Serena Imazio

Evidence of a secondary grapevine domestication centre detected by SSR analysis

Abstract. The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

Genetic relationships in Opuntia Mill. genus (Cactaceae) detected by molecular marker

The Opuntia genus includes over 181 species comprising, on the basis of morphological traits, a total of 29 series [The Cactaceae (1919)]. Starting from this classification, several authors have investigated the Opuntia genus taxonomy but the large morphological variation within different species, suggests that phenotypical characteristics will not serve to produce a stable classification. In this work chloroplastic simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP) were used to evaluate the usefulness of molecular markers in Opuntia species characterization and to study the relationships among different species. Results show that the combination of cpSSR and AFLP markers provide a quantitative estimation of genetic relationships among several Opuntia species. Both molecular analyses reveal a genetic similarity among species of series 20 and 21 [The Cactaceae (1919)] as suggested also by morphological traits. Particular attention was focused on the genetic relationship between Opuntia ficus-indica and Opuntia megacantha: individuals from different populations of the two species were analyzed with both molecular markers. A common genetic constitution of O. ficus-indica and O. megacantha was detected. On the basis of molecular data, morphological traits and biogeographical distribution, we suggest that O. ficus-indica should be considered as a domesticated form of O. megacantha. Our results suggest the importance of a revision of Opuntia genus classification using several tools: molecular, morphological and biogeographical analysis.

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.

The use of AFLP and morphological markers to study Iranian grapevine germplasm to avoid genetic erosion

Summary Molecular and morphological analyses have been used to define and clarify the relationships among the 54 most representative grapevine cultivars in Iran. A total of 459 scorable AFLP fragments, across all the accessions analysed, were detected. Among these, 185 (40.3%) were polymorphic. Values of genetic diversity, calculated by the Nei and Li index, ranged from 0 – 0.3. Our results highlight cases of different cultivars displaying the same AFLP profile and similar morphological characteristics, proving the occurrence of synonyms in Iranian germplasm. Combining AFLP results and morphological descriptions, two cases of wrong attribution were also observed: ‘Saghal Solian-2’ was renamed ‘Mosli’; and ‘Khalili Qermez’ was now called ‘Khalili Sefid’. Morphological characterisation, conducted using the IPGRI descriptors, showed large variability among the accessions analysed, mainly in young shoot and leaf colour, and in bunch size. Although synonyms were detected, and genomic similarities were verified, genetic differences and the richness observed in Iranian grapevine germplasm were not high, in contrast with their morphological characteristics. We hypothesise that many Iranian morphotypes have been derived from mutation events in a few ancient genotypes; however, the system of selection may result in the biodiversity of Iranian grapevine being endangered. For this reason, our molecular and morphological characterisation will help in the development of conservation plans and in the re-organisation of local germplasm collections.

Pedigree Reconstruction of the Italian Grapevine Aglianico ( Vitis vinifera L.) from Campania

A total of 41 accessions of Aglianico belonging to three different biotypes (Taburno, Taurasi, and Vulture) and 9 accessions of Sirica grapes were sampled from diverse areas of Campania (Italy). All accessions were first genotyped using 21 microsatellite markers (SSR) to evaluate possible homonymies, synonymies, and the genetic structure of each group. A larger dataset was then constructed adding Italian and International cultivars. On the basis of results obtained analyzing the first dataset, further investigations were carried out enlarging the number of investigated loci (up to 43). The addition of 22 SSRs was useful in the definition of likely genetic relationships linking Aglianico biotypes, Sirica and Syrah. According to their SSR allelic profiles, the monophyletic origin of the three Aglianico biotypes was confirmed. Among Aglianico Taburno accessions, eight samples (called Aglianico like-to-type) performed a different SSR allelic profile from Aglianico true-to-type. Sirica and Syrah proved to be synonyms. This work allowed to determine the genetic relationship between Aglianico and the cultivars supposed to be related. The parentage analysis was investigated. The most likely pedigree has been reconstructed; revealing a second-degree relationship between the worldwide cultivated Syrah from the Rhone Valley and Aglianico. Aglianico like-to-type appeared related to Aglianico in a parent-offspring fashion.

Chloroplast and nuclear DNA markers to characterize cultivated and spontaneous Ribes

Abstract Nine universal cpSSR markers were tested on a representative group of cultivated accessions and spontaneous ribes populations. All primer combinations, with the exception of ccmp8, underwent amplification. Monomorphic alleles were detected at the ccmp2, ccmp3, ccmp4, ccmp5, ccmp7, ccmp9 primer pairs. Two and four polymorphic alleles were observed at the ccmp6 and ccmp10 loci, respectively. These six alleles combined in five different haplotypes, two of which were found in the cultivated accessions, and three only in the spontaneous populations. AFLP analysis was also performed to better define the relationships among cultivated and spontaneous varieties, and to compare nuclear markers with chloroplast microsatellites. AFLP data were able to distinguish clearly all cultivars, while in spontaneous populations the detected polymorphisms were too low. In conclusion, cpSSR markers can be considered useful in the characterisation of ribes accessions. Thus, the combination of this tool with nuclear markers, such as AFLP, could help in distinguishing and characterising each cultivated accession, and in defining population genetic parameters in the study of spontaneous Ribes species.

Genetic relationships between Sardinian and Spanish viticulture: the case of 'Cannonau' and 'Garnacha'

Summary To evaluate the relationship between Sardinian and Spanish viticulture, Simple Sequence Repeat (SSR) markers were applied to define the genetic profiles of 29 cultivated and 48 wild grapevine (Vitis vinifera L.) accessions. SSR data confirmed synonymy between ‘Cannonau’ and several Spanish accessions of ‘Garnacha Tinta’. SSR analysis also suggested that the ‘Garnacha’ group consisted of a heterogeneous pool of cultivars displaying different morphological and genetic traits (Link coefficient = approx. 0.5), probably caused by somatic mutation or accidental breeding events between closely-related grapevine accessions. In contrast, the ‘Vernaccia’ - ‘Granaccia’ Sardinian group was different from ‘Cannonau’ (Link coefficient = 0.8) and all Spanish ‘Garnacha Tinta’ and ‘Blanca’ accessions analysed. To understand the ‘Cannonau’ - ‘Garnacha’ relationship, we studied the origin of these accessions and their relationships with spontaneous wild grapevine. Both cultivars are ancient grapes that have been cultivated for many centuries in both Sardinia and Spain. Although the name ‘Garnacha’ may derive from the Italian word ‘Vernaccia’, molecular analysis excluded any direct genetic origin of the Spanish ‘Garnacha’, or Sardinian ‘Cannonau’ from the ‘Vernaccia’ -‘Granaccia’ Sardinian group. Structure analysis split the samples analysed into three clusters (K = 3). The first two clusters corresponded to the cultivated samples, while the wild accessions were in the third cluster. Based on this information, we can exclude any direct origin of the ‘Cannonau’ - ‘Garnacha’ group from the wild grapevines analysed and distributed on Sardinia.

Evidence for a Sympatric Origin of Ribolla gialla, Gouais Blanc and Schiava cultivars (V. vinifera L.)

Ribolla gialla is an autochthonous grape variety cultivated in Friuli Venezia Giulia (Italy) and in Slovenia, and probably originated in Eastern Central Europe. Just like Ribolla gialla, the Gouais blanc and Schiava cultivars also appear to have originated in the same geographical area. To verify this hypothesis, a pool of varieties probably sharing the same historical and geographical origin were taken into account. The fingerprinting, by 35 SSR loci, of Gouais blanc, Ribolla gialla, Schiava grossa and Schiava lombarda (synonyms of Schiava bresciana), is presented and the correlation among historical, geographical and genetic information of these cultivars was investigated. Gouais blanc and Traminer, already suggested as key varieties in the development of European grape diversity and as parents for some French varieties, interestingly enough seems to be linked to Ribolla gialla. The putative parentage was verified using 58 microsatellite markers. The genetic results suggest a common geographical origin for Gouais blanc, Ribolla gialla and the Schiava group: these cultivars appear to be related through sympatric origin. The hypothesis of Ribolla gialla as a progeny of Gouais blanc and Traminer was ruled out. The data proved a second-degree relationship between Gouais blanc and Ribolla gialla and a third or more distant degree of relationships between Ribolla gialla and Traminer.

Analysis of Genetic Structure of Twelve Sicilian Grapevine Cultivars

In recent years, Sicilian viticulture has shown a significant growth with a remarkable increase both in quantity and in quality. Sicily is a large wine region in Italy and its wines are already known on the international markets. Several autoch-thonous grapevine cultivars have been preserved in Sicily, but the Island genetic resources are still almost unexploited due to the lack of genetic characterization. The aim of the present study is to investigate molecular characteristics of Sicilian cultivars; evaluate probable cases of synonymies and false attributions, and preserve Sicilian germplasm. A wide screening of 12 major Sicilian cultivars was carried out (?Carricante?, ?Catarratto comune? and ?lucido?, ?Nero d?Avola?, ?Nerello cappuccio?, ?Nerello Mascalese?, ?Perricone?, ?Grillo?, ?Grecanico?, ?Inzolia?, ?Corinto nero? and ?Frappato?). In this frame a total of 687 accessions were collected and analysed with SSR markers (6 to 21 SSR loci). Allelic profiles were obtained and compared with international databases, allowing verification of mistakes and false attributions. Results clarified several synonymies and false attributions, and highlighted the richness and variability of the Sicilian grapevine platform still present in the region. Furthermore, these data provide a base for future studies.

Proteomic analysis among different aglianico ecotypes

Although molecular markers such as SSRs are extensively used for the identification of different vines, they often fail to discriminate the clones of a given cultivar. For these reasons, many attempts are made to find devices able to achieve this purpose. In this view, we evaluated the potential of proteomics in this field comparing the skin 2-DE profiles of berries belonging to three clones of the Italian cultivar ?Aglianico? (Taurasi, Taburno and Vulture) grown in the same field and considered in different moments to reduce the impact of environmental and seasonal variability. After a preliminary and completely automated gel alignment, we tested some multivariate statistical techniques to distinguish the three clones and to isolate the variables (i.e., the spots on the gels) responsible for the clonal discrimination. Although further analyses will be necessary to refine such an approach, the results are encouraging in order to identify markers useful in discriminating clones.