Thirty-nine grape accessions, traditional and historically mentioned in Albania, were genotyped

at 13 microsatellite (SSR) markers and observed for their morphological features with the aim of
characterizing and identifying the local grape diversity relevant for economic or historical
significance and for endangered germplasm conservation. Twelve of the 45 accessions examined
were found to be synonyms or somatic mutants, leaving 33 distinct genotypes. Attempts were then
made to verify the true identities of the accessions investigated and to determine their appropriate
denominations. This entailed comparing them with published allelic profiles and morphological
features of cultivars from Apulia and from surrounding areas linked historically to the region. While
confirming the identity of the major Apulian cultivars, further matches with varieties from other
Mediterranean regions were revealed. Approximately half of the Apulian cultivars investigated were
found to have a foreign counterpart mainly along the Adriatic Sea (Croatia), in Greece, or in other
southern Italian regions. The new synonymies found with cultivars traditional to other areas shed
light on the migration of cultivars following the settlement of colonies and the historical
establishment of Mediterranean trade routes.

Introduction
Albania is situated in the middle of Balkan Peninsula, jutting between Adriatic
and Ionian Seas, toward Italy and in the north of Greece. Grapevine were
cultivated in Albania in the Bronze Age. During the first century bc

. In the eighth century bc, Greek colonists settled on the Ionian coast of and
developed the emporion, trading stations for unloading goods, including wine. Under
Roman domination, ..continued to produce and trade wheat, wine, and olive oil
so that strategic ports were built to connect the area with the eastern Mediterranean. In
the Middle Ages, the sea played a key role: the Italian cities of Venice and Genoa, both
trading in ports, increased their influence. A further link with the eastern
Mediterranean was established during the Crusades. In the early and middle 1800s,
numerous grape varieties were inventoried in the region. During the great expansion of
viticulture to 300,000 ha in the second half of the 19th century, because of the phylloxera crisis in France, the main varieties planted were Uva di Troia, Primitivo, and Negro
amaro.

Viticulture now occupies 20000 ha in Albania (Instat Albania), from which 16000
Ha for industrial grapevine production with thirty nine cultivars currently
authorizes to be grown, including thirty nine local historical varieties.
Nevertheless, there are still numerous minor varieties grown in the different regions of
the country , in moderate or very limited quantities, that several programs aim to recover
and possibly exploit for commercial use. In the last few years, ten local ancient varieties
were officially registered in the Albanian Grape Variety Catalogue (La Notte et al. 20112012). Other threatened grapes maintained in field collections are under study.
Microsatellite or simple sequence repeat (SSR) genotyping, together with vine
morphological descriptions, are the most widely used approaches to characterize and
identify grape varieties (Sefc et al. 2009, This et al. 2011) and comprise a valuable tool
for the management of collected genetic resources (Ortiz et al. 2004, Laucou et al.
2011). In grapevines, nuclear SSRs reveal highly diverse allelic patterns, codominant
inheritance, and high repeatability and interlaboratory reproducibility due to the
simplicity of allele score adjustment (This et al. 2004). SSR profiles provide direct
comparison of genotype similarity at common loci, while plant morphology observations
support synonymy hypotheses and can be related to historical references.

Viticulture now occupies ~119,000 ha in Apulia (agri.istat. it), with 59 winegrape

cultivars currently authorized to be grown, including 22 local, historical varieties.
Nevertheless, there are still numerous minor varieties grown in the region, in moderate
or very limited quantities, that several programs aim to recover and possibly exploit for
commercial use. In the last few years, six local ancient varieties were officially registered
in the Italian Grape Variety Catalogue (La Notte et al. 2011-2012). Other threatened
grapes maintained in field collections are under study.
Microsatellite or simple sequence repeat (SSR) genotyping, together with vine
morphological descriptions, are the most widely used approaches to characterize and
identify grape varieties (Sefc et al. 2009, This et al. 2011) and comprise a valuable tool
for the management of collected genetic resources (Ortiz et al. 2004, Laucou et al.
2011). In grapevines, nuclear SSRs reveal highly diverse allelic patterns, codominant
inheritance, and high repeatability and interlaboratory reproducibility due to the
simplicity of allele score adjustment (This et al. 2004). SSR profiles provide direct
comparison of genotype similarity at common loci, while plant morphology observations
support synonymy hypotheses and can be related to historical references.

Using six microsatellites Thirty-nine varieties from Albanian varieties and without
symptoms of virus and virus-like disorders had been characterized

Twenty grape varieties from Apulia had been characterized by the six microsatellites of
international use (Zulini et al. 2002); the genetic origin of the local Malvasia nera was
also demonstrated (Crespan et al. 2008a); and among a large set of Italian grape
varieties, several cultivars from Apulia were investigated by SSRs with three to five
nucleotide core repeats (Cipriani et al. 2010).

In the present study, 45 traditional, historical cultivars from Apulia were examined for
their morphological and genetic profiles. Despite their economic significance in the
region, modern table-grape varieties issued from breeding programs were excluded from
this study, which focuses on the ancient germplasm. The study aimed to help catalog,
characterize, and identify local grape varieties of relevance for their economic or
historical significance and for endangered germplasm conservation.

Since intense cultivar migration has been demonstrated for the prominent grape
Primitivo (Maleti et al. 2004, Cal et al. 2008), published allelic profiles of cultivars from
neighboring and surrounding areas were compared with their Apulian counterparts in
order to discover any new synonyms and provide historical evidence on grape circulation.

Materials and Methods

Most of the investigated accessions are maintained in the grapevine collection of the
Research, Experimentation and Education Centre in Agriculture Basile Caramia (CRSFA)
at Locorotondo (Bari, Apulia). Several accessions (including presumed bud sports) were
located in commercial vineyards. The 45 accessions are listed in Table 1.
DNA was extracted from fresh shoot tissues sampled in the field, following the procedure
described by Thomas et al. (1993) with some modifications. Samples were genotyped
using a set of 13 SSR loci, nine of them developed as common markers for international

use under the projects GenRes 081 and GrapeGen06

(www1.montpellier.inra.fr/grapegen06/ page_summary/summary.php). The 13 markers
used were VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VVMD36,
VrZAG21, VrZAG62, VrZAG64, VrZAG67, and VrZAG79 (for detailed information on
markers see www.eu-vitis.de/index.php and www.vitisdb.it/descriptors/loci_descriptors).
PCR products were then analyzed on a 3130 Genetic Analyzer (Applied Biosystems,
Foster City, CA). Data were processed using GeneMapper Software (ver. 4.0; Applied
Biosystems), and alleles were defined by their size in base pairs, by comparison with the
standard size (GeneScan 500 LIZ, Applied Biosystems).
To evaluate the markers used, the following statistical parameters were calculated from
data on the 33 genotypes that were found to be unique in the set of 45 accessions
examined, using IDENTITY software (ver. 4.0; Centre for Applied Genetics, University of
Agricultural Sciences, Wien): numbers of alleles, observed and expected heterozygosity,
estimated frequency of null alleles, and probability of identity (PI) for the analyzed loci.
The SSR profiles obtained were then compared with those provided by Vitis genetic
databases, standardizing data to common true-to-type cultivars: the European Vitis
Database (www.eu-vitis.de/index.php), the nuclear microsatellite Greek Vitis Database
(gvd.biology.uoc.gr/gvd/), the National Clonal Germplasm Repository (NCGRDavis,
www.ars.usda.gov/ Main/docs.htm?docid=13743), Pl@nt Grape (from France,
plantgrape.plantnet-project.org), as well as the 735 unique Vitis vinifera microsatellite
allelic patterns developed by the CNRInstitute of Plant Virology (unpublished data).
Bibliographic sources presenting nuclear microsatellite profiles of traditional grapes from
other southern Italian regions and other European regions surrounding or related to
Apulia by presumed historical relationships were also used for genetic data comparison.
SSR loci such as VVMD36, VrZAG21, VrZAG64, and VrZAG67, not included in the
international lists promoted by OIV and GrapeGen06, were analyzed to cover published
allelic patterns more completely. In the comparison process at least the six international
markers, but generally from 7 to 10 and often more, were in common. Probability of
identity for all loci (PItot) was calculated within the set of 735 Vitis vinifera genotypes
analyzed by the CNR Institute of Plant Virology, for the 6, 7, 10 nSSR loci found to be in
common with published profiles and for all the 13 analyzed loci. The varieties
investigated were described in terms of vine morphological features, according to the
major OIV descriptors selected from the European Vitis Database and compared with
published references.

Results and Discussion

Most of the investigated varieties have been mentioned in historical references from the
early 19th century. Approximately one-third have national or regional relevance; most are
local or neglected, but often scattered in throughout the region (Table 1).
Among the 45 accessions analyzed, 33 unique genotypes were found. Statistical
parameters on these unique profiles indicated a number of alleles/locus ranging from 6 to
10 (8 on average), a level of observed and expected heterozygosity similar to that found
in previous studies, an estimated frequency of null allele positive only for 4 loci out of 13,
and a very low overall loci probability of identity (4.93 x 10 -16). It can therefore be
assumed that the identical profiles found are mutants or synonyms (Table 2). The two
Baresana accessions differing for grape color were confirmed to be somatic mutations, as
were the four Regina clones distinguishable for berry shape and size: broad elliptic in the
Regina bianca, narrow elliptic in the mennavacca, elongated elliptic in the pizzutella,
significantly larger in the mennavaccone. Similarly, sport mutations never described
before are the two Somarello, showing black and red grapes. The other equal genotypes
were identified as internal synonyms: that is, synonyms within the set of the varieties
analyzed. Most of these synonyms refer to minor, neglected varieties. Montonico
PagadebitiUva della scala is a prolific variety that is widespread under different names

(and therefore not recognized) in commercial vineyards from central to southern Italy.
Pampanuto and Verdeca, although evidence of their synonymy has already been
reported, are still erroneously registered as distinct cultivars in the Italian catalog, thus
resulting as duplicates.
The 33 unique genetic profiles at 13 SSR markers and profiles from two international
varieties used as references are in Supplemental Table 1. For the accessions provided of
genetic and/or morphological references, hence identified, the varietal names according
to the Italian Grape Variety Catalogue are indicated. A morphological profile of these
grapes can be found (catalogoviti.politicheagricole.it/ricerca.php), except for the recently
registered Antinello, Minutolo, Marchione, Maruggio (official name Maresco), and
Somarello rosso (descriptions rossa, Santa Teresa, Uva Attina, and Uva di Angela, no
genetic or morphological matches were found among the Italian cultivars. The examined
accession Fiano di Avellino (belonging to variety Fiano B.) showed three alleles at
VrZAG67, 139, 150, and 153 bp in size, a condition not uncommon in grapes and
explained by the occurrence of a chimeric structure.
Comparison of the allelic profiles of the Apulian cultivars with those of varieties from
other regions, while confirming known identities and synonyms, also revealed new
matches, significant for reconstructing the migration of varieties over time (Table 3). The
general probability of identity (PItot) calculated on the 735 V. vinifera genotypes
(accessions from throughout Italy) included in the nSSR database of the CNR Institute of
Plant Virology (unpublished data) ranged from 5.57 x 10-9 for accessions with six loci in
common with other publications to 4.96 x 10-14 for those with 10 loci in common. It is
thus likely that identical microsatellite profiles correspond to synonyms or to mutants.
Comparison of plant morphological features (photos and descriptions) of presumed
synonyms corroborated the genetic data, except for Pampanuto (Verdeca)/Lagorthi,
where the genetic profile of Lagorthi from the Greek Vitis Database indeed matched with
that of Pampanuto (alias Verdeca) from Apulia, whose phenotype differed from Lagorthi
shown elsewhere (Kotinis 1985). The NCGRDavis database reports three different SSR
profiles relating to the name Lagorthi.
The identity of the grapes mainly grown in Apulia, such as Aglianico, Aleatico, Malvasia
nera, Negro amaro, Primitivo, and Uva di Troia, was confirmed by comparison with
published genetic profiles. The association of the unspecified Malvasia bianca (historically
mentioned in all southern Italian regions) with Malvasia del Chianti from Tuscany,
Maratina from Croatia, and Pavlos from Greece reinforces the ancient origins of this
cultivar, widespread along the Adriatic and Ionian coasts and one of the sources of Italian
grape genetic diversity (Crespan et al. 2007, 2008a). eshte Pulez-a jone
In addition to the synonyms Malvasia/Maratina/Pavlos and Primitivo/Crljenak/Kratoija,
our findings demonstrated further correspondences between grape cultivars traditional
to Apulia and varieties from other Mediterranean coastal regions, especially from Croatia
and Greece. Half of the genotypes examined were found to be synonyms of grapes from
Slovenia or Croatia (Bianco dAlessano/Beretinjok and Topol; Francavidda/Zlatarica
vrgorska; Maruggio/Posipica, Bratkovina, Popetre, and Stradunska; Santa Teresa/Frmentum), from Greece (Baresana/Kolokythas, Korithi and Roditis lefkos; Pampanuto/BampaHasan and Lagorthi; Sgarraparete/ Karystino), or from other southern Italian regions
(Baresana/Cessal; Bianco dAlessano/Iuvarello; Montonico bianco/ Greco bianco;
Pampanuto/Braca; Negro amaro/Zagarese; Susumaniello/Nerello; Uva di
Troia/Summariello) (Table 3). Although no correspondences were found among grapes
from Albania (Ladoukakis et al. 2005), Macedonia (tajner et al. 2009), or the Maltese
islands (Giannetto et al. 2010), the synonymies revealed suggest that intense movement
of grape material occurred along the trade routes of the Ionian and Adriatic Seas, with a
remarkable influence on the assortment and evolution of Apulian varieties. This reflects
what can be assumed for the entire Italian peninsula, whose grape cultivars appeared by
far the most admixed among geographic groups (Bacilieri et al. 2013). As for Apulia, it is

not yet clear whether the exchange/introduction of materials occurred during the intense
trade of the Venetians in the modern age, or whether it should be traced back to Greek
colonization. Grape movements also occurred across southern Italian regions, from Apulia
to Sicily or vice versa. Moreover, the synonymies revealed enable materials masked by
local names to be identified, such as Cessal and Braca analyzed in Sicily (Carimi et al.
2010), which have now been identified with Baresana and Pampanuto/Verdeca,
respectively. The table-grape Baresana (Baresana meaning from Bari, the largest
Apulian city), attested in the region since the mid-19th century, is considered a historical
variety in Apulia, where it is currently the object of local valorization. The white and the
pink-skinned sports have both long been present in southern Italian regions. Its white
synonyms Kolokythas, Korithi, and Roditis Lefkos are widespread in many Greek inland
regions and islands, demonstrating the historical importance of the variety. The pinkskinned variant does not seem to occur in Greece: the cultivar Roditis Rs (with pinkskinned grape) included in the Greek catalog depicted in Kotinis (1985) and described by
Branas and Truel (1965) is a variety different than Baresana.
Bombino bianco (Trevolina in Slovenia) is regarded as one of the recurrent genitors in the
origins of traditional grapes from Croatia and Apulia (Lacombe et al. 2013), giving rise in
the latter region to Uva di Troia (Bombino bianco x Bouteillan, alias Quagliano),
Moscatello selvatico (Bombino bianco x Muscat of Alexandria), and Impigno (Bombino
bianco x Bouteillan) (Cipriani et al. 2010). In addition to confirming these findings, our
data further suggest Bombino nero and Palumbo as other varieties likely belonging to the
Bombino kin group, sharing half of their alleles at each SSR locus analyzed.
Sangiovese has been shown to possibly originate in Apulia (Bergamini et al. 2013).
Among the cultivars examined in this study, Susumaniello (as also reported in Crespan et
al. 2008b), Notardomenico, and Uva di Angela are likely related to Sangiovese by half
kinship.
Cinsaut, the ancient variety traditional to Provence (southern France), also occurs in
southern Italian regions. Ottavianello is the official name in Apulia, where it is also
present as Impigno rosso, while our findings show that in Sicily it is concealed by the
name of Greca. The question thus arises as to whether Cinsaut reached southern Italian
regions from the French coast (or from the related North African colonies) or whether it
spread across southern Italy via the eastern Mediterranean.
Four cultivars, Palumbo, Passera rossa, Uva Attina, and Uva di Angela, did not show any
matches, either in SSR allelic or in vine morphological profiles. Since these are likely
unique genotypes, they are worth maintaining as endangered genetic resources,
currently neglected in Apulia. Fiano minutolo (officially named Minutolo) must be clearly
distinguished from the more popular Fiano di Avellino from Campania. Finally, few cases
of varietal misnaming (Magliocco instead of Notardomenico and Susumaniello instead of
Somarello) are reported. Conclusions
Data from genotyping several historical grape varieties from Apulia, including minor and
neglected cultivars, are presented here for the first time. Particular efforts were made to
determine the true identity of the accessions investigated and their appropriate
denominations. Cultivars from surrounding areas or from regions related to Apulia by
historical relations and analyzed in other studies were considered for this purpose.
Comparison of nuclear SSR profiles from bibliographic sources and from available
molecular databases was confirmed as a modern and reliable tool for determining
identical genotypes and synonyms. However, at least basic morphological information is
crucial in order to pair the genetic profiles obtained with the true-to-type variety and
appropriate denominations. Published references for variety morphological profiles were
therefore checked and are reported together with genetic data.

New synonymies, sometimes unexpected, found among grapes traditional to other areas
shed some light on the migration of grapes following the development of colonies and
the establishment of trade routes in ancient times. Links with Greece and the Adriatic
coastal areas, and to a lesser extent with other overseas regions of the central
Mediterranean, were evident in the traditional grape varietal assortment of Apulia, a
Mediterranean region jutting into the sea.