Multi-environment association study highlights candidate genes for robust agronomic quantitative trait loci in a novel worldwide Capsicum core collection.

Investigating crop diversity through genome-wide association studies (GWAS) on core collections helps in deciphering the genetic determinants of complex quantitative traits. Using the G2P-SOL project world collection of 10 038 wild and cultivated Capsicum accessions from 10 major genebanks, we assembled a core collection of 423 accessions representing the known genetic diversity. Since complex traits are often highly dependent upon environmental variables and genotype-by-environment (G × E) interactions, multi-environment GWAS with a 10 195-marker genotypic matrix were conducted on a highly diverse subset of 350 Capsicum annuum accessions, extensively phenotyped in up to six independent trials from five climatically differing countries. Environment-specific and multi-environment quantitative trait loci (QTLs) were detected for 23 diverse agronomic traits. We identified 97 candidate genes potentially implicated in 53 of the most robust and high-confidence QTLs for fruit flavor, color, size, and shape traits, and for plant productivity, vigor, and earliness traits. Investigating the genetic architecture of agronomic traits in this way will assist the development of genetic markers and pave the way for marker-assisted selection. The G2P-SOL pepper core collection will be available upon request as a unique and universal resource for further exploitation in future gene discovery and marker-assisted breeding efforts by the pepper community.

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.

PolyHaplotyper: haplotyping in polyploids based on bi-allelic marker dosage data.

BACKGROUND: For genetic analyses, multi-allelic markers have an advantage over bi-allelic markers like SNPs (single nucleotide polymorphisms) in that they carry more information about the genetic constitution of individuals. This is especially the case in polyploids, where individuals carry more than two alleles at each locus. Haploblocks are multi-allelic markers that can be derived by phasing sets of closely-linked SNP markers. Phased haploblocks, similarly to other multi-allelic markers, will therefore be advantageous in genetic tasks like linkage mapping, QTL mapping and genome-wide association studies. RESULTS: We present a new method to reconstruct haplotypes from SNP dosages derived from genotyping arrays, which is applicable to polyploids. This method is implemented in the software package PolyHaplotyper. In contrast to existing packages for polyploids it makes use of full-sib families among the samples to guide the haplotyping process. We show that in this situation it is much more accurate than other available software, using experimental hexaploid data and simulated tetraploid data. CONCLUSIONS: Our method and the software package PolyHaplotyper in which it is implemented extend the available tools for haplotyping in polyploids. They perform especially well in situations where one or more full-sib families are present.

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.

Multiallelic models for QTL mapping in diverse polyploid populations.

Quantitative trait locus (QTL) analysis allows to identify regions responsible for a trait and to associate alleles with their effect on phenotypes. When using biallelic markers to find these QTL regions, two alleles per QTL are modelled. This assumption might be close to reality in specific biparental crosses but is unrealistic in situations where broader genetic diversity is studied. Diversity panels used in genome-wide association studies or multi-parental populations can easily harbour multiple QTL alleles at each locus, more so in the case of polyploids that carry more than two alleles per individual. In such situations a multiallelic model would be closer to reality, allowing for different genetic effects for each potential allele in the population. To obtain such multiallelic markers we propose the usage of haplotypes, concatenations of nearby SNPs. We developed "mpQTL" an R package that can perform a QTL analysis at any ploidy level under biallelic and multiallelic models, depending on the marker type given. We tested the effect of genetic diversity on the power and accuracy difference between bi-allelic and multiallelic models using a set of simulated multiparental autotetraploid, outbreeding populations. Multiallelic models had higher detection power and were more precise than biallelic, SNP-based models, particularly when genetic diversity was higher. This confirms that moving to multi-allelic QTL models can lead to improved detection and characterization of QTLs. KEY MESSAGE: QTL detection in populations with more than two functional QTL alleles (which is likely in multiparental and/or polyploid populations) is more powerful when using multiallelic models, rather than biallelic models.

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.

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).

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.

Using probabilistic genotypes in linkage analysis of polyploids.

KEY MESSAGE: In polyploids, linkage mapping is carried out using genotyping with discrete dosage scores. Here, we use probabilistic genotypes and we validate it for the construction of polyploid linkage maps. Marker genotypes are generally called as discrete values: homozygous versus heterozygous in the case of diploids, or an integer allele dosage in the case of polyploids. Software for linkage map construction and/or QTL analysis usually relies on such discrete genotypes. However, it may not always be possible, or desirable, to assign definite values to genotype observations in the presence of uncertainty in the genotype calling. Here, we present an approach that uses probabilistic marker dosages for linkage map construction in polyploids. We compare our method to an approach based on discrete dosages, using simulated SNP array and sequence reads data with varying levels of data quality. We validate our approach using experimental data from a potato (Solanum tuberosum L.) SNP array applied to an F1 mapping population. In comparison to the approach based on discrete dosages, we mapped an additional 562 markers. All but three of these were mapped to the expected chromosome and marker position. For the remaining three markers, no physical position was known. The use of dosage probabilities is of particular relevance for map construction in polyploids using sequencing data, as these often result in a higher level of uncertainty regarding allele dosage.

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.

DNA-based genealogy reconstruction of Nebbiolo, Barbera and other ancient grapevine cultivars from northwestern Italy.

Northwestern Italy is a wine region of the world with the highest of reputations, where top quality wines of remarkable economic value are produced from traditional, long-cultivated varieties. Kinship analyses were performed using 32 microsatellite loci and more than 10 K single-nucleotide polymorphism markers on 227 traditional grapes mostly from Northwestern Italy-including those that have been neglected or are threatened. This was done to better understand the genetic grapevine origins and history of this reputable wine producing area, thus enhancing its cultural value and the marketing appeal of its wines. The work revealed a complex network of genetic relationships among varieties, with little contribution of genotypes from other areas. It revealed the major role played by a few ancient grape varieties as parents of numerous offspring, including some that are endangered today. The ancestry of many cultivars is proposed. Among these are Dolcetto, Barbera and Riesling italico. Through the inference of parent-offspring and sibling relations, marker profiles of ungenotyped putative parents were reconstructed, suggesting kinship relations and a possible parentage for Nebbiolo, one of the most ancient wine grapes worldwide. Historic and geographic implications from the resulting kinships are discussed.

Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals.

Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins' risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify Fusarium in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to Fusarium diagnosis in plants.

Parentage Atlas of Italian Grapevine Varieties as Inferred From SNP Genotyping.

The Italian grape germplasm is characterized by a high level of richness in terms of varieties number, with nearly 600 wine grape varieties listed in the Italian National Register of Grapevine Varieties and with a plethora of autochthonous grapes. In the present study an extended SNP genotyping has been carried out on Italian germplasm of cultivated Vitis vinifera subsp. sativa and Vitis hybrids. Several hundred Italian varieties maintained in the repositories of scientific Institutions and about one thousand additional varieties derived from previous studies on European, Southern Italy, Magna Graecia and Georgian germplasm were considered. The large genotyping data obtained were used to check the presence of homonyms and synonyms, determine parental relationships, and identify the main ancestors of traditional Italian cultivars and closely-related accessions. The parentage among a set of 1,232 unique varieties has been assessed. A total of 92 new parent-offspring (PO) pairs and 14 new PO trios were identified. The resulted parentage network suggested that the traditional Italian grapevine germplasm originates largely from a few central varieties geographically distributed into several areas of genetic influence: "Strinto porcino" and its offspring "Sangiovese", "Mantonico bianco" and "Aglianico" mainly as founder varieties of South-Western Italy (IT-SW); Italian Adriatic Coast (IT-AC); and Central Italy with most varieties being offsprings of "Visparola", "Garganega" and "Bombino bianco"; "Termarina (Sciaccarello)" "Orsolina" and "Uva Tosca" as the main varieties of North-Western Italy (IT-NW) and Central Italy. The pedigree reconstruction by full-sib and second-degree relationships highlighted the key role of some cultivars, and, in particular, the centrality of "Visparola" in the origin of Italian germplasm appeared clear. An hypothetical migration of this variety within the Italian Peninsula from South to North along the eastern side, as well as of "Sangiovese" from South to Central Italy along the Western side might be supposed. Moreover, it was also highlighted that, among the main founders of muscat varieties, "Moscato bianco" and "Zibibbo (Muscat of Alexandria)" have spread over the whole Italy, with a high contribution by the former to germplasm of the North-Western of the peninsula.

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.

Occurrence of Fusarium langsethiae and T-2 and HT-2 Toxins in Italian Malting Barley.

T-2 and HT-2 toxins are two of the most toxic members of type-A trichothecenes, produced by a number of Fusarium species. The occurrence of these mycotoxins was studied in barley samples during a survey carried out in the 2011-2014 growing seasons in climatically different regions in Italy. The percentage of samples found positive ranges from 22% to 53%, with values included between 26 and 787 µg/kg. The percentage of samples with a T-2 and HT-2 content above the EU indicative levels for barley of 200 µg/kg ranges from 2% to 19.6% in the 2011-2014 period. The fungal species responsible for the production of these toxins in 100% of positive samples has been identified as Fusarium langsethiae, a well-known producer of T-2 and HT-2 toxins. A positive correlation between the amount of F. langsethiae DNA and of the sum of T-2 and HT-2 toxins was found. This is the first report on the occurrence of F. langsethiae-and of its toxic metabolites T-2 and HT-2-in malting barley grown in Italy.

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.

In vitro evaluation of the activity of thiosemicarbazone derivatives against mycotoxigenic fungi affecting cereals.

With a steadily increasing world population, a more efficient system of food production is of paramount importance. One of the major causes of food spoilage is the presence of fungal pathogens and the production and accumulation of mycotoxins. In the present work we report a study on the activity of a series of functionalized thiosemicarbazones (namely cuminaldehyde, trans-cinnamaldehyde, quinoline-2-carboxyaldehyde, 5-fluoroisatin thiosemicarbazone and 5-fluoroisatin N(4)-methylthiosemicarbazone), as antifungal and anti-mycotoxin agents, against the two major genera of cereal mycotoxigenic fungi, i.e. Fusarium and Aspergillus. These thiosemicarbazones display different patterns of efficacy on fungal growth and on mycotoxin accumulation depending on the fungal species. Some of the molecules display a greater effect on mycotoxin synthesis than on fungal growth.

Metabolite profiling elucidates communalities and differences in the polyphenol biosynthetic pathways of red and white Muscat genotypes.

The chemical composition of grape berries is varietal dependent and influenced by the environment and viticulture practices. In Muscat grapes, phenolic compounds play a significant role in the organoleptic property of the wine. In the present study, we investigated the chemical diversity of berries in a Muscat collection. Metabolite profiling was performed on 18 Moscato bianco clones and 43 different red and white grape varieties of Muscat using ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS/MS) coupled with SNP genotyping. Principle component analysis and hierarchical clustering showed a separation of the genotypes into six main groups, three red and three white. Anthocyanins mainly explained the variance between the different groups. Additionally, within the white varieties mainly flavonols and flavanols contributed to the chemical diversity identified. A genotype-specific rootstock effect was identified when separately analyzing the skin of the clones, and it was attributed mainly to resveratrol, quercetin 3-O-galactoside, citrate and malate. The metabolite profile of the varieties investigated reveals the chemical diversity existing among different groups of Muscat genotypes. The distribution pattern of metabolites among the groups dictates the abundance of precursors and intermediate metabolite classes, which contribute to the organoleptic properties of Muscat berries.

Reconstituted CP29: multicomponent fluorescence decay from an optically homogeneous sample.

The multiexponential fluorescence decay of the CP29 complex in which the apoprotein and pigments were reconstituted in vitro was examined. Of the three decay components observed only the two dominant ones, with about 3 and 5 ns lifetimes, were studied. The main question addressed was whether the multicomponent decay was associated with sample optical heterogeneity. To this end, we examined the optical absorption and fluorescence of the CP29 sample by means of two different and independent experimental strategies. This approach was used as the wavelength positions of the absorption/fluorescence spectral forms has recently been shown to be a sensitive indicator of the binding site-induced porphyrin ring deformation (Zucchelli et al. Biophys J 93:2240-2254, 2007) and hence of apoprotein conformational changes. The data indicate that this CP29 sample is optically homogeneous. It is hypothesised that the different lifetimes are explained in terms of multiple detergent/CP29 interactions leading to different quenching states, a suggestion that allows for optical homogeneity.