Grapevine (Vitis vinifera L.) varietal assortment and evolution in the Marche region (central Italy)

Italy is one of the richest countries in terms of grapevine varietal assortment, and the Marche region in central Italy has an ancient winegrowing tradition. Increasing interest in autochthonous grapevine varieties prompted efforts to recover and identify local minor germplasm also in this region, and to search for pedigree relationships and determine the evolution of varietal assortment. In the present study this was done using nuclear and chloroplast microsatellite markers and SNP markers from the Vitis18kSNP chip. Eighteen new genotypes were found, of which seventeen belonged to well-known, less known and even unknown vines found to be specific to the Marche region. The rearranged pedigree highlighted the complex intertwining relationships between Marche varieties. Second-degree relationships were also derived. Some minor Marche varieties have increased the number of parent-offspring related members of Garganega, Sangiovese, Crepolino/Visparola and Sciaccarello, which are varieties already recognised as founders or recurrent parents of many Italian cultivars. Crepolino/Visparola was shown to be a key variety in the evolution of the Marche varietal assortment, having played the role of parent and grandparent, as explained by the presence of this genotype in the region in ancient times. Surprisingly, Semidano, a well-known Sardinian variety, played a clear role as parent or grandparent of three minor Marche varieties, testifying to its presence in central Italy in ancient times. Incrocio Bruni 54 was confirmed as an offspring of Sauvignon and Verdicchio, as declared by the breeder. The parentage of Lacrima was completed: this variety was found to be a spontaneous cross between Nera Rada and Aleatico. A dendrogram of genetic similarity clearly resembled the groups defined with the pedigree reconstruction and it gave an indication of the genetic similarity of the varieties excluded from the strictest parentage links. a c t


INTRODUCTION
Italy is one of the richest countries in terms of grapevine varietal assortment, with almost 600 cultivars used in wine production registered in the Italian National Catalogue of Grapevine Varieties (http://catalogoviti.politicheagricole.it). The origin of the cultivated varieties can be determined by reconstructing their kinship; indeed, in the wine supply chain, describing a wine through the synergy between cultivation, quality and cultural issues is becoming increasingly important. The Marche region in central Italy and overlooking the Adriatic Sea has an ancient winegrowing tradition. In light of increasing consumer interest in local products, research on local grapevine germplasm and its conservation and exploitation play an important role in supporting the economic sustainability of farms. Viticulture is an important sector in the Marche region, with approximately 17,300 ha of vineyards and wine production of around one million hectolitres (https://www.assoenologi.it/ wp-content/uploads/2020/09/report_previsioni_ vendemmiali.pdf). Even if fifty-six grapevine varieties are suitable for cultivation in the Marche, only six are grown in over 70 % of the region's vineyards, namely Sangiovese, Montepulciano, Verdicchio, Trebbiano Toscano/Biancame, Passerina and Pecorino. The first two varieties are used to produce the renowned Rosso Piceno wines and the third the Verdicchio dei Castelli di Jesi wines; together, these three varieties account for more than 50 % of the Marche appellation wines.
Significant efforts have been made to recover and identify local minor grapevine germplasm in many Italian regions, like Apulia, Campania, Emilia Romagna, Friuli Venezia Giulia, Liguria, Sardinia, Sicily, Tuscany (Costantini et al., 2005;De Mattia et al., 2007;Torello Marinoni et al., 2009;Carimi et al., 2010;Crespan et al., 2011;De Lorenzis et al., 2014;Schneider et al., 2014;Mercati et al., 2016;D'Onofrio et al., 2016;Gristina et al., 2017;Pastore et al., 2020), and variety pedigrees have been reconstructed for the most widespread and renowned ones and for those having local importance, helping to unravel the intricate skein of the structure of Italian grapevine populations (Di Vecchi-Staraz et al., 2007;Ruffa et al., 2016;Crespan et al., 2020;Raimondi et al., 2020;D'Onofrio et al., 2021). Similar efforts have been made in the Marche region, because the resulting knowledge is very useful in science and has cultural and marketing appeal. Since 1990, ASSAM has recovered local grapevine genetic resources for their conservation, characterisation and potential future exploitation. Over the period 1990-2012, some local, national and international grapevine varieties were evaluated and the most interesting ones were registered in the Italian National Catalogue of Grapevine Varieties, like Vernaccia nera grossa in 2008 and Garofanata in 2013 (http://catalogoviti.politicheagricole.it). In 2014, a larger recovery programme was set up: more than one hundred grapevine samples were collected in the Marche region and genotyped with microsatellite (SSR) markers to speed up their identification. Indeed, classical ampelographic identification is often difficult due to high varietal variability, often combined with poor vine health. Later, rare and unexplored genetic resources were preserved in a dedicated germplasm repository; a search for verbal and written information on these poorly known grapevines was also made. Pedigree studies were then implemented on varieties cultivated in the Marche region, in which 12 nuclear and 8 chloroplast SSRs were combined with SNPs from the Vitis18kSNP array. The very large number of point mutations in the grapevine genome make SNP (Single Nucleotide Polymorphism) markers a powerful tool with many applications. Next generation sequencing provided hundreds of thousands of SNP markers in the whole Vitis spp. genome. A first Vitis chip array, the Vitis9kSNP, was developed in 2010 (Myles et al., 2010), with 9,000 SNP detected in 11 Vitis vinifera L. varieties and six wild Vitis species. The GrapeReSeq Consortium developed a new one, the Vitis18kSNP, obtaining around 18,000 SNPs from 47 V. vinifera varieties, 12 wild Vitis species and 5 Muscadinia rotundifolia varieties (Le Paslier et al., 2013). This tool is used in pedigree studies as it contributes to finding new relationships, and it confirms or questions those found using only SSRs (Myles et al., 2011;Ruffa et al., 2016;Laucou et al., 2018;De Lorenzis et al., 2019;Crespan et al., 2020;Raimondi et al., 2020;D'Onofrio et al., 2021). Indeed, the Vitis18kSNP surpasses SSRs, because it can provide information on thousands of points in the genome in just one analysis, making up for their very low polymorphism.

Plant material obtained during a sampling campaign in the Marche region, nSSR genotyping and varietal identification
One hundred and twenty-two vines were sampled in the Marche region and preserved in the ASSAM repository (Table 1); the sampling sites are indicated when available and reported in Figure 1 for the accessions not listed or lacking an SSR profile in the Vitis International Variety Catalogue (VIVC), or for those not enrolled in the Italian catalogue.
Often these vines did not have a varietal name and thus generic names were used for the samples, referring, for example, to a vine trait, like grape colour or shape, or to the plant owner's name.

Plant material selected for pedigree study
A selection of sixty-seven grapevine varieties from two Italian repositories (CREA Viticulture and Enology and ASSAM) were analysed for pedigree relationship reconstruction (Table 2). From the results of the sampling campaign carried out on Marche Vitis germplasm, 36 Vitis vinifera varieties used in wine production were selected and analysed for the reconstruction of pedigree relationships, along with other known grapevine varieties of interest to the Marche region (Aleatico, Garnacha tinta, Lacrima, Incrocio Bruni 54, Malvasia bianca lunga and Vernaccia nera); a total of 42 varieties were thus selected (in bold in Table 2). This list also includes 25 other varieties, which were selected for possible PO relationships by screening around 4,000 unique SSR profiles present in the CREA-Viticulture and Enology molecular database (partially published).  Table 1.
Circle colours correspond to accession berry colour, black (red circles) or white (yellow circles).

Zivì F41 ASSAM repository
The recovery site is indicated, when available. The prime names of the varieties in the VIVC are shown in the "Variety name" column, and, when different, the name as registered in the IC is in brackets. The names of the 18 varieties lacking the SSR profile in the VIVC and not yet enrolled in the IC are in bold.
The search for compatible trios (parents and offspring) and duos (parent-offspring) was done based on 9 to 12 nSSRs in the CREA Viticulture and Enology database with Cervus 3.0 (Kalinowski et al., 2007) and GenAlEx 6.5 software (Peakall and Smouse, 2012), and in the VIVC using the "Relationships based on nine microsatellites" tool. The varieties which proved to be possible members of trios or duos were included in the sample set. Termarina and Verano accessions belonging to the Sciaccarello variety were both included to evaluate possible SNP polymorphisms, given their big phenotypic differences in grape morphology; Termarina is a parthenocarpic somatic variant of Sciaccarello and Verano shows the seeded, wildtype form (Crespan et al., 2016). Three parentparent-child trios with well-established parentparent-offspring relationships were added for the evaluation of Mendelian incompatibilities and statistical comparisons: Manzoni bianco = Pinot x Riesling weiss (Grando and Frisinghelli, 1998;Cipriani et al., 2010.), Raboso Veronese = Raboso Piave x Marzemina bianca (Crespan et al., 2006), and Vitouska = Malvasia bianca lunga x Glera (Crespan et al., 2007).

Genomic DNA extraction and genotyping with chloroplast SSR and SNP markers for the pedigree study
Genomic DNA was extracted from young freeze-dried leaves from the samples listed in Table 2 using the QIAGEN DNeasy 96 Plant Kit (QIAGEN GmbH, Hilden, Germany) and according to the manufacturer's protocols with the following modifications: AP1 buffer was added with 1.6 % PVP40 (Sigma Aldrich) and the samples were incubated at 65 °C for 5 min; the DNA was eluted in milliQ water at 65 °C.
A check for correct sampling was performed on these DNAs using four nSSR (VVS2, VVMD5, VrZAG79 and VVMD28). Chlorotypes were assessed with eight chloroplast SSR markers (Arroyo-Garcia et al., 2006). Two multiplex PCR were organised using fluorescent primers and SSR allele calling was performed as described for the nSSRs.
All the samples were genotyped using the Infinium® II Vitis18k SNP array, which comprises 18,071 SNPs (GrapeReSeq Consortium, Illumina), following the Infinium® HD Assay Ultra protocol (Illumina Inc., San Diego, CA). The samples were scanned using an Illumina HiScan.

Data processing for parentage relationships
For the SNP data analysis, a no-call threshold of 0.15 was applied as a GenCall cutoff using GenomeStudio Genotyping Module v2.0 of ILLUMINA. The SNP selection was performed with ASSIsT (Automatic SNP ScorIng Tool) software (Di Guardo et al., 2015) v. 1.02, applying the default parameters for germplasm material. No pedigree or map information was given. A larger set of 192 unique genotypes, mainly comprising Italian varieties, was used to obtain a more consistent SNP classification into the groups obtained with the software; only Robust, OneHomozygRare_ HWE and OneHomozygRare_notHWE SNPs were retained, as reported in Crespan et al. (2021).

Mendelian inconsistencies
Mendelian inconsistencies between pairs of samples were computed on the larger set of 192 unique genotypes by searching for PO related varieties. First degree related genotypes were then combined in all parent-parent-offspring trios and related Mendelian inconsistencies were computed. Home-made algorithms were written in Excel Visual Basic for Application (VBA) to perform these computations.

Full-sib relationships
Possible full-sib relationships were evaluated using Colony software version 2.0.6.5 (July 30, 2018), which is freely available at https:// www.zsl.org/science/research-projects/software. Both nSSR and SNP markers were used as data input, and parentage relationships were inferred by combining PLINK information, 12 SSRs and Mendelian inconsistencies. The following main settings were applied: markers error rate 0.00001, no sibship prior indicator, one medium run, FL (full likelihood) analysis method and medium precision when calculating FL.

Genetic similarity
MEGA X software version 10.1.8 (Kumar et al., 2018) was used to obtain an unrooted dendrogram of genetic similarity using the 8,770 ASSIsTselected SNP markers. Pairwise genetic distances were computed using the Kimura 2-parameter method. Missing data were removed for each sequence pair, using the 'pairwise deletion' option.
A dendrogram of genetic similarity was obtained using the Unweighted Pair-Group Arithmetic Average Method (UPGMA). A bootstrap test of 2,000 replicates was used to define the percentage of replicate trees in which the associated genotypes clustered together; these values were shown next to the branches. Only branches with bootstrap values higher than 75 were taken into consideration.

RESULTS
In the first part of this study, a sampling campaign on the minor local grapevine germplasm grown in the Marche region was performed for vine cultivar identification supported by SSR profiles.
In the second part, the pedigree relationships of 42 varieties of interest to the Marche region (17 of them being new entries identified in the first part of this study) were evaluated also in comparison with an additional 25 potentially PO-related varieties based on 9-12 SSR markers, thus totaling 67 varieties.

Identification of the vines recovered during surveys 1990-2020
The 30  , and Rosciolo (used in the nearby Lazio region), and ii) Grugnintì is a synonym for the Tuscan Boggione rosso and Tenerone; Tenerone was chosen as the prime name for this genotype due to the ampelographic description given by Bandinelli et al. (2005).
No differences were found between the SNP profiles of Verano, the seeded form of Sciaccarello, and Termarina, the partenocarpic somatic variant.

Parentage relationships
In the pedigree studies on the sixty-seven grapevine cultivars, 66 different SSR and SNP profiles were found, thus further supporting the molecular synonymy between Sciaccarello and Termarina. The nSSR profiles and related chlorotypes of these 67 varieties and of the eight additional ones are reported in Table S1. All the varieties were univocally identifiable with the 14 SNP set selected by Laucou et al., 2018, except for Sciaccarello and Termarina, whose profiles are provided in Table S2.
The Scacco accession of CREA-Viticulture and Enology shared the same SSR profile as the Tuscan Crepolino described by Armanni et al. (2008) Table S3.

Duos and trios
The same pairs of first-degree related varieties were recognised using PLINK parameters and Mendelian inconsistencies computation (Table 3). The Z1 PLINK parameter was between 0.8334 and 1.
The distribution of Mendelian inconsistencies for all pairs of genotype combinations (computed on 192 unique genotypes) is shown in Figure 2: a clear Gaussian curve is represented, referring to non-PO related varieties. However, a well separated, small group of pairs outside the Gaussian curve and located on the left side of the figure can be seen; this small group is in strong agreement with the presence of PO relationships and shows the inconsistencies found for PO related varieties, with a maximum of 22 mismatching loci, while for the pairs inside the Gaussian distribution the Mendelian inconsistencies were from 53 onwards.
A complex network of first-and second-degree relationships was found. Table 3 shows that some varieties were found to be PO related to more than one other variety: Garganega shows the highest number of PO relationships (10), then Crepolino/Visparola (6), Sciaccarello (6), Semidano (4), Sangiovese (3) and Mostosa (2); seven pairs of PO related varieties were also found.
First degree related varieties, selected according to the MI on duos, were then compared in all possible parent-parent-offspring combinations and the MI were computed accordingly. The distribution of the MI on trios is reported in Figure 3.
A group of trios with a maximum of 37 MI was shown to be well separated from the others at 268 MI onwards; the three parent-parent-offspring combinations used as reference also fall into this little group (Table 4).
One selfing and six trios were established based on PLINK parameters for PO relationships combined with 12 SSR data and MI in comparison with the three well-established parent-parentchild relationships used as references (Table 4 and Figure 4).
Chlorotypes helped in some cases to establish the sexual role played by the parents in generating their offspring. Alfredo Marchetti was found  to be derived from selfing of Maturano bianco. Bombino nero was confirmed to be the progeny of Bombino bianco × Quagliano (Bergamini et al., 2016); Ciliegiolo was a spontaneous cross between Sangiovese and Muscat rouge de Madere; Forcese derived from Garganega × Crepolino/ Visparola; Incrocio Bruni 54 was confirmed as a cross between Verdicchio and Sauvignon; Lacrima derived from Aleatico × Nera rada, and finally Morgentino was a cross between Forcese and Sciaccarello.
No reliable full-sibs were found using Colony software.

Dendrogram of genetic similarity
A dendrogram of genetic similarity was produced by applying the UPGMA method on all the genotypes selected for parentage studies, including additional reference varieties for known trios, totalling 74 unique SNP genotypes; nine clusters were found with bootstrap values higher than 75 ( Figure 5).    Group A is the largest group (16 members), nine of the members being first or second degree related and the reference variety being Crepolino/ Visparola. Group G is the second largest group (14 members), with two reference varieties, Sangiovese and Sciaccarello. The group of Garganega, F, has 11 members; the group of Semidano, D, has 8 members and the smallest groups, B, C, E, H, I, have from 5 to 2 members.

DISCUSSION
Sixteen genotypes were shown to be local Marche varieties/vines which were previously either poorly known or completely unknown: Alfredo Marchetti, Bersigana, Bianchetta marchigiana, Cocacciara, Famoso marchigiano, Fava, Forcese, Melata, Morgentino, Nera rada, Occhio nero, Pergolo, Ripanea, Scrocchiona, Torella and Vaccaro. The rearranged pedigree highlighted the complex intertwining of relationships between the Marche varieties. No putative full sibs were found, indicating not only that there is a large number of missing vines necessary to complete the puzzle, but also the liveliness of local wine growers in selecting new varieties.
Some varieties already recognised as founders or recurrent parents of many Italian cultivars, like Garganega, Sangiovese and Sciaccarello (Di Vecchi-Staraz et al., 2007;Crespan et al., 2008;Lacombe et al., 2013), increased their PO related members. The long list of varieties which are PO related with Garganega testifies and confirms the role of founder that is played by this very ancient cultivar from northern to southern Italy. The molecular data supports previous findings related to Empibotte, Malvasia bianca di Candia, Montonico bianco, Somarello rosso, Trebbiano perugino, Trebbiano toscano and adds two local Marche varieties to the list, Pergolo and Vernaccia nera grossa. Very little information is available for Pergolo. Costanzo Felici da Piobbico (1525-1585, in Felici, 1986) cites a grapevine named "la pergola"; the Ampelographic Bulletin number XVI (1883) mentions the Pergolo as being one of the varieties spread over the Appignano, Venarotta and Ascoli Piceno area; and oral sources cite Pergolo as a variety found in Montelparo and Santa Vittoria in Matenano (Fermo province).
Vernaccia nera grossa, locally named Vernaccia Cerretana, was recovered in old tree lines in the municipality of Cerreto d'Esi (Macerata province) and neighbouring areas; it was enrolled in the Italian Catalogue in 2008. The oldest available citation of this variety is as a local cultivar in the The chlorotypes (in brackets) are codified in letters according to Arroyo-García et al. (2006). Solid lines indicate the links inferred with present molecular data or the confirmation of previous findings. Arrows show the cross direction when possible. Well-known information from the literature not provided in this paper is represented as dotted lines.
Ampelographic Bulletin number X (1877) by Professor Carlo Morbelli. It spread moderately thoughout the Fabriano area towards the end of the 19th century, then its importance waned, and it was neglected in the viticulture reconstitution phase after the Second World War.
Our data confirm the following varieties to be PO related with Sangiovese: Capibianchi and Foglia tonda, and add a new one, Scrocchiona.
Six varieties were PO related with Sciaccarello, of which the new entries were Bersigana and Famoso. The only citation of Bersigana grapes is very recent and comes from the poet Umberto Piersanti (2008). Famoso marchigiano was at risk of extinction, but was recovered from an old vineyard in Pesaro area by the winemaker Giancarlo Soverchia in the 1980s. Famoso marchigiano, described as a synonym of Uva della Madonna in 1872 (De Bosis, 1873), was briefly described in the Amphelographic Bullettin number VI (1876) as being one of the main, white-berried varieties of the Pesaro Urbino province.
Crepolino/Visparola was shown to be a key variety in the evolution of the Marche varietal assortment, having played the role of parent and grandparent, which can be explained by the ancient presence of this genotype in the region. Nowadays, it is highly threatened (only one vine was found during sampling not included in this study) in Marche, but some plants are still grown in Tuscany, Sicily and Emilia Romagna. Crepolino/Visparola is also PO related with Vulpea. Vulpea was already recognised as a parent of many other varieties, especially in the Friuli Venezia Giulia region (Crespan et al., 2020), as well as in Croatia (Žulj Mihaljević et al., 2020), and its country of origin is thought to be Austria. The presumed origin of Crepolino/Visparola is ascribed to Greece, because of its full-sib relationship with the Greek cultivar Augustiatis (D'Onofrio et al., 2021). Crepolino/Visparola could then have spread along two different routes from the south of the Balkans to Austria and from Sicily to the north of the Italian peninsula. The presence of its progeny in the Marche region supports the second route.
Semidano and Nuragus are two well-known Sardinian varieties which are, to our knowledge, not grown outside of the island. The first historical report on Semidano dates back to 1870, while Nuragus was cited for the first time in 1837 (Nieddu, 2011). The first-degree relationship between them is not surprising, also given their morphological resemblance. Even if inexplicable at present, molecular data give clear evidence of the role played by Semidano in the birth of Bianchetta marchigiana and Occhio nero, which are two Marche varieties, and of Drupeggio, a variety shared by the nearby regions of Tuscany, Lazio and Umbria, and which in turn is PO related to Torella. The place of origin of some Sardinian varieties is still unclear and debated; for example, Spergola, a variety grown in the northern part of the Italian Apennine area, was discovered to be synonymous with the more renowned Sardinian Vernaccia di Oristano, and recent pedigree studies confirm that this genotype was imported into Sardinia from the Italian mainland D'Onofrio et al., 2021).
No information is available on Bianchetta marchigiana. Occhio nero may correspond to an old Marche variety called Occhietto bianco, which is briefly described in the VII Ampelographic Bulletin (1877). The cultivar Torella (meaning 'little bull') was grown for a long time in the countryside of Ortezzano (Fermo province) by the Piermarini family. Its name refers to a Piermarini ancestor called "bull" ('toro' in the Italian language) due to his strength when carrying bags of wheat and also to the large and compact clusters of the grape, which comprises big, sweet and aromatic berries suitable for being dried.
The Alfredo Marchetti vine is a rare case of selfing. Its parent is Maturano bianco, a well-known variety autochthonous of the Latium region, and also grown in the Marche region under different names, like Uva d'oro, Premotico and Ulpetta. The trunk of this vigorous, ungrafted vine grows inside the walls of a noble house in Sirolo (Ancona) up to the second floor, and the roots reach below the underground tanks. It can be assumed that a grape seed brought by some animal, probably a bird, was dropped and generated this special vine.
Incrocio Bruni 54 was confirmed as an offspring of Sauvignon and Verdicchio, which was obtained in 1936, as declared by the breeder, and enrolled in the Italian catalogue in 1971. This result invalidates the claim in Cipriani et al. (2010) that Incrocio Bruni 54 is the offspring of Aleatico × Lacrima, because the accession considered in that paper,  FIGURE 5. Unrooted optimal dendrogram for 74 varieties using the UPGMA method and a bootstrap test of 2,000 replicates (related values are shown next to the branches).
namely 'Selezione Bruni 54' from the CREA-VE repository, did not correspond to the true Incrocio Bruni 54. In 2010, only 12 ha were cultivated with Incrocio Bruni 54 throughout the Marche region (ISTAT 2010), but interest in growing this cultivar is increasing due to the excellent structure and peculiar sensorial profile (spicy hints of aromatic herbs) of its wine, which is different to that of both its parents.
Maceratino was shown to be another progeny of Verdicchio and to probably be a spontaneous cross. First cited by Rastelli (1808) and Brignoli (1809) as Maceratese, Maceratino was described by Santini (1875)  The pedigree of Lacrima was completed: Aleatico had already been identified as one parent (D'Onofrio et al., 2021) and as a result of the chlorotype polymorphism in the present study, it was possible to determine its role as father; our data also identified the mother, Nera Rada, a previously unknown vine. It is worth noting that Aleatico is locally known as Vernaccia di Pergola. The muscat flavour of Lacrima was clearly inherited from Aleatico, which, in turn, is one of the numerous offspring of Moscato bianco. The survey conducted by the ampelographic commission in the late 1800s in the Marche region showed a widespread presence of Aleatico in all the provinces of this region. No information was found for Nera Rada. Nowadays Aleatico is mainly cultivated in the Cesano Valley and more precisely in the Pergola area (Pesaro and Urbino province); it is the main grape variety used in the "Pergola" appellation wines, and is characterised by a rose and cherry flavour. Crepolino/Visparola is a variety of central and southern Italy. It is PO related to the Marche varieties Famoso marchigiano, Forcese and Sgranarella, as well as to Maiolica (central Italy), and the Apulian Minutolo. These findings are evidence that Crepolino/Visparola was more common in the past than it is today. Maiolica is widespread in Tuscany, where it was rediscovered as Sanforte and enrolled a second time with this name in the Italian Catalogue.
The Marche Gallioppo is one of the recognised synonyms of Maiolica, and it was widespread in the Marche in the 19th century. Maiolica was shown to be PO related to Negroamaro, an autochthonous Apulian variety not cultivated outside that region; this link and others already reported by D'Onofrio et al. (2021) highlight that Maiolica was more widespread in southern Italy in the past. The link between Marche and Apulia is also clear from the synonymy between Bombino bianco (Apulia) and Passerina (Marche).
The nine groups in the dendrogram clearly resemble those suggested by pedigree reconstruction and indicate a genetic similarity in the varieties excluded from the strictest parentage links. Group A refers to the varieties found to be first or second degree linked to Crepolino/Visparola, except for Morgentino, which is clustered with its second parent, Sciaccarello. The inclusion of Glera, Malvasia bianca lunga and Vitouska is easily explained, because Glera is one of the numerous offspring of Vulpea (Crespan et al., 2020). Group A also comprises varieties that, at a first glance, seem completely unrelated, such as Lacrima and its parents; their association with the group suggests a missing, still unknown link.
Group G clearly assembles the same varieties already linked by strict parentage relationships to Sangiovese or Sciaccarello; most of them are commonly assigned to central Italy grapevine germplasm. Pecorino is the only one that escaped pedigree reconstruction; its place in the dendrogram suggests this variety also belongs to central Italy, even if there is a missing link.
Cluster D, with Semidano as the most representative variety, surprisingly groups two additional varieties beyond the expected ones: Melata and Zunek. Zunek has been recovered in the Marche as Zivì, a denomination present in the Ampelographic Bulletin number XI (1879) of a variety grown in the province of Macerata, which was was not held in high esteem.
Group C is another solid group which is 100 % supported by bootstrap values. Group C links Maturano bianco and its selfing progeny Alfredo Marchetti to Cocacciara, Fava and Gabbavolpe. In the Ampelographic Bulletin number XVI (1883) Cocacciara and Uva Fava are listed as varieties of the Ascoli Piceno and Fermo provinces.
Grero is the only stand alone variety.

CONCLUSIONS
One third of the ampelographic assortment of Marche is characteristic of this region. It originated partly from already well-known founders or main parents, like Sangiovese, Garganega and Sciaccarello, and partly from a recently discovered founder for this region, Crepolino/Visparola. A surprising link with the Sardinian Semidano was revealed by the molecular analyses, but no information is available at this time to explain this finding. Incrocio Bruni 54 was confirmed to be the progeny of Verdicchio and Sauvignon, as declared by the breeder.
Given previous experiences, like for Pecorino and more recently for Garofanata, we are confident that local grapevine biodiversity, with its territorial exclusivity and qualitative characteristics, can be a valuable resource for farms that need to create new marketing spaces within an increasingly demanding and competitive market. Shortly ASSAM and CREA will proceed with the ampelographic, agronomic and oenological characterisation of the still undescribed varieties to identify the best performing ones that may eventually be enrolled in the Italian Catalogue.