedling growth stage in the greenhouse and at the adult plant stage in field experiments, under the conditions of the North Kazakhstan region. A panel consisting of 193 tetraploid wheat accessions was used in a genome-wide association study (GWAS) for the identification of quantitative trait loci (QTLs) associated with LR and SR resistance, using 16,425 polymorphic single-nucleotide polymorphism (SNP) markers in the seedling and adult stages of plant development. The investigated panel consisted of seven tetraploid subspecies (Triticum turgidum ssp. durum, ssp. turanicum, ssp. turgidum, ssp. polonicum, ssp. carthlicum, ssp. dicoccum, and ssp. dicoccoides). The GWAS, based on the phenotypic evaluation of the tetraploid collection’s reaction to the two rust species at the seedling (in the greenhouse) and adult (in the field) stages, revealed 38 QTLs (p < 0.001), comprising 17 for LR resistance and 21 for SR resistance. Ten QTLs were associated with the reaction to LR at the seedling stage, while six QTLs were at the adult plant stage and one QTL was at both the seedling and adult stages. Eleven QTLs were associated with SR response at the seedling stage, while nine QTLs were at the adult plant stage and one QTL was at both the seedling and adult stages. A comparison of these results with previous LR and SR studies indicated that 11 of the 38 QTLs are presumably novel loci. The QTLs identified in this work can potentially be used for marker-assisted selection of tetraploid and hexaploid wheat for the breeding of new LR- and SR-resistant cultivars.

Eruca sativa Mill. is an edible plant belonging to the Brassicaceae botanical family with a long story as a medicinal material, mainly linked to the presence of glucoerucin. One of the main products of this glucosinolate is erucin, a biologicallly active isothiocyanate recently recognized as a hydrogen sulfide (H 2 S) donor. In this work, an Eruca sativa extract has been obtained from a defatted seed meal (DSM), achieving a powder rich in thiofunctionalized glucosinolates, glucoerucin, and glucoraphanin, accounting for 95% and 5% of the total glucosinolate content (17% on a dry weight basis), associated with 13 identified phenolic acids and flavonoids accounting for 2.5%. In a cell-free model, Eruca sativa DSM extract slowly released H 2 S. Moreover, this extract promoted significant hypotensive effects in hypertensive rats, and evoked dose-dependent cardioprotection in in vivo model of acute myocardial infarct, obtained through a reversible coronary occlusion. This latter effect was sensitive to blockers of mitochondrial KATP and Kv7.4 potassium channels, suggesting a potential role of these mitochondrial channels in the protective effects of Eruca sativa DSM extract. Accordingly, Eruca sativa DSM extract reduced calcium uptake and apoptotic cell death in isolated cardiac mitochondria. Taken together, these results demonstrate that Eruca sativa DSM extract is endowed with an interesting nutraceutical profile on the cardiovascular system due to, at least in part, its H 2 S releasing properties. These results pave the way for future investigations on active metabolites., (© 2022 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.)

Most therapies used in patients affected by inflammatory bowel diseases are ineffective in preventing the development of chronic visceral hypersensitivity, mainly due to inflammation-induced enteric neuroplasticity. Glucosinolates, secondary metabolites mainly of Brassicaceae with anti-inflammatory and neuroprotective properties, are effective in treating both neuropathic and arthritis pain through H 2 S release and Kv7 potassium channel activation. The aim of this work was to investigate the protective and anti-hyperalgesic efficacy of a defatted seed meal from Eruca sativa Mill. (Brassicaceae), rich in glucosinolates, in a rat model of colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS). The mechanisms of action were also investigated. Visceral pain was assessed by measuring the abdominal response to colorectal distension. Fifteen days after colitis induction, the acute administration of E. sativa defatted seed meal (0.1-1 g kg - 1 p.o.) dose-dependently relieved pain. This effect was hampered by co-administering an H 2 S scavenger or a selective Kv7 blocker. Administering E. sativa (1 g kg -1 ) for 14 days, starting after DNBS injection, contributed to counteracting visceral pain persistence in the post-inflammatory phase of colitis by promoting colon healing from the damage and reducing enteric gliosis. E. sativa defatted seed meal might be employed as a nutraceutical tool for supporting abdominal pain relief in patients.

Wild plant populations show extensive genetic subdivision and are far from the ideal of panmixia which permeates population genetic theory. Understanding the spatial and temporal scale of population structure is therefore fundamental for empirical population genetics - and of interest in itself, as it yields insights into the history and biology of a species. In this study we extend the genomic resources for the wild Mediterranean grass Brachypodium distachyon to investigate the scale of population structure and its underlying history at whole-genome resolution. A total of 86 accessions were sampled at local and regional scales in Italy and France, which closes a conspicuous gap in the collection for this model organism. The analysis of 196 accessions, spanning the Mediterranean from Spain to Iraq, suggests that the interplay of high selfing and seed dispersal rates has shaped genetic structure in B. distachyon. At the continental scale, the evolution in B. distachyon is characterized by the independent expansion of three lineages during the Upper Pleistocene. Today, these lineages may occur on the same meadow yet do not interbreed. At the regional scale, dispersal and selfing interact and maintain high genotypic diversity, thus challenging the textbook notion that selfing in finite populations implies reduced diversity. Our study extends the population genomic resources for B. distachyon and suggests that an important use of this wild plant model is to investigate how selfing and dispersal, two processes typically studied separately, interact in colonizing plant species., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ 9 -tetrahydrocannabiphorol (Δ 9 -THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ 9 -tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase ( CBDAS ) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

Preservation of vascular wall integrity against degenerative processes associated with ageing, fat-rich diet and metabolic diseases is a timely therapeutical challenge. The loss of endothelial function and integrity leads to cardiovascular diseases and multiorgan inflammation. The protective effects of the H 2 S-donor erucin, an isothiocyanate purified by Eruca sativa Mill. seeds, were evaluated on human endothelial and vascular smooth muscle cells. In particular, erucin actions were evaluated on cell viability, ROS, caspase 3/7, inflammatory markers levels and the endothelial hyperpermeability in an inflammatory model associated with high glucose concentrations (25 mM, HG). Erucin significantly prevented the HG-induced decrease in cell viability as well as the increase in ROS, caspase 3/7 activation, and TNF-α and IL-6 levels. Similarly, erucin suppressed COX-2 and NF-κB upregulation associated with HG exposure. Erucin also caused a significant inhibition of p22phox subunit expression in endothelial cells. In addition, erucin significantly prevented the HG-induced increase in endothelial permeability as also confirmed by the quantification of the specific markers VE-Cadherin and ZO-1. In conclusion, our results assess anti-inflammatory and antioxidant effects by erucin in vascular cells undergoing HG-induced inflammation and this protection parallels the preservation of endothelial barrier properties.

Plant diseases are responsible for substantial crop losses each year and affect food security and agricultural sustainability. The improvement of crop resistance to pathogens through breeding represents an environmentally sound method for managing disease and minimizing these losses. The challenge is to breed varieties with a stable and broad-spectrum resistance. Different approaches, from markers to recent genomic and 'post-genomic era' technologies, will be reviewed in order to contribute to a better understanding of the complexity of host-pathogen interactions and genes, including those with small phenotypic effects and mechanisms that underlie resistance. An efficient combination of these approaches is herein proposed as the basis to develop a successful breeding strategy to obtain resistant crop varieties that yield higher in increasing disease scenarios.

Mounting evidence indicates the key role of nitrogen (N) on diverse processes in plant, including development and defense. Using a combined transcriptomics and metabolomics approach, we studied the response of seedlings to N starvation of two different tetraploid wheat genotypes from the two main domesticated subspecies: emmer and durum wheat. We found that durum wheat exhibits broader and stronger response in comparison to emmer as seen from the expression pattern of both genes and metabolites and gene enrichment analysis. They showed major differences in the responses to N starvation for transcription factor families, emmer showed differential reduction in the levels of primary metabolites while durum wheat exhibited increased levels of most of them to N starvation. The correlation-based networks, including the differentially expressed genes and metabolites, revealed tighter regulation of metabolism in durum wheat in comparison to emmer. We also found that glutamate and γ-aminobutyric acid (GABA) had highest values of centrality in the metabolic correlation network, suggesting their critical role in the genotype-specific response to N starvation of emmer and durum wheat, respectively. Moreover, this finding indicates that there might be contrasting strategies associated to GABA and glutamate signaling modulating shoot vs. root growth in the two different wheat subspecies.

Traits such as plant height (PH), juvenile growth habit (GH), heading date (HD), and tiller number are important for both increasing yield potential and improving crop adaptation to climate change. In the present study, these traits were investigated by using the same bi-parental population at early (F 2 and F 2 -derived F 3 families) and late (F 6 and F 7 , recombinant inbred lines, RILs) generations to detect quantitative trait loci (QTLs) and search for candidate genes. A total of 176 and 178 lines were genotyped by the wheat Illumina 25K Infinium SNP array. The two genetic maps spanned 2486.97 cM and 3732.84 cM in length, for the F 2 and RILs, respectively. QTLs explaining the highest phenotypic variation were found on chromosomes 2B, 2D, 5A, and 7D for HD and GH, whereas those for PH were found on chromosomes 4B and 4D. Several QTL detected in the early generations (i.e., PH and tiller number) were not detected in the late generations as they were due to dominance effects. Some of the identified QTLs co-mapped to well-known adaptive genes (i.e., Ppd-1 , Vrn-1, and Rht-1 ). Other putative candidate genes were identified for each trait, of which PINE1 and PIF4 may be considered new for GH and TTN in wheat. The use of a large F 2 mapping population combined with NGS-based genotyping techniques could improve map resolution and allow closer QTL tagging.

Heading time in barley is considered a key developmental stage controlling adaptation to the environment and it affects grain yield; with the combination of agronomy (planting dates) and genetics being some of the determinants of adaptation to environmental conditions in order to escape late frost, heat, and terminal drought stresses. The objectives of this study are (i) to apply a gene-based characterization of 118 barley doubled haploid recombinants for vernalization, photoperiod, and earliness per se; (ii) use such information to quantify the optimal combination of genotype/sowing date that escapes extreme weather events; and (iii) how water and nitrogen management impact on grain yield. The doubled haploid barley genotypes with different allelic combinations for vernalization, photoperiod, and earliness per se were grown in eight locations across the Mediterranean basin. This information was linked with the crop growth model parameters. The photoperiod and earliness per se alleles modify the length of the phenological cycle, and this is more evident in combination with the recessive allele of the vernalization gene VRN-H2. In hot environments such as Algeria, Syria, and Jordan, early sowing dates (October 30 and December15) would be chosen to minimize the risk of exposing barley to heat stress. To maintain higher yields in the Mediterranean basin, barley breeding activities should focus on allelic combinations that have recessive VRN-H2 and EPS2 genes, since the risk of cold stress is much lower than the one represented by heat stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cammarano, Ronga, Francia, Akar, Al-Yassin, Benbelkacem, Grando, Romagosa, Stanca and Pecchioni.)

Durum wheat (Triticum turgidum L. ssp. durum) is one of the top crops in Kazakhstan, where it is cultivated in different ecological niches, mainly at higher latitudes in the steppe zone of the northern region. Therefore, local breeding programs for durum wheat are primarily focused on selection for high productivity in Northern Kazakhstan based on the introduction of promising foreign germplasm and the adoption of marker-assisted selection. In this study, a world tetraploid wheat collection consisted of 184 primitive and domesticated accessions, which were previously genotyped using 16,425 polymorphic SNP markers, was field-tested in Northern and South-eastern Kazakhstan. The field tests have allowed the identification of 80 durum wheat promising lines in Northern Kazakhstan in comparison with a local standard cultivar. Also, GGE (Genotype and Genotype by Environment) biplot analyses for yield performance revealed that accessions of T. dicoccum, T. carthlicum, and T. turanicum also have potential to improve durum wheat yield in the region. The genome-wide association study (GWAS) has allowed the identification of 83 MTAs (marker-trait associations) for heading date, seed maturation time, plant height, spike length, number of fertile spikes, number of kernels per spike, and thousand kernel weight. The comparison of the 83 identified MTAs with those previously reported in GWAS for durum wheat suggests that 38 MTAs are presumably novel, while the co-localization of a large number of MTAs with those previously published confirms the validity of the results of this study. The MTAs reported herewith will provide the opportunity to implement marker-assisted selection in ongoing durum wheat breeding projects targeting higher productivity in the region., Competing Interests: The authors have declared that no competing interests exist.

The first breeding program in the world for durum wheat was conceived in Italy in the early 1900s. Over the decades, pressure exerted by natural and artificial selection could have progressively reduced the genetic diversity of the durum wheat germplasm. In the present study, a large panel of Italian durum wheat accessions that includes landraces, old and modern cultivars was subjected to genotyping using the Illumina iSelect 15K wheat SNP array. The aim was to assess the impact that selection has in shaping Italian durum wheat genetic diversity and to exploit the patterns of genetic diversity between populations to identify molecular signatures of divergence and selection. Relatively small differences in genetic diversity have been observed among accessions, which have been selected and cultivated in Italy over the past 150 years. Indeed, directional selection combined with that operated by farmers/breeders resulted in the increase of linkage disequilibrium (LD) and in changes of the allelic frequencies in DNA regions that control important agronomic traits. Results from this study also show that major well-known genes and/or QTLs affecting plant height (RHT), earliness (VRN, PPD) and grain quality (GLU, PSY, PSD, LYC, PPO, LOX3) co-localized with outlier SNP loci. Interestingly, many of these SNPs fall in genomic regions where genes involved in nitrogen metabolism are. This finding highlights the key role these genes have played in the transition from landraces to modern cultivars. Finally, our study remarks on the need to fully exploit the genetic diversity of Italian landraces by intense pre-breeding activities aimed at introducing a new source of adaptability and resistance in the genetic background of modern cultivars, to contrast the effect of climate change. The list of divergent loci and loci under selection associated with useful agronomic traits represents an invaluable resource to detect new allelic variants for target genes and for guiding new genomic selection programs in durum wheat., (Copyright © 2020 Taranto, D’Agostino, Rodriguez, Pavan, Minervini, Pecchioni, Papa and De Vita.)

By selecting for prostrate growth habit of the juvenile phase of the cycle, durum wheat cultivars could be developed with improved competitive ability against weeds, and better soil coverage to reduce the soil water lost by evaporation. A panel of 184 durum wheat ( Triticum turgidum subsp. durum ) genotypes, previously genotyped with DArT-seq markers, was used to perform association mapping analysis of prostrate/erect growth habit trait and to identify candidate genes. Phenotypic data of plant growth habit were recorded during three consecutive growing seasons (2014-2016), two different growth conditions (field trial and greenhouse) and two sowing periods (autumn and spring). Genome-wide association study revealed significant marker-trait associations, twelve of which were specific for a single environment/year, 4 consistent in two environments, and two MTAs for the LSmeans were identified across all environments, on chromosomes 2B and 5A. The co-localization of some MTAs identified in this study with known vernalization and photoperiod genes demonstrated that the sensitivity to vernalization and photoperiod response are actually not only key components of spring/winter growth habit, but they play also an important role in defining the magnitude of the tiller angle during the tillering stage. Many zinc-finger transcription factors, such as C2H2 or CCCH-domain zinc finger proteins, known to be involved in plant growth habit and in leaf angle regulation were found as among the most likely candidate genes. The highest numbers of candidate genes putatively related to the trait were found on chromosomes 3A, 4B, 5A and 6A. Moreover, a bioinformatic approach has been considered to search for functional ortholog genes in wheat by using the sequence of rice and barley tiller angle-related genes. The information generated could be used to improve the understanding of the mechanisms that regulate the prostrate/erect growth habit in wheat and the adaptive potential of durum wheat under resource-limited environmental conditions.

Resequencing in resistant cultivar 'Nure' and structural comparison with the same region of susceptible 'Morex' was performed in order to gain a better insight into barley Frost-resistance-H2 locus. Accurate annotation showed copy number variation (CNV) in the proximal part of the locus. In 'Nure', two exact copies of the HvCBF4-HvCBF2A region and one of the HvCBF4-HvCBF2B segment were observed, while in 'Morex' the corresponding region harboured a single HvCBF4-HvCBF2A (22 kb) segment. Abundance and diversity of repetitive element classes, gene function gain/losses, regulatory motifs and SNPs in gene sequences were identified. An expression study of key HvCBFs with/without CNV on selected genotypes contrasting for frost resistance and estimated HvCBF4-HvCBF2B copy number (2-10 copies) was also performed. Under light stimulus at warm temperature (23 °C), CNV of HvCBF2A and HvCBF4 correlated with their expression levels and reported frost resistance of genotypes; moreover, expression levels of HvCBF2A and HvCBF14 were strongly correlated (r = 0.908, p < 0.01). On the other hand, frost resistance correlated to HvCBF14 expression (r = 0.871, p < 0.01) only after cold induction (6°C) in the dark. A complex interplay of HvCBFs expression levels under different light/temperature stimuli is discussed in light of CNV and presence/number of regulatory elements that integrate different signal transduction pathways., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cadmium (Cd) and lead (Pb) are two toxic heavy metals (HMs) whose presence in soil is generally low. However, industrial and agricultural activities in recent years have significantly raised their levels, causing progressive accumulations in plant edible tissues, and stimulating research in this field. Studies on toxic metals are commonly focused on a single metal, but toxic metals occur simultaneously. The understanding of the mechanisms of interaction between HMs during uptake is important to design agronomic or genetic strategies to limit contamination of crops. To study the single and combined effect of Cd and Pb on durum wheat, a hydroponic experiment was established to examine the accumulation of the two HMs. Moreover, the molecular mechanisms activated in the roots were investigated paying attention to transcription factors (bHLH family), heavy metal transporters and genes involved in the biosynthesis of metal chelators (nicotianamine and mugineic acid). Cd and Pb are accumulated following different molecular strategies by durum wheat plants, even if the two metals interact with each other influencing their respective uptake and translocation. Finally, we demonstrated that some genes ( bHLH 29, YSL2, ZIF1, ZIFL1, ZIFL2, NAS2 and NAAT ) were induced in the durum wheat roots only in response to Cd.

Background: The presence and persistence of water on the leaf can affect crop performance and thus might be a relevant trait to select for or against in breeding programmes. Low-cost, rapid and relatively simple methods are of significant importance for screening of large populations of plants for moisture analysis of detached leaves. Leaf moisture can be detected using an electric circuit, where the resistance changes are proportional to the moisture of the measured surface. In this study, we present a protocol to analyse genotypic differences through the electrical properties of living or stored tissues, performed using a commercial device. Expanded and non-expanded leaves were compared to determine the effects of leaf maturity on these data. Two wheat genotypes that differ in tissue affinity for bound water were used to define the influence of water status., Results: The device indirectly estimates leaf moisture content using two electrodes applied to the leaf lamina of fresh and stored samples. Single moisture readings using this moisture meter had mean execution time of ~ 1.0 min. Exponential associations provided good fits for relationships between the moisture meter reading (MMR) and the electrical resistance applied to the electrodes. MMR normalised for the water/ dry matter ratio (MMRnorm) was lower for mature leaves of the water-mutant than those of wild-type, for the fully hydrated fresh leaves. MMR of fully mature leaves when partially dehydrated and measured after 10 min at 27 °C and 40% relative humidity was greater for the water-mutant than the wild-type., Conclusions: This case study provides a low-cost tool to compare electrical-resistance estimates of leaf moisture content, together with a promising and rapid phenotyping protocol for genotypic screening of wheat under standard environmental conditions. Measurement of changes in MMR with time, of fresh and partially dehydrated leaves, or of MMR normalised to tissue water content allowed for differentiation between the genotypes. Furthermore, the differences observed between genotypes that here relate particular to tissue affinity for bound water suggest that not only the free-water fraction, but also other water fractions, can affect these electrically estimated leaf moisture measures.

Old wheat genotypes are perceived by consumers as healthier than modern ones. The release of gluten peptides with in vitro digestion and the content of potentially prebiotic carbohydrates (i.e. resistant fraction of starch and cell-wall associated dietary fiber) were evaluated in tetraploid wheats, namely 9 old and 3 modern Triticum turgidum ssp. genotypes. Simulated digestion of wholemeal flours yielded 152 major peptides, 59 of which were attributed a sequence. Principal component analysis revealed that peptide profiles were variable in old genotypes, unlike in modern ones. Digestion of old genotypes generally yielded peptides in greater concentration. In particular, 5 peptides of γ-gliadin, known to trigger the adaptive immune reaction, and two peptides of α-gliadin, known to be toxic to celiac patients, were particularly abundant in some old varieties. Resistant starch (RS) was negligible in modern genotypes (<0.6%), but it was remarkably abundant in some old varieties, reaching the highest value in Dauno III (8.5%, P < 0.05). Dauno III also presented the highest amount of soluble fiber (4.2%, P < 0.05). Pasta was made with an old and a modern genotype (Dauno III and PR22D89, respectively) with opposite RS content. Pasta making and cooking affected starch digestibility, overtaking differences between genotypes and yielding the same amount of RS for both the varieties (approx. 1.7%). The data herein presented suggest that the wholemeal flours of old tetraploid wheat genotypes could not boast particular claims associated to a lower exposure to gluten peptides and, if cooked, to a prebiotic potential., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

Durum wheat ( Triticum turgidum L. subsp. durum (Desf.) Husn) is a major food source in Mediterranean countries since it is utilized for the production of pasta, leavened and unleavened breads, couscous, and other traditional foods. The technological and nutritional properties of durum wheat semolina depend mainly on the type of gluten proteins and on their amount, which is a genotype- and environment-dependent trait. Gluten proteins are also responsible for celiac disease (CD), an autoimmune enteropathy with a prevalence of about 0.7-2% in the human population. At this purpose, two Italian durum wheat cultivars, Saragolla and Cappelli, currently used for monovarietal pasta, were chosen to compare (i) the reserve and embryo proteome, (ii) the free and bound phenolics, antioxidant activity, and amino acid composition, and (iii) the content of immunogenic peptides produced after a simulated gastrointestinal digestion. The results obtained from 2 years of field cultivation on average showed a higher amount of gluten proteins, amino acids, and immunogenic peptides in Cappelli. Saragolla showed a higher abundance in bound phenolics, antioxidant enzymes, and stress response proteins in line with its higher antioxidant activity. However, the impact of the year of cultivation, largely depending on varying rainfall regimes through the wheat growth cycle, was significant for most of the parameters investigated. Differences in technological and nutritional characteristics observed between the two cultivars are discussed in relation to the influence of genetic and environmental factors.

Stem rust, caused by Puccinia graminis f. sp. tritici ( Pgt ), is a major biotic constraint to wheat production worldwide. Disease resistant cultivars are a sustainable means for the efficient control of this disease. To identify quantitative trait loci (QTLs) conferring resistance to stem rust at the seedling stage, an association mapping panel consisting of 230 tetraploid wheat accessions were evaluated for reaction to five Pgt races under greenhouse conditions. A high level of phenotypic variation was observed in the panel in response to all of the races, allowing for genome-wide association mapping of resistance QTLs in wild, landrace, and cultivated tetraploid wheats. Twenty-two resistance QTLs were identified, which were characterized by at least two marker-trait associations. Most of the identified resistance loci were coincident with previously identified rust resistance genes/QTLs; however, six regions detected on chromosomes 1B, 5A, 5B, 6B, and 7B may be novel. Availability of the reference genome sequence of wild emmer wheat accession Zavitan facilitated the search for candidate resistance genes in the regions where QTLs were identified, and many of them were annotated as NOD (nucleotide binding oligomerization domain)-like receptor (NLR) genes or genes related to broad spectrum resistance.

Low temperature is a major factor limiting rice growth and yield, and seedling is one of the developmental stages at which sensitivity to chilling stress is higher. Tolerance to chilling is a complex quantitative trait, so one of the most effective approaches to identify genes and pathways involved is to compare the stress-induced expression changes between tolerant and sensitive genotypes. Phenotypic responses to chilling of 13 Japonica cultivars were evaluated, and Thaibonnet and Volano were selected as sensitive and tolerant genotypes, respectively. To thoroughly profile the short-term response of the two cultivars to chilling, RNA-Seq was performed on Thaibonnet and Volano seedlings after 0 (not stressed), 2, and 10 h at 10 °C. Differential expression analysis revealed that the ICE-DREB1/CBF pathway plays a primary role in chilling tolerance, mainly due to some important transcription factors involved (some of which had never been reported before). Moreover, the expression trends of some genes that were radically different between Thaibonnet and Volano (i.e., calcium-dependent protein kinases OsCDPK21 and OsCDPK23, cytochrome P450 monooxygenase CYP76M8, etc.) suggest their involvement in low temperature tolerance too. Density of differentially expressed genes along rice genome was determined and linked to the position of known QTLs: remarkable co-locations were reported, delivering an overview of genomic regions determinant for low temperature response at seedling stage. Our study contributes to a better understanding of the molecular mechanisms underlying rice response to chilling and provides a solid background for development of low temperature-tolerant germplasm.

Free, soluble conjugated, and insoluble bound phenolic acids and some of the main antioxidant phytochemicals (i.e., total polyphenols, proanthocyanidins, carotenoids) were investigated in 20 genotypes of colored barley. These included 16F8 recombinant inbred lines (RILs) obtained from crosses of four parental lines: '2005 FG', 'K4-31, 'L94', and 'Priora'. The aim of this work was these to promote the introduction of new barley genotypes with high contents of such natural antioxidants, and of dietary fiber. These new genotypes will enlarge the market of novel functional foods. Large variations were seen in the contents of phytochemicals and β-glucans across these barley genotypes. The highest protein (14.4%) and β-glucan (4.6%) contents were in the blue naked parental genotype '2005 FG'. Overall, insoluble bound phenolic acids represented 88.3% of the total phenolic acids, and ferulic acid was the main conjugated phenolic acid. Salicylic and gallic acids were the most represented among the free phenolic acids, with no p-coumaric and cinnamic acids detected. Total polyphenols and proanthocyanidins were highest in the RILs '3009' (2917 μg g -1 ) and '1997' (1630 μg g -1 ). The barley line with high total polyphenols (RIL '3009') also showed the highest antioxidant capacities (by both DPPH and ABTS methods: 13.4 μmol g -1 and 15.6 μmol g -1 , respectively). Among the RILs examined, '3004', '3008', and '3009' showed 30% higher antioxidant capacities than their parentals, thus providing potential health-promoting benefits., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The concentrations of 10 minerals were investigated in the grain of 12 durum wheat genotypes grown under free air CO 2 enrichment conditions, and in four of their derived pasta samples, using inductively coupled plasma mass spectrometry. Compared to ambient CO 2 (400ppm; AMB), under elevated CO 2 (570ppm; ELE), the micro-element and macro-element contents showed strong and significant decreases in the grain: Mn, -28.3%; Fe, -26.7%; Zn, -21.9%; Mg, -22.7%; Mo, -40.4%; K, -22.4%; and Ca, -19.5%. These variations defined the 12 genotypes as sensitive or non-sensitive to ELE. The pasta samples under AMB and ELE showed decreased mineral contents compared to the grain. Nevertheless, the contributions of the pasta to the recommended daily allowances remained relevant, also for the micro-elements under ELE conditions (range, from 18% of the recommended daily allowance for Zn, to 70% for Mn and Mo)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Increasing grain yield potential in wheat has been a major target of most breeding programs. Genetic advance has been frequently hindered by negative correlations among yield components that have been often observed in segregant populations and germplasm collections. A tetraploid wheat collection was evaluated in seven environments and genotyped with a 90K SNP assay to identify major and stable quantitative trait loci (QTL) for grain yield per spike (GYS), kernel number per spike (KNS) and thousand-kernel weight (TKW), and to analyse the genetic relationships between the yield components at QTL level. The genome-wide association analysis detected eight, eleven and ten QTL for KNS, TKW and GYS, respectively, significant in at least three environments or two environments and the mean across environments. Most of the QTL for TKW and KNS were found located in different marker intervals, indicating that they are genetically controlled independently by each other. Out of eight KNS QTL, three were associated to significant increases of GYS, while the increased grain number of five additional QTL was completely or partially compensated by decreases in grain weight, thus producing no or reduced effects on GYS. Similarly, four consistent and five suggestive TKW QTL resulted in visible increase of GYS, while seven additional QTL were associated to reduced effects in grain number and no effects on GYS. Our results showed that QTL analysis for detecting TKW or KNS alleles useful for improving grain yield potential should consider the pleiotropic effects of the QTL or the association to other QTLs.

Background: In plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding., Results: Twenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait., Conclusions: The availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat.

The scientific presentations at the First International Brachypodium Conference (abstracts available at http://www.brachy2013.unimore.it) are evidence of the widespread adoption of Brachypodium distachyon as a model system. Furthermore, the wide range of topics presented (genome evolution, roots, abiotic and biotic stress, comparative genomics, natural diversity, and cell walls) demonstrates that the Brachypodium research community has achieved a critical mass of tools and has transitioned from resource development to addressing biological questions, particularly those unique to grasses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance in barley, yet its molecular basis is still not clearly understood. To gain a better insight into the structural characterization of the locus, a high-resolution linkage map developed from the Nure × Tremois cross was initially implemented to map 13 loci which divided the 0.602 cM total genetic distance into ten recombination segments. A PCR-based screening was then applied to identify positive bacterial artificial chromosome (BAC) clones from two genomic libraries of the reference genotype Morex. Twenty-six overlapping BACs from the integrated physical-genetic map were 454 sequenced. Reads assembled in contigs were subsequently ordered, aligned and manually curated in 42 scaffolds. In a total of 1.47 Mbp, 58 protein-coding sequences were identified, 33 of which classified according to similarity with sequences in public databases. As three complete barley C-repeat Binding Factors (HvCBF) genes were newly identified, the locus contained13 full-length HvCBFs, four Related to AP2 Triticeae (RAPT) genes, and at least five CBF pseudogenes. The final overall assembly of Fr-H2 includes more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. We believe that this gold-standard sequence for the Morex Fr-H2 will be a useful genomic tool for structural and evolutionary comparisons with Fr-H2 in winter-hardy cultivars along with Fr-2 of other Triticeae crops.

Background: Frost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola (Italy) and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure., Results: GWAS analyses identified twelve and seven positive SNP associations at Foradada and Fiorenzuola, respectively, using Eigenstrat and six and four, respectively, using kinship. Linkage disequilibrium analyses of the significant SNP associations showed they are genetically independent. In the kinship analysis, two of the significant SNP associations were tightly linked to the Fr-H2 and HvBmy loci on chromosomes 5H and 4HL, respectively. The other significant kinship associations were located in genomic regions that have not previously been associated with cold stress., Conclusions: Haplotype analysis revealed that most of the significant SNP loci are fixed in the winter or facultative types, while they are freely segregating within the un-adapted spring barley genepool. Although there is a major interest in detecting new variation to improve frost tolerance of available winter and facultative types, from a GWAS perspective, working within the un-adapted spring germplasm pool is an attractive alternative strategy which would minimize statistical issues, simplify the interpretation of the data and identify phenology independent genetic determinants of frost tolerance.

As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present, domesticated crops encountered considerable ecological and environmental change. Spring-sown crops that flowered without the need for an extended period of cold to promote flowering and day length-insensitive crops able to exploit the longer, cooler days of higher latitudes emerged and became established. To investigate the genetic consequences of adaptation to these new environments, we identified signatures of divergent selection in the highly differentiated modern-day spring and winter barleys. In one genetically divergent region, we identify a natural variant of the barley homolog of Antirrhinum CENTRORADIALIS (HvCEN) as a contributor to successful environmental adaptation. The distribution of HvCEN alleles in a large collection of wild and landrace accessions indicates that this involved selection and enrichment of preexisting genetic variants rather than the acquisition of mutations after domestication.

The potential of the model grass Brachypodium distachyon L. (Brachypodium) for studying grass-pathogen interactions is still underexploited. We aimed to identify genomic regions in Brachypodium associated with quantitative resistance to the false brome rust fungus Puccinia brachypodii . The inbred lines Bd3-1 and Bd1-1, differing in their level of resistance to P. brachypodii, were crossed to develop an F(2) population. This was evaluated for reaction to a virulent isolate of P. brachypodii at both the seedling and advanced growth stages. To validate the results obtained on the F(2), resistance was quantified in F(2)-derived F(3) families in two experiments. Disease evaluations showed quantitative and transgressive segregation for resistance. A new AFLP-based Brachypodium linkage map consisting of 203 loci and spanning 812 cM was developed and anchored to the genome sequence with SSR and SNP markers. Three false brome rust resistance QTLs were identified on chromosomes 2, 3, and 4, and they were detected across experiments. This study is the first quantitative trait analysis in Brachypodium. Resistance to P. brachypodii was governed by a few QTLs: two acting at the seedling stage and one acting at both seedling and advanced growth stages. The results obtained offer perspectives to elucidate the molecular basis of quantitative resistance to rust fungi.

An association panel consisting of 185 accessions representative of the barley germplasm cultivated in the Mediterranean basin was used to localise quantitative trait loci (QTL) controlling grain yield and yield related traits. The germplasm set was genotyped with 1,536 SNP markers and tested for associations with phenotypic data gathered over 2 years for a total of 24 year × location combinations under a broad range of environmental conditions. Analysis of multi-environmental trial (MET) data by fitting a mixed model with kinship estimates detected from two to seven QTL for the major components of yield including 1000 kernel weight, grains per spike and spikes per m(2), as well as heading date, harvest index and plant height. Several of the associations involved SNPs tightly linked to known major genes determining spike morphology in barley (vrs1 and int-c). Similarly, the largest QTL for heading date co-locates with SNPs linked with eam6, a major locus for heading date in barley for autumn sown conditions. Co-localization of several QTL related to yield components traits suggest that major developmental loci may be linked to most of the associations. This study highlights the potential of association genetics to identify genetic variants controlling complex traits.

Several molecular evidences have been gathered in Poaceae that point out a central role of the CBF/DREB1 transcription factors in the signal transduction pathways leading to low-temperature tolerance, although to a quite different extent between crops originating from either temperate or tropical climates. A common feature of the CBF/DREB1 genes in Poaceae is their structural organization at the genome level in clusters of tandemly duplicated genes. In temperate cereals such as barley and wheat, expansion of specific multigene phylogenetic clades of CBFs that map at the Frost Resistance-2 locus has been exclusively observed. In addition, copy number variants of CBF genes between frost resistant and frost sensitive genotypes raise the question if multiple copies of the CBF/DREB1s are required to ensure freezing tolerance. On the other hand, in crops of tropical origin such as rice and maize, a smaller or less-responsive CBF regulon may have evolved, and different mechanisms might determine chilling tolerance. In this review, recent advances on the organization and diversity at the CBF cluster locus in the grasses are provided and discussed., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Frost Resistance-1 (FR-1) and FR-2 are two loci affecting freezing tolerance and winter hardiness of the temperate-climate cereals. FR-1 is hypothesized to be due to the pleiotropic effects of VRN-1. FR-2 spans a cluster of C-Repeat Binding Factor (CBF) genes. These loci are genetically and functionally linked. Recent studies indicate CBF transcripts are downregulated by the VRN-1 encoded MADS-box protein or a factor in the VRN-1 pathway. Here, we report that barley genotypes 'Dicktoo' and 'Nure' carrying a vrn-H1 winter allele at VRN-H1 harbor increased copy numbers of CBF coding sequences relative to Vrn-H1 spring allele genotypes 'Morex' and 'Tremois'. Sequencing bacteriophage lambda genomic clones from these four genotypes alongside DNA blot hybridizations indicate approximately half of the eleven CBF orthologs at FR-H2 are duplicated in individual genomes. One of these duplications discriminates vrn-H1 genotypes from Vrn-H1 genotypes. The vrn-H1 winter allele genotypes harbor tandem segmental duplications through the CBF2A-CBF4B genomic region and maintain two distinct CBF2 paralogs, while the Vrn-H1 spring allele genotypes harbor single copies of CBF2 and CBF4. An additional CBF gene, CBF13, is a pseudogene interrupted by multiple non-sense codons in 'Tremois' whereas CBF13 is a complete uninterrupted coding sequence in 'Dicktoo' and 'Nure'. DNA blot hybridization with wheat DNAs reveals greater copy numbers of CBF14 also occurs in winter wheats than in spring wheats. These data indicate that variation in CBF gene copy numbers is widespread in the Triticeae and suggest selection for winter hardiness co-selects winter alleles at both VRN-1 and FR-2.

The basal resistance of barley to powdery mildew (Blumeria graminis f. sp. hordei) is a quantitatively inherited trait that is based on nonhypersensitive mechanisms of defense. A functional genomic approach indicates that many plant candidate genes are involved in the defense against formation of fungal haustoria. It is not known which of these candidate genes have allelic variation that contributes to the natural variation in powdery mildew resistance, because many of them may be highly conserved within the barley species and may act downstream of the basal resistance reaction. Twenty-two expressed sequence tag or cDNA clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcriptional profiling, gene silencing, or overexpression data, as well as mlo, Ror1, and Ror2, were mapped and considered candidate genes for contribution to basal resistance. We mapped the quantitative trait loci (QTL) for powdery mildew resistance in six mapping populations of barley at seedling and adult plant stages and developed an improved high-density integrated genetic map containing 6,990 markers for comparing QTL and candidate gene positions over mapping populations. We mapped 12 QTL at seedling stage and 13 QTL at adult plant stage, of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTL identified for basal resistance to powdery mildew. This co-localization justifies giving priority to those six candidate genes to validate them as being responsible for the phenotypic effects of the QTL for basal resistance.

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT((R)) markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r (2) of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT((R)) markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.

Barley is an economically important model for the Triticeae tribe. We recently developed a new resource: the 'Nure' x 'Tremois' mapping population. Two low temperature QTLs were found to segregate on the long arm of chromosome 5H (Fr-H1, distal; Fr-H2, proximal). With the final aim of positional cloning of the genetic determinants of Fr-H1 and Fr-H2, a large segregating population of 1,849 F(2) plants between parents 'Nure' and 'Tremois' was prepared. These two QT loci were first validated by using a set of F(3) families, marker-selected to harbor pairs of reciprocal haplotypes, with one QTL fixed at homozygosity and the alternate one in heterozygous phase. The study was then focused towards the isolation of the determinant of Fr-H2. Subsequent recombinant screens and phenotypic evaluation of F(4) segregants allowed us to estimate (P < or = 0.01) a refined genomic interval of Fr-H2 (4.6 cM). Several barley genes with the CBF transcription factor signature had been already roughly mapped in cluster at Fr-H2, and they represent likely candidate genes underlying this QTL. Using the large segregating population (3,698 gametes) a high-resolution genetic map of the HvCBF gene cluster was then constructed, and after fine mapping, six recombinations between the HvCBFs were observed. It was therefore possible to genetically divide seven HvCBF subclusters in barley, in a region spanning 0.81 cM, with distances among them varying from 0.03 to 0.32 cM. The few recombinants between the different HvCBF subclusters are being marker-selected and taken to homozygosity, to phenotypically separate the effects of the single HvCBF genes.

Genetic analyses have identified two loci in wheat and barley that mediate the capacity to overwinter in temperate climates. One locus co-segregates with VRN-1, which affects the vernalization requirement. This locus is known as Frost resistance-1 (Fr-1). The second locus, Fr-2, is coincident with a cluster of more than 12 Cbf genes. Cbf homologs in Arabidopsis thaliana play a key regulatory role in cold acclimatization and the acquisition of freezing tolerance. Here we report that the Hordeum vulgare (barley) locus VRN-H1/Fr-H1 affects expression of multiple barley Cbf genes at Fr-H2. RNA blot analyses, conducted on a 'Nure'x'Tremois' barley mapping population segregating for VRN-H1/Fr-H1 and Fr-H2, revealed that transcript levels of all cold-induced Cbf genes at Fr-H2 were significantly higher in recombinants harboring the vrn-H1 winter allele than in recombinants harboring the Vrn-H1 spring allele. Steady-state Cbf2 and Cbf4 levels were also significantly higher in recombinants harboring the Nure allele at Fr-H2. Additional experiments indicated that, in vrn-H1 genotypes requiring vernalization, Cbf expression levels were dampened after plants were vernalized, and dampened Cbf expression was accompanied by robust expression of Vrn-1. Cbf levels were also significantly higher in plants grown under short days than under long days. Experiments in wheat and rye indicated that similar regulatory mechanisms occurred in these plants. These results suggest that VRN-H1/Fr-H1 acts in part to repress or attenuate expression of the Cbf at Fr-H2; and that the greater level of low temperature tolerance attributable to the Nure Fr-H2 allele may be due to the greater accumulation of Cbf2 and Cbf4 transcripts during normal growth and development.

It is generally assumed that compounds are emitted from flowers in order to attract and guide pollinators. Due to the invisibility and the highly variable nature of floral scent, no efficient and reliable methods to screen for genetic variation have been developed. Moreover, no convenient plant model systems are available for flower scent studies. In the past decade, several floral fragrance-related genes have been cloned; the biosynthesis and metabolic engineering of floral volatiles have been studied with the development of biotechnology. This review summarizes the reported floral fragrance-related genes and the biosynthesis of floral scent compounds, introduces the origin of new modification enzymes for flower scent, compares different methods for floral fragrance-related gene cloning, and discusses the metabolic engineering of floral scent. Finally, the perspectives and prospects of research on floral fragrance are presented.

Cereal crop yield is greatly affected in many growing areas by abiotic stresses, mainly low temperature and drought. In order to find candidates for the tolerance genes for these stresses, 13 genes encoding for transcription factors and upstream regulators were screened by amplification and SSCP on six parental genotypes of three barley mapping populations ('Nure' x 'Tremois', 'Proctor' x 'Nudinka', and 'Steptoe' x 'Morex'), and mapped as newly developed STS, SNP, and SSCP markers. A new consensus function map was then drawn using the three maps above, including 16 regulatory candidate genes (CGs). The positions of barley cold and drought tolerance quantitative trait loci (QTLs) presently described in the literature were added to the consensus map to find positional candidates from among the mapped genes. A cluster of six HvCBF genes co-mapped with the Fr-H2 cold tolerance QTL, while no QTLs for the same trait were positioned on chromosome 7H, where two putative barley regulators of CBF expression, ICE1 and FRY1, found by homology search, were mapped in this work. These observations suggest that CBF gene(s) themselves, rather than their two regulators, are at present the best candidates for cold tolerance. Four out of 12 drought tolerance QTLs of the consensus map are associated with regulatory CGs, on chromosomes 2H, 5H, and 7H, and two QTLs with effector genes, on chromosomes 5H and 6H. The results obtained could be used to guide MAS applications, allowing introduction into an ideal genotype of favourable alleles of tolerance QTLs.

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits - winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele.

Barley ( Hordeum vulgare subsp. vulgare) is an economically important diploid model for the Triticeae; and a better understanding of low-temperature tolerance mechanisms could significantly improve the yield of fall-sown cereals. We developed a new resource for genetic analysis of winter hardiness-related traits, the 'Nure' x 'Tremois' linkage map, based on a doubled-haploid population that is segregating for low-temperature tolerance and vernalization requirement. Three measures of low-temperature tolerance and one measure of vernalization requirement were used and, for all traits, QTLs were mapped on chromosome 5H. The vernalization response QTL coincides with previous reports at the Vrn-1/Fr1 region of the Triticeae. We also found coincident QTLs at this position for all measures of low-temperature tolerance. Using Composite Interval Mapping, a second proximal set, of coincident QTLs for low-temperature tolerance, and the accumulation of two different COR proteins (COR14b and TMC-Ap3) was identified. The HvCBF4 locus, or another member of the CBF loci clustered in this region, is the candidate gene underlying this QTL. There is a CRT/DRE recognition site in the promoter of cor14b with which a CBF protein could interact. These results support the hypothesis that highly conserved regulatory factors, such as members of the CBF gene family, may regulate the stress responses of a wide range of plant species.

Leaf stripe caused by the fungus Pyrenophora graminea represents a serious threat to grain yield in organically grown barley and in conventional Nordic and Mediterranean districts, for which resistant cultivars are necessary. A medium-density, molecular marker map derived from a 'Steptoe' (partially resistant) x 'Morex' (susceptible) spring barley cross and its derived doubled-haploid mapping population inoculated with the fungus made it possible to identify QTLs of resistance to leaf stripe. In order to investigate isolate-specificity of partial resistance, the 'Steptoe' x 'Morex' segregating population was inoculated with two highly virulent P. graminea isolates, Dg2 and Dg5. The present study demonstrates that partial resistance to leaf stripe of cv 'Steptoe' is governed in part by shared loci and in part by isolate-specific ones. One QTL is common to the resistance for the two isolates, on the long arm of chromosome 2 (2H), two QTLs are linked on chromosome 3 (3H), and the remaining two are isolate-specific, respectively for isolate Dg2 on chromosome 2 (2H) and for isolate Dg5 on chromosome 7 (5H). The QTL in common is that with the major effect on the resistance for each isolate, explaining 18.3% and 30.9% R(2) respectively for Dg2 and Dg5. The isolate-specific QTLs mapped in the 'Steptoe' x 'Morex' barley reference map support the assumption of Parlevliet and Zadoks (1977) that partial resistance may be due to minor gene-for-minor-gene interactions. Map comparisons of the QTLs with the known qualitative resistance genes to leaf stripe, Rdg1 (2H) and Rdg2 (7H), as well as with other QTLs of partial resistance in barley, show that the QTL for resistance to both isolates mapped on the long arm of chromosome 2 (2H) does not coincide with the qualitative Rdg1 gene but is linked to it at about 30 cM. One isolate-specific QTL of resistance to P. graminea, mapped on the short arm of chromosome 2 (2H), is coincident with a QTL for resistance to Pyrenophora teres previously mapped in the 'Steptoe' x 'Morex' cross.

Leaf stripe is a seed-borne disease of barley (Hordeum vulgare) caused by Pyrenophora graminea. Little is known about the genetics of resistance to this pathogen. In the present work, QTL analysis was applied on two recombinant inbred line (RIL) populations derived from two- and six-rowed barley genotypes with different levels of partial resistance to barley leaf stripe. Quantitative trait loci for partial resistance were identified using the composite interval mapping (CIM) method of PLABQTL software, using the putative QTL markers as cofactors. In the L94 x 'Vada' mapping population, one QTL for resistance was detected on chromosome 2H; the same location as the leaf-stripe resistance gene Rdg1 mapped earlier in 'Alf', where it confers complete resistance to the pathogen. An additional minor-effect QTL was identified by further analyses in this segregating population on chromosome 7H. In L94 x C123, two QTLs for resistance were mapped, one each on chromosomes 7H and 2H.

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plant's adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5'-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.

A major gene underlying quantitative resistance of barley against Pyrenophora graminea, a seedborne pathogen causing leaf stripe, was mapped with molecular markers in a barley doubled haploid (DH) population derived from the cross 'Proctor' x 'Nudinka'. This quantitative trait locus (QTL) accounts for r (2)= 58.5% and was mapped on barley chromosome 1, tightly linked to the "naked" gene. A second resistance QTL accounting for 29.3% of the variation in the trait was identified on the P arm of barley chromosome 2. Another two minor QTLs were detected by further analysis. None of the QTLs was found in the barley chromosome 2 "Vada" region studied by Giese et al. (1993).

Barley middle-repeat sequences were screened for their ability to discriminate 51 barley commercial varieties. Two hordein clones, a clone encoding a leaf-specific thionin, a desiccation induced cDNA clone, a clone coding for 5S-rRNA and one corresponding to ubiquitin genes were tested. A very sensitive RFLP technique including four cutter restriction enzymes and denaturing 4% polyacrylamide gels were used to evidence the highest level of polymorphism.The RFLP data were analyzed by computer. Some probe/enzyme combinations were able to differentiate a large number of the cultivars tested, whereas three probe/enzyme combinations succeeded in identifying all the varieties. The use of this RFLP method can thus be suggested for cultivar identification in barley.