QTL Analysis of ß-Glucan Content and Other Grain Traits in a Recombinant Population of Spring Barley.

Barley with high grain ß-glucan content is valuable for functional foods. The identification of loci for high ß-glucan content is, thus, of great importance for barley breeding. Segregation mapping for the content in ß-glucan and other barley grain components (starch, protein, lipid, ash, phosphorous, calcium, sodium) was performed using the progeny of the cross between Glacier AC38, a mutant with high amylose, and CDC Fibar, a high ß-glucan waxy cultivar. The offspring of this cross showed transgressive segregation for ß-glucan content. Linkage analysis based on single-nucleotide polymorphism (SNP) molecular markers was used for the genotyping of the parents and recombinant inbred lines (RILs). Two Quantitative Trait Loci (QTL) for ß-glucan content and several QTL for other grain components were found. The former ones, located on chromosomes 1H and 7H, explained 27.9% and 27.4% of the phenotypic variance, respectively. Glacier AC38 provided the allele for high ß-glucan content at the QTL on chromosome 1H, whereas CDC Fibar contributed the allele at the QTL on chromosome 7H. Their recombination resulted in a novel haplotype with higher ß-glucan content, up to 18.4%. Candidate genes are proposed for these two QTL: HvCslF9, involved in ß-glucan biosynthesis, for the QTL on chromosome 1H; Horvu_PLANET_7H01G069300, a gene encoding an ATP-Binding Cassette (ABC) transporter, for the QTL on chromosome 7H.

Double Gamers-Can Modified Natural Regulators of Higher Plants Act as Antagonists against Phytopathogens? The Case of Jasmonic Acid Derivatives.

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum, Neofusicoccum parvum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium poae, F. culmorum, F. graminearum, F. oxysporum f. sp. lactucae,F. sporotrichioides, Aspergillus flavus, Rhizoctonia solani,Sclerotinia spp. and Verticillium dahliae. The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides, also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi.

Quantification of Botrytis cinerea in Grapevine Bunch Trash by Real-Time PCR.

Quantification of colonization of grape bunch trash by Botrytis cinerea is crucial for Botrytis bunch rot (BBR) control. A previously developed quantitative polymerase chain reaction (qPCR) method was adapted to quantify B. cinerea DNA in grape bunch trash, and a colonization coefficient (CC) was calculated as the ratio between the DNA concentrations of B. cinerea and of Vitis vinifera. CC values increased linearly with the number of conidia of B. cinerea or the quantity of mycelium of B. cinerea added to the bunch trash increased. CC values also increased linearly in bunch trash samples containing increasing percentages of B. cinerea-colonized bunch trash; in the latter samples, CC values were correlated with subsequent assessments of B. cinerea colonization of trash (as determined by plating on agar) and sporulation on the trash (as determined by spore counts after incubation in humid chambers). The qPCR assay was also validated using trash collected from bunches treated or not treated with fungicides in three vineyards in two seasons. CC values reflected the reduction in sporulation and in latent infections of mature berries caused by fungicide application. The qPCR assay enables rapid, specific, sensitive, and reliable quantification of the degree of colonization of bunch trash by B. cinerea, which makes it a useful tool for studies of the epidemiology and management of BBR.

Effect of temperature on growth, wheat head infection, and nivalenol production by Fusarium poae.

Fusarium poae is one of the Fusarium species commonly detected in wheat kernels affected by Fusarium Head Blight. Fusarium poae produces a wide range of mycotoxins including nivalenol (NIV). The effect of temperature on colony growth and NIV production was investigated in vitro at 5-40 °C with 5 °C intervals. When the data were fit to a Beta equation (R2 ≥ 0.97), the optimal temperature was estimated to be 24.7 °C for colony growth and 27.5 °C for NIV production. The effects of temperature on infection incidence, fungal biomass, and NIV contamination were investigated by inoculating potted durum wheat plants at full anthesis; inoculated heads were kept at 10-40 °C with 5 °C intervals for 3 days and then at ambient temperature until ripening. Temperature significantly affected the incidence of floret infection and fungal biomass (as indicated by DNA amount) in the affected heads but did not affect NIV content in the head tissue. Inoculation of potted plants with F. poae did not reduce yield.

In Vitro Evaluation of Sub-Lethal Concentrations of Plant-Derived Antifungal Compounds on FUSARIA Growth and Mycotoxin Production.

Phytopathogenic fungi can lead to significant cereal yield losses, also producing mycotoxins dangerous for human and animal health. The fungal control based on the use of synthetic fungicides can be complemented by "green" methods for crop protection, based on the use of natural products. In this frame, the antifungal activities of bergamot and lemon essential oils and of five natural compounds recurrent in essential oils (citronellal, citral, cinnamaldehyde, cuminaldehyde and limonene) have been evaluated against three species of mycotoxigenic fungi (Fusarium sporotrichioides, F. graminearum and F. langsethiae) responsible for Fusarium Head Blight in small-grain cereals. The natural products concentrations effective for reducing or inhibiting the in vitro fungal growth were determined for each fungal species and the following scale of potency was found: cinnamaldehyde > cuminaldehyde > citral > citronellal > bergamot oil > limonene > lemon oil. Moreover, the in vitro mycotoxin productions of the three Fusaria strains exposed to sub-lethal concentrations of the seven products was evaluated. The three fungal species showed variability in response to the treatments, both in terms of inhibition of mycelial growth and in terms of modulation of mycotoxin production that can be enhanced by sub-lethal concentrations of some natural products. This last finding must be taken into account in the frame of an open field application of some plant-derived fungicides.

Copy number variation at the HvCBF4-HvCBF2 genomic segment is a major component of frost resistance in barley.

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.

Population structure and genome-wide association analysis for frost tolerance in oat using continuous SNP array signal intensity ratios.

KEY MESSAGE: Infinium SNP data analysed as continuous intensity ratios enabled associating genotypic and phenotypic data from heterogeneous oat samples, showing that association mapping for frost tolerance is a feasible option. Oat is sensitive to freezing temperatures, which restricts the cultivation of fall-sown or winter oats to regions with milder winters. Fall-sown oats have a longer growth cycle, mature earlier, and have a higher productivity than spring-sown oats, therefore improving frost tolerance is an important goal in oat breeding. Our aim was to test the effectiveness of a Genome-Wide Association Study (GWAS) for mapping QTLs related to frost tolerance, using an approach that tolerates continuously distributed signals from SNPs in bulked samples from heterogeneous accessions. A collection of 138 European oat accessions, including landraces, old and modern varieties from 27 countries was genotyped using the Infinium 6K SNP array. The SNP data were analyzed as continuous intensity ratios, rather than converting them into discrete values by genotype calling. PCA and Ward's clustering of genetic similarities revealed the presence of two main groups of accessions, which roughly corresponded to Continental Europe and Mediterranean/Atlantic Europe, although a total of eight subgroups can be distinguished. The accessions were phenotyped for frost tolerance under controlled conditions by measuring fluorescence quantum yield of photosystem II after a freezing stress. GWAS were performed by a linear mixed model approach, comparing different corrections for population structure. All models detected three robust QTLs, two of which co-mapped with QTLs identified earlier in bi-parental mapping populations. The approach used in the present work shows that SNP array data of heterogeneous hexaploid oat samples can be successfully used to determine genetic similarities and to map associations to quantitative phenotypic traits.

Triticale in Italy.

Triticale is currently grown throughout the world with a wider diffusion in Europe, with Poland, Belarus, Germany, France and Spain as major producers. Although triticale occupies a very small fraction of the Italian cultivated land (16,000 ha of harvested area, mean value of the past 5 years), a continuous interest for this crop and its possible uses explains the work and progress made by breeding activities in different periods. The aim of this review is to report some experiences related to the cultivation of triticale in Italy. A general long-term view of the performance of triticale varieties in Italy has been distilled from a large amount of data derived from the pluri-decennial Italian national variety trials network. This activity, historically coordinated by CREA-GB, extends over several decades and examines the agronomic performance, in different Italian environments, of the most widespread and emerging varieties of triticale. Indications on new breeding targets can be deduced from the elaborations in the frame of both climatic change and market demands.

A Molecular Toolbox to Identify and Quantify Grape Varieties: On the Trace of "Glera".

A pillar of wine authenticity is the variety/ies used. Ampelographic descriptors and SSR markers, included in several national and international databases, are extensively used for varietal identification purposes. Recently, SNP markers have been proposed as useful for grape varietal identification and traceability. Our study has been directed toward the development of a molecular toolbox able to track grape varieties from the nursery to the must. Two complementary approaches were developed, exploiting SNP markers with two different technologies, i.e., a high-throughput platform for varietal identification and a digital PCR system for varietal quantification. As proof-of-concept, the toolbox was successfully applied to the identification and quantification of the "Glera" variety along the Prosecco wine production chain. The assays developed found their limits in commercial, aged wines.

Long-Term In Situ Conservation Drove Microevolution of Solina d'Abruzzo Wheat on Adaptive, Agronomic and Qualitative Traits.

Solina is an example of a bread wheat landrace that has been conserved in situ for centuries in Central Italy. A core collection of Solina lines sampled in areas at different altitudes and climatic conditions was obtained and genotyped. A clustering analysis based on a wide SNP dataset generated from DArTseq analysis outlined the existence of two main groups, which, after Fst analysis, showed polymorphism in genes associated with vernalization and photoperiod response. Starting from the hypothesis that the different pedoclimatic environments in which Solina lines were conserved may have shaped the population, some phenotypic characteristics were studied in the Solina core collection. Growth habit, low-temperature resistance, allelic variations at major loci involved in vernalization response, and sensitivity to photoperiod were evaluated, together with seed morphologies, grain colour, and hardness. The two Solina groups showed different responses to low temperatures and to photoperiod-specific allelic variations as well as the different morphology and technological characteristics of the grain. In conclusion, the long-term in situ conservation of Solina in environments sited at different altitudes has had an impact on the evolution of this landrace which, despite its high genetic diversity, remains clearly identifiable and distinct so as to be included in conservation varieties.

Hierarchical Effects of Lactic Fermentation and Grain Germination on the Microbial and Metabolomic Profile of Rye Doughs.

A multi-omics approach was adopted to investigate the impact of lactic acid fermentation and seed germination on the composition and physicochemical properties of rye doughs. Doughs were prepared with either native or germinated rye flour and fermented with Saccharomyces cerevisiae, combined or not with a sourdough starter including Limosilactobacillus fermentum, Weissella confusa and Weissella cibaria. LAB fermentation significantly increased total titrable acidity and dough rise regardless of the flour used. Targeted metagenomics revealed a strong impact of germination on the bacterial community profile of sprouted rye flour. Doughs made with germinated rye displayed higher levels of Latilactobacillus curvatus, while native rye doughs were associated with higher proportions of Lactoplantibacillus plantarum. The oligosaccharide profile of rye doughs indicated a lower carbohydrate content in native doughs as compared to the sprouted counterparts. Mixed fermentation promoted a consistent decrease in both monosaccharides and low-polymerization degree (PD)-oligosaccharides, but not in high-PD carbohydrates. Untargeted metabolomic analysis showed that native and germinated rye doughs differed in the relative abundance of phenolic compounds, terpenoids, and phospholipids. Sourdough fermentation promoted the accumulation of terpenoids, phenolic compounds and proteinogenic and non-proteinogenic amino acids. Present findings offer an integrated perspective on rye dough as a multi-constituent system and on cereal-sourced bioactive compounds potentially affecting the functional properties of derived food products.

Combining Native and Malted Triticale Flours in Biscuits: Nutritional and Technological Implications.

Triticale-based biscuits were formulated with increasing substitution levels (i.e., 0, 25, 50, 75, and 100% w/w) of malted triticale flour (MTF). The products were analyzed for technological and nutritional characteristics, including the evaluation of the in vitro starch digestion. The results indicated that the substitution of triticale flour with MTF increased (p < 0.05) the total dietary fiber and ash contents. Total starch decreased (p < 0.05) when the level of MTF increased in the formulation, causing an increase in reducing sugars and an increase in the starch hydrolysis index and in the in vitro predicted glycemic index (pGI). The hardness and spread ratio values of biscuits decreased (p < 0.05) with increasing levels of MTF in the recipe. The lightness of doughs and biscuits decreased (p < 0.05) with increasing MTF levels. Overall, MTF could be used to formulate biscuits with higher dietary fiber content than native triticale flour and a medium to high in vitro glycemic index value as a function of the substitution level.

Indirect Measurement of ß-Glucan Content in Barley Grain with Near-Infrared Reflectance Spectroscopy.

ß-Glucan is a component of barley grains with functional properties that make it useful for human consumption. Cultivars with high grain ß-glucan are required for industrial processing. Breeding for barley genotypes with higher ß-glucan content requires a high-throughput method to assess ß-glucan quickly and cheaply. Wet-chemistry laboratory procedures are low-throughput and expensive, but indirect measurement methods such as near-infrared reflectance spectroscopy (NIRS) match the breeding requirements (once the NIR spectrometer is available). A predictive model for the indirect measurement of ß-glucan content in ground barley grains with NIRS was therefore developed using 248 samples with a wide range of ß-glucan contents (3.4%-17.6%). To develop such calibration, 198 unique samples were used for training and 50 for validation. The predictive model had R2 = 0.990, bias = 0.013% and RMSEP = 0.327% for validation. NIRS was confirmed to be a very useful technique for indirect measurement of ß-glucan content and evaluation of high-ß-glucan barleys.

Exploration of the Genetic Diversity of Solina Wheat and Its Implication for Grain Quality.

Different Solina wheat accessions (n = 24) collected in the Abruzzo region (Italy) were studied using 45,000 SNP markers generated from the DarTseq platform. The structure of genetic data was analyzed by Principal Component Analysis and Hierarchical Cluster analysis that revealed the existence of two main clusters (Clu1 and Clu2) characterized by samples with different geographical origin. The Solina genetic dataset was further merged and analyzed with a public genetic one provided by CIMMYT containing 25,963 genotypes from all over the world. The Solina accessions occupied a vast space, thus confirming a high heterogeneity of this landrace that, nevertheless, is considerably unique and placed quite far from other clusters. Clu1 and Clu2 divergence were clearly visible. Solina clusters were genetically closer to landraces from Turkey and the central fertile crescent than to the Italian genotypes present in the dataset. Selected commercial quality traits of accessions of the two Solina clusters were analyzed (yield, thousand kernel weight, test weight, and protein content), and significant differences were found between clusters. The results of this investigation did not highlight any relationships of Solina with Italian genotypes, and confirmed its wide genetic diversity by permitting to identify two genetic groups with distinct origin and quality traits.

Muscat Flavor in Grapevine: A Digital PCR Assay to Track Allelic Variation in VvDXS Gene.

The aroma of grapes and derived wines has long been one of the major traits considered in the selection of grapevine varieties through the centuries. In particular, Muscat aromatic grapes have been highly appreciated and widespread since ancient times. Monoterpenes are the key compounds responsible for the Muscat flavor. A major QTL affecting monoterpene level has been found to co-localize with the 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) gene, encoding for the 1-deoxy-D-xylulose 5-phosphate synthase enzyme involved in the plastidial pathway of terpene biosynthesis. In more detail, a single nucleotide polymorphism (SNP 1822) in the coding region of the gene causes a "gain of function" mutation, which is involved in Muscat flavor. In this work, we have developed a digital PCR-based assay to target allelic variations in the VvDXS gene, SNP1822, with the aim to propose a fast and sensitive analytical tool for targeting Muscat-flavored grapevine genotypes. The assay accurately predicts the genetic structure at 1822 SNP, critical for the development of the aroma in the great majority of Muscats. In the case of grapes in which the aromatic component is due to mutations other than SNP 1822 (e.g., Chasselas Musqué and Chardonnay Muscat), further specific assays can be developed.

Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain.

Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseqTM).

A Digital PCR Assay to Quantify the Percentages of Hulled vs. Hulless Wheat in Flours and Flour-Based Products.

Several food products, made from hulled wheats, are now offered by the market, ranging from grains and pasta to flour and bakery products. The possibility of verifying the authenticity of wheat species used at any point in the production chain is relevant, in defense of both producers and consumers. A chip digital PCR assay has been developed to detect and quantify percentages of hulless (i.e., common and durum wheat) and hulled (i.e., einkorn, emmer and spelt) wheats in grains, flours and food products. The assay has been designed on a polymorphism in the miRNA172 target site of the AP2-5 transcription factor localized on chromosome 5A and involved in wheat spike morphogenesis and grain threshability. The assay has been evaluated even in a real-time PCR system to assess its applicability and to compare the analytical costs between dPCR and real-time PCR approaches.

A Chip Digital PCR Assay for Quantification of Common Wheat Contamination in Pasta Production Chain.

Pasta, the Italian product par excellence, is made of pure durum wheat. The use of Triticum durum derived semolina is in fact mandatory for Italian pasta, in which Triticum aestivum species is considered a contamination that must not exceed the 3% maximum level. Over the last 50 years, various electrophoretic, chemical, and immuno-chemical methods have been proposed aimed to track the possible presence of common wheat in semolina and pasta. More recently, a new generation of methods, based on DNA (DeoxyriboNucleic Acid ) analysis, has been developed to this aim. Species traceability can be now enforced by a new technology, namely digital Polymerase Chain Reaction (dPCR) which quantify the number of target sequence present in a sample, using limiting dilutions, PCR, and Poisson statistics. In our work we have developed a duplex chip digital PCR (cdPCR) assay able to quantify common wheat presence along pasta production chain, from raw materials to final products. The assay was verified on reference samples at known level of common wheat contamination and applied to commercial pastas sampled in the Italian market.

Digital PCR: What Relevance to Plant Studies?

Digital PCR (dPCR) is a breakthrough technology that able to provide sensitive and absolute nucleic acid quantification. It is a third-generation technology in the field of nucleic acid amplification. A unique feature of the technique is that of dividing the sample into numerous separate compartments, in each of which an independent amplification reaction takes place. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for reading the digital output data. The dPCR assays developed so far in the plant science sector were identified in the literature, and the major applications, advantages, disadvantages, and applicative perspectives of the technique are presented and discussed in this review.

A Fast, Naked-Eye Assay for Varietal Traceability in the Durum Wheat Production Chain.

The development of a colorimetric mono-varietal discriminating assay, aimed at improving traceability and quality control checks of durum wheat products, is described. A single nucleotide polymorphism (SNP) was identified as a reliable marker for wheat varietal discrimination, and a rapid test for easy and clear identification of specific wheat varieties was developed. Notably, an approach based on the loop-mediated isothermal amplification reaction (LAMP) as an SNP discrimination tool, in combination with naked-eye visualization of the results, was designed and optimized. Our assay was proven to be effective in the detection of adulterated food products, including both substitution and mixing with different crop varieties.