Two main distinct evolutionary stories describe the Italian grapevine assortment

A dataset of high-quality 7k SNP profiles of 1,038 unique Eurasian grapevine varieties was used to infer the most likely grapevine migration events, a spatial ancestry estimation, and a model about the origin of Eurasian grapevine germplasm. The comparison of putative gene flow scenarios from Caucasus throughout Europe aided to fit the more reliable spreading routes around the Mediterranean Basin. The same dataset was also used to assess the population genetic diversity, structure, and relatedness of Italian varieties. These data suggested a different history between Northern and Southern Italian grapevines, appearing clearly split into two different clusters. Interestingly, the Italian genotypes were shown to be distinguishable from all the other Eurasian populations for the first time. The same SNP panel was used to determine parental relationships, identifying the main parents of traditional Italian and closely related cultivars. The parentage network suggested that Italian germplasm largely originated from a few key parents distributed into several geographical areas of genetic influence, with more or less large overlaps. These key cultivars are ‘Bombino bianco’, ‘Garganega’/‘Grecanico’, ‘Mantonico bianco’, ‘Orsolina’/‘Coccalona nera’, ‘Muscat à petits grains blanc’, ‘Malvasia odorosissima’, ‘Sangiovese’, ‘Sciaccarello’, ‘Visparola’ and ‘Vulpea’. The pedigree reconstruction by fullsib and second-degree relationships highlighted the pivotal role of some cultivars, such as ‘Visparola’, until now scarcely known. A hypothetic migration of this variety from Southern to Northern Italy along the Eastern side, as well as ‘Sangiovese’ migration from Southern to Central Italy along the Western side might be supposed. Moreover, ‘Muscat à petits grains blanc’, mainly through its offspring ‘Malvasia odorosissima’, furnished a consistent contribution to the development of many aromatic grapes grown in the Northern-Western part of the Italian Peninsula. These results represent the most complete study of grapevine Italian population genetics that has been carried out until now on the Italian germplasm

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

Grapevine field experiments reveal the contribution of genotype, the influence of environment and the effect of their interaction (GxE) on berry transcriptome

Changes in the performance of genotypes in different environments are defined as genotype x environment (GxE) interactions. In grapevine (Vitis vinifera), complex interactions between different genotypes and climate, soil, and farming practices yield unique berry qualities. However, the molecular basis of this phenomenon remains unclear. To dissect the basis of grapevine GxE interactions, we characterized berry transcriptome plasticity, genome methylation landscape, and within-genotype allelic diversity in two genotypes, cultivated in three different environments, over two vintages. We identified, through a novel data-mining pipeline, genes with expression profiles that were unaffected by genotype or environment, genotype-dependent but unaffected by the environment, environmentally-dependent regardless of genotype, and GxE-related. The GxE-related genes showed different degrees of within-cultivar allelic diversity in the two genotypes and were enriched for stress responses, signal transduction and secondary metabolism categories. Our study unraveled the mutual relationships between genotypic and environmental variables during GxE interaction in a woody perennial species, providing a reference model to explore how cultivated fruit crops respond to diverse environments. Also, the pivotal role of vineyard location in determining the performance of different varieties, by enhancing berry quality traits, was unraveled

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification

Dissecting the susceptibility/resistance mechanism of Vitis vinifera for the future control of downy mildew

23CO.NA.VI. 2020 – 8° Convegno Nazionale di Viticoltura, Udine, Italy, July 5-7, 2021openInternationalBothThe Eurasian grapevine (Vitis vinifera), a species cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. Nevertheless, germplasm from Georgia (Southern Caucasus, the first grapevine domestication centre), characterized by a high genetic variability, showed resistance traits to P. viticola. The cultivar Mgaloblishvili exhibited the most promising phenotype in terms of resistance against P. viticola. Its defence response results in: i) low disease intensity; ii) low sporulation; iii) damaged mycelium; iv) production of antimicrobial compounds such as volatile organic compounds (VOCs), whose effectiveness on the pathogen was evaluated by leafdisc assays. At the transcriptomic level, its resistance mechanism is determined by the differential expression of both resistance and susceptible genes. The resistance genes are related to: i) pathogen recognition through PAMP, DAMP and effector receptors; ii) ethylene signalling pathway; iii) synthesis of antimicrobial compounds (VOCs) and fungal wall degrading enzymes; iv) development of structural barriers (cell wall reinforcement). The first putative susceptible gene was the transcription factor VviLBDIf7 gene, whose validation was carried out by dsRNA (double-stranded RNA) assay. In this work, these unique results on plant-pathogen interaction are reviewed with the aim of developing new strategies to control the disease.openRicciardi, Valentina; Marcianò, Demetrio; Sargolzaei, Maryam; Marrone Fassolo, Elena; Fracassetti, Daniela; Brilli, Matteo; Moser, Mirko; Vahid, Shariati J.; Tavakole, Elahe; Maddalena, Giuliana; Passera, Alessandro; Casati, Paola; Pindo, Massimo; Cestaro, Alessandro; Costa, Alex; Bonza, Maria Cristina; Maghradze, David; Tirelli, Antonio; Failla, Osvaldo; Bianco, Piero Attilio; Quaglino, Fabio; Toffolatti, Silvia Laura; De Lorenzis, GabriellaRicciardi, V.; Marcianò, D.; Sargolzaei, M.; Marrone Fassolo, E.; Fracassetti, D.; Brilli, M.; Moser, M.; Vahid, S.J.; Tavakole, E.; Maddalena, G.; Passera, A.; Casati, P.; Pindo, M.; Cestaro, A.; Costa, A.; Bonza, M.C.; Maghradze, D.; Tirelli, A.; Failla, O.; Bianco, P.A.; Quaglino, F.; Toffolatti, S.L.; De Lorenzis, G

Effectiveness of AFLPs and retrotransposon-based markers for the identification of portuguese grapevine cultivars and clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces

Studio di differenti marcatori molecolari nell'identificazione varietale della vite.

La corretta identificazione varietale riveste una rilevante importanza scientifica e pratica in tutti i settori dell’arboricoltura. Tali problemi interessano in modo particolare specie come la vite che presentano aspetti di grande complessità, determinati sia dall’elevato numero di cultivar, sia dall’esistenza di frequenti casi di omonimia e sinonimia. L'identificazione delle varietà attraverso le loro caratteristiche morfologiche (ampelografia e ampelometria) può essere soggetta ad errore, poiché le caratteristiche fenotipiche sono influenzate a diversi livelli dall'ambiente. Per ottimizzare la conservazione e l'utilizzazione delle varietà autoctone è estremamente utile un'analisi della diversità genetica di queste, mediante l'uso di marcatori molecolari basati sull'analisi del DNA, sia per il loro notevole potenziale discriminante fra genotipi, sia per la loro “neutralità” rispetto alle condizioni ambientali. Grazie a queste tecniche possono essere evidenziate differenze, a livello di sequenza nucleotidica, con estrema precisione. Il lavoro di questa tesi ha lo scopo di realizzare una caratterizzazione molecolare di ventidue genotipi appartenenti al genere Vitis, avvalendosi di differenti tecniche di marcatura molecolare: REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), IRAP (Inter-Retrotransposon Amplified Polymorphism) I-SSR (Inter-Simple Sequence Repeat) e SSR (Simple Sequence Repeats). I retrotrasposoni sono elementi genetici mobili, di origine retrovirale, molto ripetuti (generalmente da 1000 a oltre 100.000 copie, per genoma aploide), interspersi nel genoma e ubiquitari negli eucarioti, sia animali sia vegetali. Nel corso dell’evoluzione, essi hanno occupato siti casuali nel genoma attraverso un intermedio a RNA, convertito in DNA extracromosomico grazie ad enzimi, prodotti autonomamente dal retrotrasposone stesso, prima di reinserirsi in un nuovo sito. La loro azione quindi ha generato una enorme variabilità genetica anche tra individui della stessa specie. I metodi di marcatura molecolare basata sui retroelementi consistono nell'amplificazione, mediante PCR, di sequenze di DNA fiancheggianti i retrotrasposoni, sfruttando l'elevato numero di questi nel genoma e la possibilità che in individui diversi i retrotrasposoni possano essersi inseriti in siti diversi del genoma. In particolare la tecnica REMAP consiste nell’amplificazione delle porzioni del genoma comprese tra un retroelemento e un microsatellite, mentre la tecnica IRAP prevede una amplificazione dei frammenti di DNA situati tra due retrotrasposoni vicini. Nel nostro lavoro sono stati progettati oligonucleotidi in base alla sequenza di tre retrotrasposoni fin qui isolati in vite: Vine-1, Tvv1 e Gret1. Questi oligonucleotidi sono stati utilizzati insieme ad altri primers microsatellitari in reazioni di PCR. Il più alto grado di polimorfismi è stato registrato utilizzando primers costruiti sul retrotrasposone Tvv1. Il numero di bande polimorfiche che è stato ottenuto dalle tecniche REMAP, IRAP e I-SSR è risultato essere sufficientemente elevato per discriminare con buona certezza tra le cultivar di V. vinifera e le specie di Vitis. La possibilità di ottenere dei prodotti di amplificazione anche in genotipi differenti dalla specie V. vinifera, suggerisce che questo retroelemento sia presente in tutto il genere Vitis e che abbia svolto un ruolo importante nella definizione e nella funzione della struttura del genoma delle differenti specie di vite. Analizzando i frammenti di PCR mediante un sequenziatore automatico è stato possibile anche mettere in evidenza alcune differenze tra cloni di una stessa varietà. Questo costituisce una novità importante ed incoraggia, per il futuro, la ricerca di ulteriori primer microsatellitari per ottenere un sufficiente numero di polimorfismi tra cloni. Per le analisi SSR sono stati utilizzati oligonucleotidi già usati con successo per evidenziare polimorfismi a carico di loci microsatellitari di vite. Con questa analisi è stato possibile distinguere, in parte, genotipi di cultivar o specie differenti di Vitis, tuttavia i profili delle lunghezze degli amplificati sono risultati uguali per i cloni della cultivar Sangiovese che è stata testata. In conclusione, questo lavoro mostra che i retrotrasposoni, ed in particolare il Tvv1, possono essere utilizzati come marcatori molecolari nell’identificazione varietale della vite, e che questi elementi probabilmente hanno giocato un ruolo importante nell’evoluzione del genoma della vite. L’utilizzo di tecniche di marcatura molecolare basata sui retroelementi e sui microsatelliti, si è dimostrato utile per discriminare tra genotipi di vite anche essi vicini tra loro dal punto di vista genetico, ed incoragggia la ricerca di ulteriori oligonucleotidi per arricchire la banca dati internazionale, che custodisce la lista delle lunghezze dei loci microsatellitari di differenti cultivar del genere Vitis

CHARACTERIZATION OF AROMA BIOSYNTHETIC PATHWAYS IN VITIS VINIFERA

Varietal aroma plays a significant role in defining the wine aroma, but there are insufficient knownledges about their biosynthesis and evolution during berries development, especially in herbaceous phase. To know the evolution of aromatic compounds during all phases of berries development it is crucial for understanding the way to manage the grapes aromatic potential in the vineyard for improving wine aromatic profile. The present work describes the changes in terpenes (monoterpenes and sesquiterpenes), C13-noriprenoids, C6-alcohols and benenoids levels in flower buds, flowers and during berries development from fruit set to ripening, of two different Vitis vinifera L. cultivars: “Moscato bianco” and “Sangiovese”. In the same samples the expression patterns of some flavour candidate genes were measured in order to study the correlation between these putative genes and volatile compounds accumulation. Another aspect of this reaserch has been focused on the effects of vine canopy microclimate on berries aroma composition: in “Ciliegiolo” and “Sangiovese” cultivars basal leaves have been removed at fruit set or at véraison to valuate possible differences in grape flavour conten. The aromatic compounds were detected in flower buds, flowers and in all stages of Moscato bianco and Sangiovese berry development. Aromatic compounds content reached the highest value during flowering and at full maturation. The flavor profile of Moscato bianco grapes appeared mainly characterized to compounds monoterpenes, while the presence of other classes of aroma identified (sesquiterpenes, norisoprenoids, aliphatic alcohols and benzene derivatives) was not very relevant; during flowering we observed the highest value of sesquiterpenes content. The flavor profile of Sangiovese grapes appeared mainly characterized by monoterpenes and norisoprenoids; such as in Moscato bianco, we have been observed the highest accumulation sesquiterpenes in flowers. The terpenes content in flowers and during berry ripening coincided with expression increase of genes involved in the biosynthesis of monoterpenes and sesquiterpenes. Abundance of terpene synthases transcripts (monoterpenes and sesquiterpenes synthase), detected in flower buds and open flowers and showed hight correlation with monoterpenes and sesquiterpenes contents in preflowering and during flowering, could be involved in the formation of flower volatile compounds. While terpene synthases transcripts detected after flowering could be involved in the formation of berry volatile compounds. Absence of sesquiterpenes detected during ripening suggested a specific role of these terpene synthase in moterpenes formation of ripe berry. Based on gene expression results, among gene showing a good correlation between aromatic content and gene expression, we selected 3 genes to focus functional characterization analysis. Leaf removal during fruit set in both Sangiovese and Ciliegiolo lead and increase in monoterpenes and especially in C13-norisoprenoids aroma. C13-norisoprenoids increase following leaf removal confirmed the hypothesis that these compounds derive from carotenoids degratation, favoured by sunshine. These results contribuited to establish that vine canopy management is effective in the modulation of aroma berry composition and consequently in wine sensory quality

Genetic Diversity and Population Structure in a Vitis spp. Core Collection Investigated by SNP Markers

Single nucleotide polymorphism (SNP) genotyping arrays are powerful tools to measure the level of genetic polymorphism within a population. The coming of next-generation sequencing technologies led to identifying thousands and millions of SNP loci useful in assessing the genetic diversity. The Vitis genotyping array, containing 18k SNP loci, has been developed and used to detect genetic diversity of Vitis vinifera germplasm. So far, this array was not validated on non-vinifera genotypes used as grapevine rootstocks. In this work, a core collection of 70 grapevine rootstocks, composed of individuals belonging to Vitis species not commonly used in the breeding programs, was genotyped using the 18k SNP genotyping array. SNP results were compared to the established SSR (Simple Sequence Repeat) markers in terms of heterozygosity and genetic structure of the core collection. Genotyping array has proved to be a valuable tool for genotyping of grapevine rootstocks, with more than 90% of SNPs successfully amplified. Structure analysis detected a high degree of admixed genotypes, supported by the complex genetic background of non-vinifera germplasm. Moreover, SNPs clearly differentiated non-vinifera and vinifera germplasm. These results represent a first step in studying the genetic diversity of non-conventional breeding material that will be used to select rootstocks with high tolerance to limiting environmental conditions