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

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

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

Integrated Bayesian Approaches Shed Light on the Dissemination Routes of the Eurasian Grapevine Germplasm

The domestication and spreading of grapevine as well as the gene flow history had been described in many studies. We used a high-quality 7k SNP dataset of 1,038 Eurasian grape varieties with unique profiles to assess the population genetic diversity, structure, and relatedness, and to infer the most likely migration events. Comparisons of putative scenarios of gene flow throughout Europe from Caucasus helped to fit the more reliable migration routes around the Mediterranean Basin. Approximate Bayesian computation (ABC) approach made possible to provide a response to several questions so far remaining unsolved. Firstly, the assessment of genetic diversity and population structure within a well-covered dataset of ancient Italian varieties suggested the different histories between the Northern and Southern Italian grapevines. Moreover, Italian genotypes were shown to be distinguishable from all the other Eurasian populations for the first time. The entire Eurasian panel confirmed the east-to-west gene flow, highlighting the Greek role as a “bridge” between the Western and Eastern Eurasia. Portuguese germplasm showed a greater proximity to French varieties than the Spanish ones, thus being the main route for gene flow from Iberian Peninsula to Central Europe. Our findings reconciled genetic and archaeological data for one of the most cultivated and fascinating crops in the world

A Point-of-Care Assay Based on Reflective Phantom Interface (RPI) Technology for Fast, Multi-Toxin Screening in Wheat

Mycotoxigenic fungi can colonize small grain cereals causing severe yield losses and grain contaminations. Fusaria can be responsible for the contamination of wheat grains and derived products via several classes of mycotoxins, negatively impacting human and animal health. Among the strategies to control mycotoxins are analytical tools for their identification and quantification from field to food and feed. A fast multi-toxin assay based on reflective phantom interface (RPI) technology was developed to identify and quantify deoxynivalenol, zearalenone, and T-2/HT-2 toxins. The PRX analytical workflow was organized as follows: a fast mycotoxins extraction step followed by an analytical step carried out in a system composed of three elements: (I) a universal reader able to read a series of (II) cartridges that incorporate the RPI technology and (III) a software that analyzes data and gives feedback on the results. The assay was evaluated in wheat reference samples at known levels of toxin contaminations and on naturally contaminated grain samples. The results obtained suggest that the assay can be considered a useful screening tool for point-of-care and point-of-sale control of toxins contamination along wheat production and transformation chains

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

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