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.

Materials and methods

1. 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.

Table 1. Samples obtained during a sampling campaign in the Marche region. VIVC: Vitis International Variety Catalogue; IC: Italian Catalogue.

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Sample name	Recovery or Sampling site	Variety name	VIVC variety number	IC code
Bianca (D'Onofrio)	Pedaso	Afus Ali (Regina)	122	527
Uva bianca (Zaghi)	Sirolo	Alfredo Marchetti
Negrì o Neretto di Piobbico (Ulivello)	Sarnano	Barbera	974	19
Rossa acino ovale (Rossi)	Falerone
Bersigana	ASSAM repository	Bersigana
Cacciù nero	ASSAM repository	Besgano nero	1284
Bianchetta	ASSAM repository	Bianchetta marchigiana
Pagadebito (Spreca)	Lapedona	Bombino bianco/Passerina	1533	32/181
Passerina F9	ASSAM repository
Uva bianca 1907 (Amadio)	Campofilone
Uva D'oro	ASSAM repository
Moscato nero (Ubaldi)	Montefalcone App.	Cardinal	2091	507
Bianca fienile (Silvestri)	Cossignano	Chasselas blanc (Chasselas dorato)	2473	509
Morettone (Remia)	Montegiorgio	Ciliegiolo	2660	62
Cimiciola F38	ASSAM repository
Gaglioppa (Capecci)	Ripatransone
Morettone	ASSAM repository
Morettone (Capecci)	Ripatransone
Centenaria (Sgariglia)	Acquaviva	Cornichon blanc (Pizzutello bianco)	16448	524
Cocacciara	ASSAM repository	Cocacciara
Fondazione fico (o pozzo?)	Montefiore dell'Aso
Pianta 2 porcile (Beato)	Montelparo
Famoso	Urbino (PU)	Famoso marchigiano
Fava	ASSAM repository	Fava
Forcese (Capecci)	Ripatransone	Forcese
Forcese (Ubaldi)	Montefalcone App.
Forconese	ASSAM repository
Cotrognone	ASSAM repository	Garofanata	24957	463
Moscatellone (Duri)	Serrapetrona
Uva Bianca (Serboni)	Serrapetrona
S. Maria/Luglia (Ulivello)	Monte Urano	Italia	5582	514
Tintorino (Montalbini)	Arcevia	Jacquez	5627
Uva Luglia (Ulivello)	Montelparo	Koenigin der weingarden (Regina dei vigneti)	6350	528
Luglia/Lugliola/S. Maria (Milanesi)	Montefalcone App.	Luglienga bianca (Sant’Anna di Lipsia)	6982	544
Bianca strada (Orlandi)	Villa Tara	Maceratino	7023	124
Fondazione strada grande	Montefiore dell'Aso
Trebbiano (Spreca)	Lapedona
Gallioppo (Silvestri)	Cossignano	Maiolica	7136	126
Gallioppo 75	ASSAM repository
Gallioppo GE	ASSAM repository
Gallioppo GE F12 I9 P2	ASSAM repository
Malvasia bianca lunga (La Pila)	Montegiorgio	Malvasia bianca di Candia	23555	131
Malvasia? (Montalbini)	Arcevia
P1 (Scarabotti )	Recanati
P2 (Scarabotti)	Recanati
San Niccolò TOR 1 (ITAS Vivarelli)	Fabriano
San Niccolò TOR 2 (ITAS Vivarelli)	Fabriano
San Niccolò TOR 4 (ITAS Vivarelli)	Fabriano
Sangiovese (ITAS Macerata)	Macerata	Malvasia di Casorzo	7264	134
Bianca resistente (Anselmi)	Monte San Martino	Malvasia istriana	7269	138
Premotico (Milanesi)	Montefalcone App.	Maturano bianco	23347	424
Ulpetta nuova (Sgariglia)	Acquaviva
Ulpetta vecchia (Sgariglia)	Acquaviva
Uva cane	ASSAM repository
Melata Savini 2 (Silvestri)	Cossignano	Melata
(Uva) Melata	ASSAM repository
Granarello	ASSAM repository	Merlot	7657	146
Rossa foglia (Mattei Verde)	Arcevia	Montepulciano	7949	150
Chiapparù	ASSAM repository	Montonico bianco	7960	151
Uva Regno	ASSAM repository
(Uva) Moie	ASSAM Petritoli	Morgentino
Uva Moscata p 2 Rossi	Falerone	Moscato Cerletti
Bianca (Zanoni)	Pedaso	Muscat of Alexandria (Zibibbo)	8241	343
Moscatello Francese (Ulivello)	Monte Urano
Pianta secolare (Maranesi)	Petritoli
Bianca Chiesa S. Domenico	Ancona	Moscato di Terracina	8053	281
Malvasia Candia (La Pila)	Montegiorgio	Moscato giallo	8056	154
Moscatello N.	Montefalcone loc. Faveto	Muscat rouge de Madere	8249
Moscianino	ASSAM repository	Moscianello	26683	889
Bianca	ASSAM repository	Mostosa	8075	157
Cacciù bianco 78/6	ASSAM repository
Cacciù bianco (Botticelli)	Montottone
Cacciu bianco (Spreca)	Lapedona
Fondazione casa	Montefiore dell'Aso
Mostosa 49	ASSAM repository
(Uva) nera rada	ASSAM repository	Nera rada
Uva rossa (Iena)	Porto S. Elpidio	Nerello mascalese	8480	165
Occhio nero	ASSAM repository	Occhio nero
Vissanello (Orlandi)	Villa Tara	Pecorino	9072	184
Pergolo (Vitali)	Montalparo	Pergolo
Pianta 4 (Giammarini)	Marina Palmense	Petit Bouschet	1619
Rossa foglia (Mattei Rossa)	Arcevia
Cornetta? (Ulivello)	Magliano di Tenna	Pizzutello nero	9524
Pianta 1 (Giammarini)	Marina Palmese	Plavina crna	9557	893
Pianta 2 (Giammarini)	Marina Palmese
Cacciù nero	ASSAM repository	Ripanea
Uva Rossa (Serboni)	Serrapetrona
Balsamina	ASSAM repository	Sangiovese	10680	218
Pianta 1 entrata (Beato)	Montelparo
Sangiovese	Ripatransone
Uva Rosata pianta 3 (Rossi)	Falerone
Rossa croccante	ASSAM repository	Scrocchiona
Ciciu	ASSAM repository	Sgranarella	26656	900
Ciciu F42	ASSAM repository
Cimicino	ASSAM repository
Pianta 3 bosco 1 (Beato)	Montelparo
Pianta 4 bosco 2 (Beato)	Montelparo
Pianta 5 bosco 3 (Beato)	Montelparo
Grugnintì (Fattobene) campo	Pollenza	Tenerone
Grugnintì (Fattobene) argine	Pollenza
Tinturina (Ubaldi)	Montefalcone App.	Terrano	12374	233
Uva nera (Virgili)	Monte San Martino
(Uva) torella (Piermarini)	Ortezzano	Torella
San Niccolò DA 1 (ITAS Vivarelli)	Fabriano	Trebbiano toscano	12628	244
Uva Fico	ASSAM repository
Vite Bastarda (Ulivello)	Monte Urano	Uva Tosca	12836	249
Cacciù (Capecci)	Ripatransone	Vaccaro
Vaccaro	ASSAM repository
Vaccù	ASSAM repository
Bianca precoce (Colasanti)	ASSAM repository	Verdicchio	12963	254
Vummì	ASSAM repository
Bordoletto (Duri)	Serrapetrona	Vernaccia di S. Gimignano	12998	261
Brugentile	ASSAM repository	Vernaccia nera grossa	23042	415
Brugentile N1	Cerreto d’Esi
Brugentile Casanao 2	Coccore
Uva francese (Ulivello)	Fermo	Villard blanc	13081
Uva Bianca pianta 1 (Rossi)	Falerone
Bianca resistente (Cossignani)	Montefiore dell'Aso
Bianca (Potentini)	Porto Recanati
Uva Bianca (Marchetti)	Cupra Marittima	Zunek	17739
Zivì (Milanesi)	Montefalcone App.
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.

Figure 1. Sites of recovery in Marche region of the 18 grapevine varieties lacking the SSR profile in the VIVC, which are not yet enrolled in the Italian catalogue and in bold in Table 1.

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Circle colours correspond to accession berry colour, black (red circles) or white (yellow circles).

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.

Genomic DNA was roughly extracted and genotyped with 12 nuclear SSR (nSSR) markers, including the nine used internationally for grapevine identification (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) (Maul et al., 2012), plus ISV2 (VMC6e1), ISV4 (VMC6g1) and VMCNG4b9 (Migliaro et al., 2013). Fluorescent primers and an ABI3130xl genetic analyser (Applied Biosystems, Foster City, CA) were used to produce the nSSR profiles. Allele calling was performed with GeneMapper software version 5.0, with a homemade bin set obtained from reference varieties. The SSR profiles of all genotyped samples were identified using the CREA Viticulture and Enology molecular database, the available literature and the Vitis International Variety Catalogue (VIVC, http://www.vivc.de).

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.

2. 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) and the Sicilian Visparola (Carimi et al., 2010; De Lorenzis et al., 2014). Scacco was also analysed by Pastore et al. (2020) as a cultivar grown in Emilia Romagna where it is known under the synonym Rossola (Tebano). Therefore, this genotype was shown to have a range spreading from southern to northern Italy. Given that the only available ampelographic description for this variety is Crepolino, and that previous pedigree relationships are related to Visparola (D'Onofrio et al., 2021), the combination of these two names, Crepolino/Visparola, is used hereafter for this genotype.

Excluding the eight additional varieties listed in Table S1 from the computation, three chlorotypes, A, C and D, were found with large differences in frequency: type D was prevalent (75.8 %; 50/66), followed by type A (21.2 %; 14/66) and type C (only 3.0 % 2/66).

SNP pruning performed with ASSIsT software retained 8,770 SNP out of 18,071 (48.53 %): 3,407 were classified as Robust (18.9 %), 2,784 as OneHomozygRare_HWE (15.4 %) and 2,579 as OneHomozygRare_NotHWE (14.3 %). The 8,770 SNP profiles related to the 74 unique varieties are reported in Table S3.

2.1. 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.

Table 3. Duos: parent-offspring (PO) relationships inferred with PLINK parameters and Mendelian inconsistencies (MI).

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First variety	Second variety	PLINK parameters				MI	Reference literature
		Z0	Z1	Z2	PI_HAT
Crepolino/Visparola	Famoso marchigiano	0.0109	0.9853	0.0038	0.4964	6	present paper
	Forcese	0.0237	0.8722	0.1041	0.5402	13	present paper
	Maiolica	0.018	0.982	0	0.491	10	D'Onofrio et al. (2021)
	Minutolo	0.0146	0.9551	0.0303	0.5079	8	Lacombe et al. (2013)
	Vesprino	0.0218	0.9594	0.0188	0.4985	12	present paper
	Vulpea	0.0218	0.8395	0.1387	0.5584	12	D'Onofrio et al. (2021)
Famoso marchigiano	Garofanata	0.0091	0.942	0.0489	0.5199	5	present paper
Fava	Gabbavolpe	0.0218	0.9506	0.0275	0.5028	12	present paper
Forcese	Moscianello	0.0218	0.9612	0.017	0.4976	12	present paper
Garganega	Empibotte	0.0255	0.8927	0.0818	0.5282	14	Crespan et al. (2008)
	Forcese	0.0164	0.91	0.0736	0.5286	9	present paper
	Malvasia bianca di Candia	0.0109	0.925	0.0641	0.5266	6	Di Vecchi-Staraz et al. (2007)
	Marzemina bianca	0.0309	0.9613	0.0077	0.4884	17	Crespan et al. (2008)
	Montonico bianco	0.0109	0.9279	0.0611	0.5251	6	Crespan et al. (2008)
	Pergolo	0.0091	0.9202	0.0707	0.5308	5	present paper
	Somarello rosso	0.0109	0.8704	0.1187	0.5539	6	Di Vecchi-Staraz et al. (2007)
	Trebbiano perugino	0.0291	0.9517	0.0192	0.495	16	present paper
	Trebbiano toscano	0.0182	0.9815	0.0003	0.4911	10	Di Vecchi-Staraz et al. (2007)
	Vernaccia nera grossa	0	1	0	0.5	9	present paper
Maiolica	Negroamaro	0.0146	0.9577	0.0277	0.5066	8	D'Onofrio et al. (2021)
Montepulciano	Bombino bianco	0.0218	0.9393	0.0389	0.5085	12	Lacombe et al. (2013)
Sciaccarello	Bersigana	0.0146	0.9633	0.0222	0.5038	8	present paper
	Caloria	0.0237	0.95	0.0263	0.5013	13	Di Vecchi-Staraz et al. (2007)
	Famoso	0	1	0	0.5	7	D'Onofrio et al. (2021)
	Tenerone	0	1	0	0.5	13	present paper
	Livornese/Rollo	0.0127	0.9348	0.0524	0.5198	7	Di Vecchi-Staraz et al. (2007)
	Muscat rouge de Madere	0.0109	0.9891	0	0.4946	6	Di Vecchi-Staraz et al. (2007)
Sangiovese	Capibianchi	0.0218	0.9513	0.0268	0.5025	12	Di Vecchi-Staraz et al. (2007)
	Foglia tonda	0.02	0.9207	0.0593	0.5197	11	Crespan et al. (2008)
	Scrocchiona	0.0197	0.9803	0	0.4901	11	present paper
Semidano	Bianchetta marchigiana	0.0164	0.9016	0.082	0.5328	9	present paper
	Drupeggio	0.0146	0.9646	0.0208	0.5031	8	present paper
	Nuragus	0.0146	0.9349	0.0505	0.518	8	present paper
	Occhio nero	0.0091	0.9485	0.0424	0.5167	5	present paper
Torella	Drupeggio	0.0255	0.9252	0.0493	0.5119	14	present paper
Mostosa	Trebbiano abruzzese	0.0182	0.8962	0.0856	0.5337	10	present paper
	Vaccaro	0.0273	0.8334	0.1393	0.556	15	present paper
Verdicchio	Maceratino	0.02	0.976	0.0039	0.492	11	Lacombe et al. (2013)

Expected values for PO relationships: Z0 = 0, Z1 = 1, Z2 = 0, PI_HAT = 0.5.

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.

Figure 2. Distribution of Mendelian inconsistencies computed on pairs of samples in the larger set of 192 unique genotypes.

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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.

Figure 3. Distribution of Mendelian inconsistencies computed on first degree related genotypes combined in all possible parent-parent-offspring trios.

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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-parent-child relationships used as references (Table 4 and Figure 4).

Table 4. Trios selected by combining PLINK parameters data for PO relationships using 8770 SNP ASSIsT-selected, 12 SSRs and Mendelian inconsistencies (MI).

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Offspring	First candidate	Second candidate	PLINK parameters				MI	Reference literature
			Z0	Z1	Z2	PI_HAT
Alfredo Marchetti	Maturano bianco	Maturano bianco	0.0036	0.2891	0.7073	0.8518	2	present paper
Bombino nero	Bombino bianco		0.0091	0.9459	0.045	0.518		Bergamini et al. (2016)
		Quagliano	0.0237	0.9461	0.0302	0.5033
	Bombino bianco	Quagliano					18
Ciliegiolo	Sangiovese		0.0218	0.978	0.0002	0.4892		Di Vecchi-Staraz et al. (2007)
		Muscat rouge de Madere	0.0237	0.9596	0.0168	0.4965
	Sangiovese	Muscat rouge de Madere					24
Forcese	Garganega		0.0164	0.91	0.0736	0.5286		present paper
		Crepolino	0.0237	0.8722	0.1041	0.5402
	Garganega	Crepolino					22
Incrocio Bruni 54	Verdicchio		0.0382	0.9361	0.0257	0.4937		present paper
		Sauvignon	0.0255	0.9672	0.0072	0.4909
	Verdicchio	Sauvignon					35
Lacrima	Aleatico		0.0182	0.8999	0.0819	0.5319		D'Onofrio et al. (2021)
		Nera rada	0.0219	0.9064	0.0718	0.5249		present paper
	Aleatico	Nera rada					22
Morgentino	Forcese		0	1	0	0.5		present paper
		Sciaccarello	0.0255	0.9275	0.047	0.5108
	Forcese	Sciaccarello					26
Reference trios
Manzoni bianco	Pinot		0.0109	0.9871	0.002	0.4955		Grando and Frisighelli, 1998
		Riesling weiss	0.0401	0.8672	0.0927	0.5263
	Pinot	Riesling weiss					22
Raboso veronese	Raboso Piave		0	1	0	0.5		Crespan et al. (2006)
		Marzemina bianca	0.0252	0.9748	0	0.4874
	Raboso Piave	Marzemina bianca					31
Vitouska	Malvasia bianca lunga		0.0273	0.8543	0.1184	0.5456		Crespan et al. (2007)
		Glera	0.0164	0.9155	0.0682	0.5259
	Malvasia bianca lunga	Glera					24

Figure 4. Reconstruction of the pedigree of the Marche varieties.

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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.

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.

3. 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).

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).

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Pairwise distances were computed using the Kimura 2-parameter method. The analyses were conducted in MEGA X.

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 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 (Raimondi et al., 2020; 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, 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) as having a long list of synonyms, like Montecchiese, Greco Maceratese, Greco Montecchiese, Matelicano, Ribona and Verdicchio sirolese among others. This long list testifies to its ancient and diffuse presence in the Marche region. Its resemblance to Verdicchio had already been noted both for its ampelographic and oenological traits; therefore, the molecular data confirm previous observations. Maceratino is currently quite successful as a result of new interest in the “Ribona” appellation.

Mostosa and Trebbiano abruzzese are morphologically very similar and shown to be PO related; Mostosa is also first degree related to Vaccaro. Little is known about Vaccaro: it was cited in the Ampelographic Bulletin number XVI (1883) as being one of the varieties of the Ascoli Piceno province, and oral testimonies have referred to this variety as being grown in the Campofilone and Ortezzano areas.

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.

Combined clusters and second-degree relationships are the result of Garganega, Crepolino/Visparola and Sciaccarello crossed in different combinations - often with still unknown vines - that gave rise to new varieties; for example, Forcese is the offspring of Garganega and Crepolino/Visparola; in turn Forcese was crossed with Sciaccarello to produce Morgentino. Morgentino is listed in the Ampelographic Bulletin number XVI (1883) as Brugnentino, Moglia or Uva Moglia in Montelparo area, Servigliano, Santa Vittoria in Matenano and other synonyms in Ascoli Piceno province.

Garofanata, described during the ampelographic exhibition held in Ancona in 1872 (De Bosis, 1873), was shown to be second degree-related to Crepolino/Visparola through Famoso marchigiano.

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.

Acknowledgements

This research was funded by ASSAM, Marche Region Agency for Agri-food Sector, under the Regional Law of 3 June 2003, no. 12 “Protection of animal and plant genetic resources of the Marche region”.

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