plants Article Recovery, Molecular Characterization, and Ampelographic Assessment of Marginal Grapevine Germplasm from Southern Umbria (Central Italy) Alessandra Zombardo 1,* , Paolo Storchi 1 , Paolo Valentini 1, Alice Ciofini 1, Daniele Migliaro 2 and Manna Crespan 2 1 Council for Agricultural Research and Economics, Research Centre for Viticulture and Enology, Viale Santa Margherita 80, 52100 Arezzo, AR, Italy; [email protected] (P.S.); [email protected] (P.V.); alice.ciofi[email protected] (A.C.) 2 Council for Agricultural Research and Economics, Research Centre for Viticulture and Enology, Viale 28 Aprile 26, 31015 Conegliano, TV, Italy; [email protected] (D.M.); [email protected] (M.C.) * Correspondence: [email protected]; Tel.: +39-057-535-3021 Abstract: The protection of grapevine biodiversity and the safeguarding of genetic variability are certainly primary and topical objectives for wine research, especially in territories historically devoted to viticulture. To assess the autochthonous germplasm of three different districts of Southern Umbria (Central Italy), the plant material of 70 grapevines retrieved from reforested land plots or old vineyards was collected, and their genetic identity was investigated using 13 microsatellite markers (SSR). The results revealed the presence of 39 unique genotypes, divided into 24 already-known cultivars and 15 never-reported SSR profiles. Most of the grapevine accessions were then vegetatively Citation: Zombardo, A.; Storchi, P.; propagated and cultivated in a vineyard collection both to be protected from extinction and to be Valentini, P.; Ciofini, A.; Migliaro, D.; evaluated at the ampelographic level. Overall, this work emphasizes the need for recovering the Crespan, M. Recovery, Molecular threatened genetic variability that characterizes minor neglected grapevine cultivars or biotypes of Characterization, and Ampelographic Southern Umbria germplasm, and the requirement to revalue and exploit the more valuable genetic Assessment of Marginal Grapevine resources to enhance the local agri-food economy. Germplasm from Southern Umbria (Central Italy). Plants 2021, 10, 1539. Keywords: Vitis vinifera; germplasm recovery; genetic identification; SSR markers; ampelography https://doi.org/10.3390/plants10081539 Academic Editor: Mario Ciaffi 1. Introduction Received: 28 June 2021 Umbria is one of the smallest regions in the central part of the Italian Peninsula and is Accepted: 19 July 2021 Published: 27 July 2021 characterized by a landlocked landscape of rolling hills. Because of its pivotal position, Um- bria always had important historical roles, often being a territory of disputes between the Publisher’s Note: MDPI stays neutral different neighboring domains, but above all, it was a key hub for major commercial routes with regard to jurisdictional claims in and agricultural trades, in both north–south (from and to Rome) and east–west directions published maps and institutional affil- (between the Adriatic and Tyrrhenian Seas). Umbria has developed a well-established iations. winemaking tradition that dates to the Etruscan period and persists to this day due to its favorable climate [1]. Until the recent past, wine production in this region was exclusively for family use and was primarily represented by mixed viticulture [2]. Throughout the 1950s, in fact, the most common training system still used was “viti maritate” (or “arbusta” according to Plinio il Vecchio), in which the vines are grown on other supporting trees Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. (e.g., poplars) within a promiscuous cropping system [3]. Afterward, starting from the This article is an open access article 1960s, there was a decisive change that oriented viticulture towards intensive monoculture distributed under the terms and and quality winemaking, thanks to the adoption of modern agronomic and oenological conditions of the Creative Commons techniques [4]. Attribution (CC BY) license (https:// Currently, the wine-growing acreage of Umbria is about 12,000 hectares [5] with two creativecommons.org/licenses/by/ denomination of controlled and guaranteed origin (DOCG) and 13 denomination of con- 4.0/). trolled origin (DOC) wines, which are rather appreciated on the national and international Plants 2021, 10, 1539. https://doi.org/10.3390/plants10081539 https://www.mdpi.com/journal/plants Plants 2021, 10, 1539 2 of 18 Plants 2021, 10, 1539 2 of 16 Currently, the wine‐growing acreage of Umbria is about 12,000 hectares [5] with two denomination of controlled and guaranteed origin (DOCG) and 13 denomination of con‐ trolled origin (DOC) wines, which are rather appreciated on the national and international markets. The main cultivated variety is Sangiovese (20% of the totaltotal area),area), followedfollowed byby TrebbianoTrebbiano ToscanoToscano (12%), (12%), Grechetto Grechetto (11%),(11%), MerlotMerlot (10%),(10%), SagrantinoSagrantino (7%),(7%), andand CabernetCabernet Sauvignon (4%).(4%). Despite the limited availability ofof autochthonousautochthonous cultivarscultivars inin thethe officialofficial productiveproductive panorama, the the Umbrian Umbrian viticulture viticulture retains retains a huge a huge biodiversity biodiversity heritage heritage [6]. [ As6]. Asa matter a matter of offact, fact, several several minor minor grapevine grapevine varieties varieties (belonging (belonging to to known genotypes oror showingshowing aa uniqueunique fingerprinting)fingerprinting) cancan easilyeasily bebe foundfound inin abandonedabandoned reforestedreforested landland plotsplots or inin oldold vineyards,vineyards, andand somesome historicalhistorical documentsdocuments describedescribe certaincertain spontaneousspontaneous vinesvines varietiesvarieties inin wooded wooded areas areas called called “Viti “Viti vicciute” vicciute” [7 ].[7]. Moreover, Moreover, there there is evidenceis evidence that that several sev‐ autochthonouseral autochthonous varieties varieties are still are grownstill grown locally, locally, although although unnamed unnamed and neitherand neither identified iden‐ nortified described nor described [8]. The [8]. real The origins real oforigins the different of the grapevinedifferent grapevine varieties historically varieties historically cultivated incultivated Umbria arein Umbria still unclear, are still and unclear, the presence and the of presence sparse wild of sparse vines (aswildVitis vines vinifera (as VitisL. subsp. vinif‐ sylvestrisera L. subsp.) is reported sylvestris [)9 is]. reported [9]. InIn thethe period 2013–2020, a research project fundedfunded by the Umbria Region was devel-devel‐ oped to protect the traditional germplasmgermplasm ofof Umbria.Umbria. Several inspections werewere carriedcarried outout in 12 municipalities included in Alto Orvietano (southwest), Colli Martani (south-central), in 12 municipalities included in Alto Orvietano (southwest), Colli Martani (south‐central), and Valnerina (southeast) territories of both the provinces of Perugia and Terni in Southern and Valnerina (southeast) territories of both the provinces of Perugia and Terni in South‐ Umbria (Figure1) to find vines of possible interest in private farms, in old and abandoned ern Umbria (Figure 1) to find vines of possible interest in private farms, in old and aban‐ vineyards, or in wooded areas no longer devoted to agriculture. This work aimed to search, doned vineyards, or in wooded areas no longer devoted to agriculture. This work aimed identify, rescue from genetic erosion, and revive some local grapevine varieties belonging to search, identify, rescue from genetic erosion, and revive some local grapevine varieties to fragile and disadvantaged agricultural districts that are slowly recovering thanks to the belonging to fragile and disadvantaged agricultural districts that are slowly recovering economic boost of food and wine tourism [10]. thanks to the economic boost of food and wine tourism [10]. Figure 1. Geographical location and list of the 12 municipalities of Southern Umbria where the 70 grapevine samples Figure 1. Geographical location and list of the 12 municipalities of Southern Umbria where the 70 grapevine samples included in the study were recovered. The color refers to the three territories considered: Alto Orvietano (in red), Colli includedMartani (in in thegreen), study and were Valnerina recovered. (in light The blue). color In refers brackets: to the abbreviation three territories of the considered: two provinces Alto of Orvietano Umbria, (inPG red), = Perugia; Colli MartaniTR = Terni. (in green), and Valnerina (in light blue). In brackets: abbreviation of the two provinces of Umbria, PG = Perugia; TR = Terni. 2. Results 2. Results 2.1. Genetic Characterization of Grapevine GermplasmGermplasm The molecularmolecular analysesanalyses werewere performedperformed onon 7070 grapevinegrapevine samplessamples collectedcollected inin threethree different districtsdistricts located located in Southernin Southern Umbria Umbria (Alto (Alto Orvietano, Orvietano, Colli Martani,Colli Martani, and Valner- and ina). Thirty-nine genotypes were found (Table1), with 52 samples belonging to 24 known grapevine varieties and 18 samples showing 15 never-reported SSR profiles (Table2). Plants 2021, 10, 1539 3 of 16 Table 1. List of the 70 grapevine samples grouped by their genotype. Original locations (municipality and province), berry color, true-to-type prime name, SSR profile ID number, and Vitis International Variety Catalogue [11]