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Wild grapes of Armenia:
unexplored source of genetic
diversity and disease resistance
Kristine Margaryan
1,2
*, Reinhard Töpfer
3
, Boris Gasparyan
4
,
Arsen Arakelyan
1
, Oliver Trapp
3
, Franco Röckel
3
and Erika Maul
3
1
Research Group of Plant Genomics, Institute of Molecular Biology of National Academy of Sciences
Republic of Armenia (RA), Yerevan, Armenia,
2
Department of Genetics and Cytology, Yerevan State
University, Yerevan, Armenia,
3
Julius Kuehn-Institute (JKI), Institute for Grapevine Breeding
Geilweilerhof, Siebeldingen, Germany,
4
Institute of Archaeology and Ethnography, National Academy
of Sciences Republic of Armenia (RA), Yerevan, Armenia
The present study is the first in-depth research evaluating the genetic diversity
and potential resistance of Armenian wild grapes utilizing DNA-based markers to
understand the genetic signature of this unexplored germplasm. In the proposed
research, five geographical regions with known viticultural history were explored.
A total of 148 unique wild genotypes were collected and included in the study
with 48 wild individuals previously collected as seed. A total of 24 nSSR markers
were utilized to establish a fingerprint database to infer information on the
population genetic diversity and structure. Three nSSR markers linked to the
Ren1 locus were analyzed to identify potential resistance against powdery
mildew. According to molecular fingerprinting data, the Armenian V. sylvestris
gene pool conserves a high genetic diversity, displaying 292 different alleles with
12.167 allele per loci. The clustering analyses and diversity parameters supported
eight genetic groups with 5.6% admixed proportion. The study of genetic
polymorphism at the Ren1 locus revealed that 28 wild genotypes carried three
R-alleles and 34 wild genotypes carried two R-alleles associated with PM
resistance among analyzed 107 wild individuals. This gene pool richness
represents an immense reservoir of under-explored genetic diversity and
breeding potential. Therefore, continued survey and research efforts are
crucial for the conservation, sustainable management, and utilization of
Armenian wild grape resources in the face of emerging challenges in viticulture.
KEYWORDS
wild grapevine, genetic diversity, SSR marker, resistance, Armenia
1 Introduction
The landscape diversity of Armenia and the peculiarities of the relief are pivotal factors
that enrich the plant diversity. Being located in the largely volcanic Armenian Highlands
with elevations ranging from 450 to 4,096 m above sea level and situated at the crossroads
of two completely different floristic regions, namely, mesophyllous Caucasian and the
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Raul De La Rosa,
Spanish National Research Council (CSIC),
Spain
REVIEWED BY
Annalisa Marchese,
University of Palermo, Italy
Angjelina Belaj,
IFAPA Centro Alameda del Obispo, Spain
*CORRESPONDENCE
Kristine Margaryan
kristinamargaryan@ysu.am
RECEIVED 14 August 2023
ACCEPTED 16 November 2023
PUBLISHED 08 December 2023
CITATION
Margaryan K, Töpfer R, Gasparyan B,
Arakelyan A, Trapp O, Röckel F
and Maul E (2023) Wild grapes of Armenia:
unexplored source of genetic diversity
and disease resistance.
Front. Plant Sci. 14:1276764.
doi: 10.3389/fpls.2023.1276764
COPYRIGHT
© 2023 Margaryan, Töpfer, Gasparyan,
Arakelyan, Trapp, Röckel and Maul. This is an
open-access article distributed under the
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 08 December 2023
DOI 10.3389/fpls.2023.1276764
largely woodless, arid, Armeno-Iranian, gave rise to varied and
contrasting environments that support an extremely floristic
richness. Armenia is a well-known hotspot of cultivated crop
diversity defined by Vavilov (Vavilov, 1926). On a relatively small
territory of the country, the flora is represented by approximately
3,800 species of vascular plants from 160 families and 913 genera,
including 146 endemic species (Nanagulyan et al., 2020). According
to archaeobotanical studies, wheat, barley, rye, oat, pea, melon,
watermelon, apricot, pomegranate, and grapes have been cultivated
in Armenia since ancient times, and the country is also
outstandingly rich in wild relatives of cultivated plants. Crop wild
relatives are valuable genetic resources and represent a large pool of
genetic diversity for new allelic variation required in breeding
programs capable of coping with the major biotic and abiotic
stresses (Margaryan et al., 2019).
The cultivated grapevine, V. vinifera L. subsp. sativa (DC.) Hegi
is closely related to, and fully inter-fertile with, an aggregate of wild
forms commonly referred to as V. vinifera L. subsp. sylvestris (C. C.
Gmelin) Hegi [= V. sylvestris C. C. Gmelin]. In Armenia, V. vinifera
subsp. sylvestris occurs both in the northern and the southern parts
of the country, growing in relatively mild subtropical niches in Lori
and Tavush provinces, in pre-mountainous areas of Vayots Dzor
and Syunik and in floristic regions of Artsakh.
Grapevine has had a unique religious and cultural importance
for the Armenians through millennia. In the cuneiform inscriptions
of the era of the Van Kingdom, the planting of grapevines, the
construction of wine cellars, and other agronomical activities are
mentioned. The improvement of viticulture and wine-making
traditions in Armenia records significant progress in the Middle
Ages, as evidenced by archaeological excavations and bibliographic
sources. In the works of Armenian historians and epigraphic
inscriptions, there are numerous references to vineyards, wine
presses, and wine. The Armenian Church played an important
role in the development of viticulture and wine-making as
important economic sectors. Almost all monasteries and famous
churches had vineyards and wine presses, and wine had an essential
role in religious and spiritual life. Grapes were also an important
source of food and medicine. Fruit of wild grapevines was harvested
to make wine and juice, and the leaves and roots were used for
different medicinal purposes. In the book “Haybusak,”the author
reports the existence and in detail characterized wild grapes as liana
growing in mountains with small, sour, and red berries (Alishan,
1895). In 2007, a quasi-industrial complex for wine production was
discovered in Areni-1 cave, built in the limestone rock formations at
Arpa River canyon in Vayots Dzor province. Due to the ideal
microclimate inside the cave, Areni-1 has yielded large quantities of
exceptionally well-preserved organic remains, including grape
seeds. Chemical analysis of the excavated material indicates
millennia lasting tradition of wine making dating back to 4230–
3790 BC. The oldest and best-preserved monument is a testament to
the 6,000 years of wine-making tradition in Armenian Highlands
(Smith et al., 2014;Hobosyan et al., 2021).
In recent years, in parallel with the wine industry renaissance in
the country, only a limited number of traditional varieties were used
for wine production with a serious shift to single variety vineyards.
The intensive cultivation of a small number of commercial cultivars
has resulted in an alarming reduction in genetic diversity, since only
30–35 of 400 native grapevine varieties are used in wine and brandy
production. Minor autochthonous cultivars having only a local
importance in the different wine-growing regions are under-
exploited. Their ignorance might be related to the lack of
comprehensive characterization of native neglected varieties,
especially to missing data on oenological and agronomical traits
and, partially, due to demands of the wine/brandy market. All of
these arguments prove the necessity and importance of collection,
conservation, characterization, and efficient use of grape germplasm
resources, and knowledge of genetic diversity and genetic
relationships between grape genotypes. Actually, very little is
known about the magnitude of grape germplasm in Armenia.
Until now, there have been only a few studies forwarded on
characterization of native Armenian grape varieties using
molecular-genetic approaches (Dallakyan et al., 2020;Margaryan
et al., 2021;Nebish et al., 2021).
Until recently, the exact location of grapevine domestication
remains debated. Most lines of evidence point to a primary
domestication event in the Near East and Armenian-Persian
refuge, but the critical details of grapevine domestication were
often inconsistent (McGovern, 2003;Myles et al., 2011;Wan
et al., 2013). The large-scale study launched recently elucidates
grapevine evolution and domestication history with 3,525 cultivated
and wild European grapevines. According to the study,
domestication occurred concurrently approximately 11,000 years
ago in Western Asia and the Caucasus parallel to yield table and
wine grapes, respectively (Dong et al., 2023).
During domestication, essential morphological shifts occurred
including larger berry and bunch sizes, higher content of sugar, and
altered seed morphology (Zhou et al., 2017). Domestication resulted
in most drastic changes in the reproductive biology of the
grapevine. Critical were the shift from sexual reproduction to
vegetative propagation and the change from a dioecious into a
hermaphroditic crop, which is able to pollinate itself and thus set
fruit without the need for cross-pollination, leading to bottle necks,
limiting genetic diversity. In contrast, dioecious V. vinifera subsp.
sylvestris maintained considerable genetic polymorphism and
manifest wide variability. Consequently, seedlings raised from
mother plants segregate widely in numerous traits, including size,
shape, color, juiciness, sweetness, and palatability of the grape
berries (Zohary et al., 2012).
Genetic diversity of wild species is threatened by genetic erosion
and extinction due to urbanization, desertification, drought,
agricultural development, habitat destruction by overgrazing and
forest clearing, and the negative impact of climate change
worldwide (Lala et al., 2018;Giovino et al., 2022). V. vinifera
subsp. sylvestris is a unique and valuable genetic resource for the
improvement of cultivars in terms of the wide range of tolerance
and resistance against biotic and abiotic factors and the high level of
adaptation potential in the context of global climate changes (Ocete
et al., 1995;Arnold et al., 1998). Thus, the conservation of the
existing genetic diversity of wild grapes is essential to safeguard the
potential of wild germplasm to be used in future breeding programs.
Domestication is an evolutionary process where strong selection for
specific traits, combined with population bottlenecks, greatly alter
Margaryan et al. 10.3389/fpls.2023.1276764
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the genetic structure of populations and the underlying genetic
architecture of phenotypic traits (Pusadee et al., 2009). Loss of
genetic diversity has profound implications for crop improvement.
Therefore, potentially valuable traits may be lost in vine cultivars,
thereby reducing the set of phenotypes that can, later on, be used by
breeders for variety improvement. In this context, the frequent
insufficiency of new traits in grapevine germplasm collections has
led to increased efforts to preserve wild relatives of domesticated
plants, as a reservoir of phenotypes for future crop improvement.
The richness of extensive natural biodiversity of V. vinifera L. in
Armenia is an unexplored source with great potential to regulate
undesirable pests and diseases. The first records pointed to the
potential of wild grapes done by Sosnovskiy (1947), who mentioned
resistance of V. sylvestris from Lori against powdery mildew by
observing plants for many years in the forests in natural conditions
(Sosnovskiy, 1947). He observed also that these plants might be
tolerant against Phylloxera. However, during the Soviet Era,
breeding programs, even being quite active, have been focused
mainly on creation of table grapes and bred cultivars for brandy and
vodka production. It is only in 2017 in the frame of Armenian-
German bilateral cooperation that the large-scale research was
initiated focusing on comprehensive characterization of grapevine
genetic resources of Armenia. In their research, Margaryan et al.
(2019) described and characterized wild genotypes collected from
Syunik province (Margaryan et al., 2019). Authors studied the
genetic diversity of wild grapevines concerning their capacity for
stilbene biosynthesis, since phytoalexins, as the stilbenes, are the
central element of basal immunity, which might be exploited as a
genetic resource for resistance breeding. Among analyzed wild
individuals, potential genotypes have been selected for
future studies.
The study of Riaz et al. (2020) confirmed the resistant potential
of Armenian wild grapes against powdery mildew. The authors
discovered and characterized an additional gene pool that shared a
Ren1-like local haplotype. The results endorse the hypothesis that
mildew resistance was present in wild plants and potentially evolved
through sexual recombination. Authors suggested a notion that
wild progenitor V. sylvestris may have developed PM resistance over
a long time. It is also possible that resistance was introduced into
cultivated V. vinifera ssp. sativa in certain regions at the time of
domestication. Being selected, the Ren1 haplotype stayed intact in
V. vinifera ssp. sativa accessions due to the practice of clonal
propagation (Riaz et al., 2020). Recently, Possamai et al. (2021)
described the Ren1.2 loci, a new variant of Ren1, was located in the
same chromosomal region as Ren1. The loci mapped in the
Georgean variety ‘Shavtsitka’, which were classified as a variant of
the previously mentioned locus. Both the loci Ren1 and Ren1.2 were
found in cultivars of different origins, proposing that Caucasus
grapevines have independently developed their resistance loci in the
exact same location as Ren1. Based on results provided by authors,
the Ren1.2 locus shows partial resistance to E. necator, reducing
hyphal proliferation and sporulation (Possamai et al., 2021).
Recent studies, strongly increase the interest in Caucasian grape
germplasm not only for the richness of genetic diversity but also for
the breeding resistant grape varieties and have shown that the
resistance trait appears to be widely diffused in such germplasm
(Possamai et al., 2021;Vezzulli et al., 2022).
Hence, the aim of the present study was to analyze genetic
diversity and structure among Armenian wild grapevine
populations prospected from the different regions of the
subspecies’rangeinArmeniatoexplorethelevelofgenetic
signature of this germplasm and its genetic pattern. The deeper
study of Armenian grape germplasm required to uncover further
source of resistance. Obtained results will guarantee baseline
information for the development of suitable conservation
strategies for better management and maintenance of wild
grapevine populations in Armenia.
2 Materials and methods
2.1 Plant material
The present study on natural populations of wild grapes started
by investigating historical bibliography and existing records about
the presence of wild grapes in the territory of Armenia and Artsakh
located at the northeastern end of the Armenian Highlands in the
Lesser Caucasus mountain system. Wild Vitis germplasm was
collected from the following four administrative regions of
Armenia (Syunik, southernmost province; Vayots Dzor,
southeastern province; Tavush, northeast province and Lori,
north province) during surveys between 2018 and 2022 in their
natural habitat: main mountainous areas, climbing the rocks, and
embracing the trees, riverbanks, and forests (Figure 1). During
material sampling, one of the important criteria was to collect wild
grapes from isolated areas, located as far as possible from vineyards
and home gardens (the nearest sampling area was 10–15 km from
vineyards, and in the Syunik region, the material was collected from
areas approximately 40–50 km distance from villages). Considering
the phenotypic similarity of wild and cultivated grapevines, the wild
grapevine sampling strategy for each selected V. sylvestris candidate
was based on the main differentiating reference traits to distinguish
wild from domesticated grapevines in order to reduce as much as
possible the risk of collecting plants derived from hybridization with
a cultivated grapevine. Each selected sample was analyzed
ampelographically, and only samples that met the basic
phenotypic profile of wild grapes were subjected to further
genetic analysis. During surveys, the following OIV descriptors
were used: OIV151, OIV 001, OIV 202, OIV 204, OIV 223, and
OIV 225.
A total of 148 unique V. sylvestris genotypes were included and
analyzed in this study using 24 nSSR markers. Initially, 220 putative
wild individuals were collected. The excluded accessions were
redundant genotypes, accessions with more than 40% missing
microsatellite data, escaped cultivars from vineyards, and feral
types. To identify feral types, we performed a parentage analysis
between Armenian V. sylvestris and autochthonous cultivated
accessions; the dataset from Caucasian wild and cultivated grapes
also was used (unpublished data). Cervus 3.0.7 was applied as one of
the approaches to screen the material (Margaryan et al., 2021).
Margaryan et al. 10.3389/fpls.2023.1276764
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Among the analyzed set, 61 samples were collected from pre-
mountainous areas of Syunik province, 46 samples from Artsakh, 15
samples from Vayots Dzor province, 15 samples from Tavush
province, and 11 samples from Lori. GPS coordinates and habitat
notes were recorded when the samples were collected from their
natural habitats. A part of the accessions was also photo documented
to aid with subspecies identification. Young leaves were collected
from actively growing shoots, placed in filter paper, air-dried, and
stored in paper envelopes for DNA extraction and further use.
Additionally, a subset of 48 wild genotypes collected in the
beginning of 2000s and analyzed by Riaz et al., 2018 were added to
the study to discover their relationship to the set of recently
collected material (Riaz et al., 2018). According to the
information provided by authors, these 48 wild accessions were
collected from Alaverdi (Lori province) as seeds from female vines.
Seedling plants from seed lots were maintained in the USDA
National Clonal Germplasm Repository in Davis, CA, USA. Thus,
the following analysis will generate results from a total of 196 wild
genotypes. For 48 seedlings, nSSR marker data have been extracted
and adapted. To distinguish the two data sources, the 148 samples
collected for the present study and the 48 seedlings from Riaz et al.
(2018) will be designated as genepool A and genepool
B, respectively.
2.2 DNA extraction and nSSR analysis
Total genomic DNA was extracted from dried leaf tissue after
grinding with MM 300 Mixer Mill system (Retsch, Haan,
Germany). DNA extraction was performed, employing the
DNeasy 96 plant mini kit (QIAGEN, Düsseldorf, Germany)
following the manufacturer’s protocol. DNA concentration and
quality were checked by spectrophotometric analysis and
electrophoresis in 1% agarose gel. Microsatellite fingerprinting of
genotypes was performed on 24 microsatellite loci (nSSRs) well
distributed across the 19 grape chromosomes as previously
described (Sefc et al., 1999;Laucou et al., 2011) [i.e., VVS2,
VVMD5, VVMD7, VVMD21, VVMD24, VVMD25, VVMD28,
VVMD27, and VVMD32; four of the VrZAG series (VrZAG62,
VrZAG79, VrZAG67, VrZAG83); VMC4f3.1; VMC1b11; and nine
of the VVI series VVIb01, VVIn16, VVIh54, VVIn73, VVIp31,
VVIp60, VVIv37, VVIv67, and VVIq52]. Nine polymorphic
microsatellite markers proposed by the GrapeGen06 project,
namely, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28,
VVMD32, VVS2, VrZAG62, and VrZAG79, were used for
comparison of genetic profiles with the SSR-marker database of
the Julius Kühn-Institut (JKI), maintaining approximately 8,000
genetic profiles from distinct sources.
For fragment length determination by capillary electrophoresis
on ABI 3130xl Genetic Analyzer (Applied Biosystems, Life
Technologies, Waltham, MA, USA), all forward primers were 5′-
labeled with a fluorescent dye (FAM, HEX, TAMRA, ROX, and
PET). The combination of markers with different labels and diverse
fragment lengths allows one to perform the polymerase chain
reaction (PCR) and grouped markers in seven multiplex pools,
comprising two to five SSR markers characterized by similar
annealing temperatures (Supplementary Table S1). The 2x
KAPA2G Fast PCR Kit (Düren, Germany) was used to set up 5-
FIGURE 1
Geographic distribution of the sampled V. vinifera L. subsp. sylvestris populations in Armenia and Artsakh (Nagorno Karabakh).
Margaryan et al. 10.3389/fpls.2023.1276764
Frontiers in Plant Science frontiersin.org04
mL reaction mixtures containing master mix, 100 pmol of each
primer, and 1 ng of template DNA. A GeneAmp PCR system 9700
thermal cycler (Applied Biosystems, Schwerte, Germany) was used
for the amplification starting with 3 min initial denaturation at 95°
C, followed by 30 cycles for 30 s. A final extension was performed at
72°C for 7 min. One microliter of the PCR product was used for
fragment length determination, and the results were processed with
GeneMapper 5.0 software (Applied Biosystems, Life Technologies,
Waltham, MA, USA) recorded in base pairs. Allele size was
determined by comparing the fragment peaks with the internal
size standard, using the Microsatellite default method for size
calling with SSR and the expected repeat size. To correct the
amplification shifts among different multiplexes, SSR profiles were
adapted by including in each PCR amplification run the DNA of
standard cultivars Cabernet franc and Muscat à petits grains blancs.
2.3 Flower phenotype analysis
The determination of flower sex was carried out for all
genotypes collected throughout Armenia and was analyzed by a
specifically designed APT3 marker from adenine phosphoribosyl
transferase gene capable to distinguish flower sex: female (F), male
(M), or hermaphrodites (H) (Fechter et al., 2012). Field phenotypic
screening was done only for part of accessions: 46 accessions from
Artsakh, 45 accessions from Syunik, 15 accessions from Vayots
Dzor, 12 accessions from Tavush, and 4 accessions from Lori.
Because of geographic location and relief, it was not always possible
to screen flower phenotypes in natural habitats.
2.4 Powdery mildew (Erysiphe
necator) evaluations
A total of 107 non-redundant wild genotypes from five natural
populations in Armenia (in situ) were analyzed in this study at the
Ren1 locus. PM symptoms were observed on wild individuals in situ
during an inventory of V. sylvestris germplasm using a five-class
scale (1–9) OIV 455 descriptor. The natural habitats of the studied
species cover diverse geographic origins southeastern province and
the pre-mountainous area of Syunik in southern Armenia, Artsakh,
in the north-eastern Armenian Highlands, northeast Tavush
province, and northern Lori province. The overall health status of
each plant was evaluated; symptomatic plants were excluded from
the analyzed set. Additional three microsatellite markers, SC47-18,
SC8-0071-14, and SC175-1, that are associated with the Ren1 gene
for PM resistance were analyzed (Riaz et al., 2013;Riaz et al., 2018;
Possamai et al., 2021;Luksicet al., 2022). The SSR markers were
multiplexed within two runs. All forward primers were labeled on
the 5′end with fluorescent dyes (HEX, 6-FAM). The following
steps, including mpxPCR and fragment length determination by
capillary electrophoresis, were carried out as described above. To
correct the amplification shifts among multiplexes, SSR profiles
were adapted by including in each PCR amplification run the DNA
of Kishmish Vatkana, 2010-007-0027 and Vitis Syl. Geo
W31 accessions.
2.5 Data analysis
The genetic diversity among groups and over all the groups of
wild grapes was estimated. The standardized nSSR genotyping data
were used to determine the number of different alleles (Na), the
number of effective alleles (Ne), Shannon’s Information Index (I),
observed heterozygosity (Ho), expected heterozygosity (He),
fixation index (F), also called inbreeding coefficient, and private
alleles (PA). The allele frequency for each nSSR locus was calculated
as well. GenAlEx software version 6.5 was used to compute genetic
diversity statistics for each nSSR locus (Nei, 1973;Peakall and
Smouse, 2006).
Clustering was performed by MEGA 7 software, version 7.0.26,
which was used to generate a distance tree by the neighbor-joining
(N-J) hierarchical clustering method using the codominant
genotypic distances between all pairwise combinations calculated
by the GenAlEx 6.5 (Saitou and Nei, 1987;Kumar et al., 2016).
Principal coordinates analysis (PCoA) was performed by GenAlEx
6.5 via covariance matrix with data standardization (Kalinowski
et al., 2007). Analysis of molecular variance (AMOVA) was
performed to characterize the partition of the observed genetic
variation among and within populations and genetic groups using
GenAlEx 6.5 software (Excoffier et al., 1992;Kalinowski et al., 2007).
The significance test was performed over 999 permutations.
Bayesian clustering was applied on the 24 nSSR genotype data
for the wild distinct genotypes. The admixture model in Structure
2.3.4 was employed to infer the number of genetic populations (K)
existing in the samples and to assign genotypes to populations of
origin, with no prior information (Pritchard et al., 2000). The
Structure configuration was set to ignore population information
and use an admixture model with correlated allele frequencies.
Various numbers of putative populations (K) were tested, ranging
from 1 to 10. Burning time and replication number were set to
100,000 and 100,000, respectively, in each independent run with 10
iterations. The choice of the most likely number of clusters (best K)
was evaluated following the ad hoc statistic delta K (DK) as
described Evanno et al. (2005) using Structure Harvester (Evanno
et al., 2005;Earl and vonHoldt, 2012). The Structure bar plot was
visualized by running the clump file obtained by Structure
Harvester, in Structure Plot v 2.0 (Ramasamy et al., 2014).
3 Results
3.1 Flower characterization
The key trait distinguishing sativa vs. sylvestris subspecies is the
flower sex, since wild grapevine is dioecious, whereas flowers of
cultivars are usually hermaphroditic. During our surveys, the search
for wild plants was focused on collecting dioecious accessions. For
122 wild individuals, the flower morphology was analyzed in situ,
and for the whole material, the flower phenotype was analyzed by
APT3 marker. Sex expression in Vitis flower is thought to be
controlled by a major locus with three alleles, male M,
hermaphrodite H, and female F, with an M > H > F allelic
dominance (Picq et al., 2014). In wild individuals, males are MM
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and MF and females are FF, and in cultivars, hermaphrodites can be
HF or HH. According to the obtained data among 196 sylvestris,five
different allelic patterns were determined at APT3 loci classifying
genotypes as F: 39 genotypes have shown 268/268 (F), 20 genotypes
268/397 (F), 33 genotypes 268/336 (F), 3 genotypes 336/336 (F), and
4 genotypes 268/336/397 (F).
Six different allelic patterns were determined at APT3 loci
describing genotypes as M: 37 genotypes 268/466 (MF), 15
genotypes 336/466 (MF), 16 genotypes 268/397/466 (MF), 13
genotypes 268/336/397/466 (MF), 8 genotypes 268/336/466 (MF),
and 4 genotypes 466/466 (MM). Thus, among the analyzed samples
based on molecular phenotyping, 51.04% were female and 48.95%
were male individuals. For four genotypes out of 196, DNA analysis
was not applicable. Flower phenotypes estimated by DNA-based
flower sex marker and field phenotyping of V. sylvestris are
presented in Supplementary Table S2.
3.2 Genetic diversity of wild grapes
in Armenia
Table 1 displays the diversity parameters across all wild
accessions for 24 nSSR markers. A total of 292 alleles were
detected across all markers with an average of 12.167 alleles
across 196 accessions. The number of alleles ranged from 4
(VrZAG83, VVIn16) to 22 (VMC4f3.1). The number of effective
TABLE 1 Diversity indices calculated for 196 distinct genotypes of wild grapevines determined from 24 nuclear microsatellite data.
Locus Ra (bp) Na Ne I Ho He F
VVS2 125–157 13 7.447 2.189 0.793 0.866 0.084
VVMD5 226–248 11 6.017 1.991 0.733 0.834 0.121
VVMD7 235–263 13 6.922 2.146 0.799 0.856 0.066
VVMD25 237–271 11 3.598 1.522 0.691 0.722 0.043
VVMD27 176–198 12 5.548 1.907 0.740 0.820 0.098
VVMD28 218–282 18 7.290 2.292 0.790 0.863 0.084
VVMD32 240–292 15 6.600 2.157 0.771 0.848 0.092
VrZAG62 188–204 8 5.595 1.812 0.850 0.821 −0.035
VrZAG79 237–261 12 6.991 2.158 0.830 0.857 0.032
VVIv67 348–401 18 5.315 2.095 0.761 0.812 0.063
VrZAG67 122–159 14 5.815 2.046 0.797 0.828 0.037
VrZAG83 188–201 4 3.692 1.344 0.710 0.729 0.026
VVIn16 147–155 4 1.978 0.879 0.443 0.495 0.104
VVIn73 258–272 6 2.241 0.974 0.510 0.554 0.078
VVIp60 274–331 15 6.661 2.163 0.701 0.850 0.176
VVMD24 204–218 8 4.211 1.613 0.747 0.763 0.020
VVMD21 244–267 9 4.316 1.619 0.626 0.768 0.186
VMC4f3.1 163–217 22 9.828 2.579 0.802 0.898 0.107
VVIb01 289–317 11 3.203 1.433 0.660 0.688 0.041
VVIh54 139–179 15 5.917 1.991 0.747 0.831 0.101
VVIq52 70–86 9 3.305 1.468 0.655 0.697 0.061
VVIv37 148-182 13 8.309 2.231 0.758 0.880 0.138
VMC1b11 167–203 16 4.947 1.957 0.663 0.798 0.169
VVIp31 157–195 15 9.692 2.411 0.864 0.897 0.037
Total 292
Min. 4 1.978 0.879 0.443 0.495 −0.035
Max 22 9.828 2.579 0.864 0.898 0.186
Mean 12.167 5.643 1.874 0.727 0.791 0.080
Ra, range of allele size (bp); Na, number of different alleles; Ne, effective alleles; I, Shannon’s information index; Ho, observed heterozygosity; He, expected heterozygosity; F, fixation index.
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alleles ranged from 1.978 (VVIn16) to 9.828 for VMC4f3.1. The
highest Shannon’s information index (I) was observed in VMC4f3.1
locus (2.579) and lowest in VVIn16 (0.879), while the average
among SSR loci was 1.874. Shannon’s information index is an
important parameter mirroring the level of polymorphism.
For microsatellite markers efficiency, the observed and the
expected heterozygosity (Ho, He) are considered to evaluate the
genetic variability among the samples analyzed. Both observed and
expected heterozygosity varied among loci. Observed
heterozygosity of the analyzed set was lower than the expected
heterozygosity for the majority of the analyzed markers, with a
mean value of 0.727 and 0.791, respectively. Lower values of
observed heterozygosity in conjunction with the results of the
fixation index point to the existence of inbreeding, as F-values are
expected to be close to zero in case of random mating. Based on our
results, the differences between Ho and He were not significant,
pointing to random mating promotion. The locus with the lowest F-
value was VrZAG62 (−0.035), while the highest was VVMD21
(0.186). The mean F-value for the dataset was 0.080.
The SSR marker data were divided into five genetic groups/
populations based on geographic origin or habitat of collected
samples, and the allelic profiles were used to calculate statistical
indices to determine diversity within and among the
groups (Figure 2).
The mean value of number of alleles for all populations is
12.167. The Vayots Dzor and Tavush groups were the least diverse
with an average of 5.958 alleles, and the Syunik group was the most
diverse with 9.333 alleles. The other two genetic groups (Artsakh
and Lori) showed a comparable number of alleles that ranged from
9.167 to 8.292 (Supplementary Table S9). The observed
heterozygosity (Ho) was lower than the expected heterozygosity
(He) for Artsakh, and no difference was observed for the Syunik and
Alaverdi groups. For the groups Lori, Tavush, and Vayots Dzor, the
expected heterozygosity (He) was slightly higher than the observed
heterozygosity (Ho). Expected heterozygosity ranged from 0.569 to
0.760 between the groups. From all groups, the highest He was
registered for the Artsakh group with a mean of 0.760, and the
lowest He was the Alaverdi group with 0.569.
A total of 65 private alleles were found across all markers within
five groups. Private alleles for every group and allelic patterns across
V. sylvestris are presented in Supplementary Table S3 and Figure 2.
The wild genotypes from Lori, Syunik, and Artsakh had by far the
most private alleles compared with the other two groups. Overall,
statistical indices found these three groups to be the most diverse
and Tavush and Vayots Dzor the least diverse. The highest number
of private alleles was identified for VMC4f3.1 and VMC1b11
markers in wild populations. Analysis summary of allele size (AS)
and frequencies (AF) for each of the microsatellites is presented
in Supplementary Table S4. Genetic profiles of analyzed
non-redundant 196 wild grape genotypes are provided in
Supplementary Table S5.
3.3 Cluster analysis
The neighbor-joining (NJ) distance tree was constructed to
study genetic relationships among 196 wild grape genotypes based
on allele frequencies of 24 nSSR loci. Two major clusters with sub-
clusters were clearly distinguished. The first cluster contained all
samples of genepool A collected in different geographical regions
across Armenia and the second cluster grouped genepool B that
originated from Lori, Alaverdi, which were seedling plants from
seed lots (Figure 3). Cluster 2 with two clearly separated sub-clusters
is the most divergent clade and formed a separate genepool, not
related to genepool A. In this case, we can hypothesize that
genotypes involved in genepool B derived from one or closely
related mother plants. Thus, the wild individuals of genepool A
and genepool B from Lori were not assigned to the same cluster.
The geographic clustering as single separate clades can be seen in
the sylvestris originating from Syunik, Artsakh, Tavush, and Vayots
Dzor province, while some of the wild individuals from Lori and
Vayots Dzor were represented in blend groups as well.
FIGURE 2
Allelic patterns across wild grapes collected from five geographical locations.
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3.4 Population structure analysis
and differentiation
To identify the structure of analyzed populations and the
correlations among wild genotypes from diverse geographical
origins, two different analyses were performed. PCoA based on
the genetic distance matrix obtained by the 24 nSSR genotype
profiles revealed a similar pattern to that observed in the neighbor-
joining dendrogram (Figure 4).
The two PCoA axes explained a total of 22.57% of the observed
variance. The Alaverdi (blue) genepool B from Lori province forms
an isolated gene pool on the left side of the main axis, suggesting
genetic dissimilarity with genepool A collected for the present
study, which groups on the right side of the main axis. Because
precise information about the origin of samples included in
genepool B is lacking, based on obtained results, we can
hypothesize that these accessions derived from a reduced number
of genetically related mother plants. The Syunik population (labeled
in green) forms a sister group with the accessions from Vayots
Dzor (labeled in yellow), indicating that they are genetically similar
and partly also match the accessions from Artsakh (labeled in red).
V. sylvestris from Lori and Tavush are closely associated to the right
cluster. In contrast, genepool B is clearly different from samples of
genepool A recently collected from Lori province. Only few wild
accessions from Syunik and Tavush are placed between two
main clusters.
The second method used to estimate genetic relationships
among the 196 wild grapes from different origins was a clustering
algorithm implemented in the program Structure. The results of the
Bayesian analysis of genetic structure were roughly analogous to
those of the NJ cluster analysis and PCoA. However, subtle
population subdivisions have been clearly detected by Structure.
The DK value suggested two possibilities, namely, K = 2 and K = 8,
as the second order rate of change of likelihood distribution
(Figure 5). The statistic of Evanno et al. (2005) showed the
highest probability for K = 2 (Supplementary Table S7). The K = 2
structure simulation splits the wild populations into two groups in
absolute correspondence to the genepool A and genepool B. Here, it
is important to note that, probably, the origin of genotypes grouped
in genepool B could have some impact structuring of analyzed
population. Nonetheless, interpreting the value of K should be
carried out with care because it provides an ad hoc
FIGURE 3
Neighbor-joining dendrogram demonstrating genetic relationships among 196 Armenian wild grape accessions based on 24 nSSR loci. Wild grapes
originated from Syunik province are marked in green, wild grapes from Artsakh in violet, wild grapes from Lori province in blue, wild grapes from
Tavush province in orange, and wild grapes from Vayots Dzor province in yellow.
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approximation, and in some cases, population structure may be
missed by STRUCTURE. Hence, we used an ad hoc statistic DKto
choose the optimum number of clusters (K) based on the second-
order rate of change in the log probability of data between
successive K-values as proposed by Evanno et al. (2005). To gain
insight into the nature of wild germplasm, further analyses of
population structure beyond K =8 are required to better
understand the genetic background of these populations. With
K=8 structure, the simulation differentiates the populations
according to the geographic origin of the material, and at the
same time, the separation of southern and northern gene pools
into two main clades becomes clear.
The program also splits three sub-populations within the Lori
group, where two of subpopulations belong to the genepool B and
the third one have been collected within our study.
Plotting the Q matrix values as the estimated membership
coefficients for each individual in each K clusters, for K = 2,
disclosed clusters corresponding to their habitats. Most
individuals showed an average estimated major membership
proportion ≥0.70 and therefore could be classified as mainly
belonging to one of the two distinct genetic groups according to
their largest ancestry membership fraction (Figure 5).
Supplementary Table S6 presents the Q-value assignments of 196
accessions to two groups. Accessions with Q-values below 0.70 were
FIGURE 5
Barplot displaying the admixture proportions of 196 wild genotypes as estimated by STRUCTURE analysis at K = 2 and K=8. Each accession is
represented by a single vertical bar divided into K color segments representing its proportions in the two, and eight inferred genetic clusters using
STRUCTURE software. Groups were named according to the geographic locations.
FIGURE 4
Principal coordinate analysis (PCoA) represented by two axes using a covariance matrix of 24 nSSR locus of 196 accessions. Genepool A (right) and
genepool B (left) are clearly differentiated. Wild genotypes of genepool A are labeled with the corresponding color for the group to which
they belong.
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called admixed. The proportion of admixed genotypes for the
analyzed set was 5.6%.
The AMOVA analysis is presented in Table 2. When the total
genetic variation was partitioned, 11% was attributed to the
differences among populations, 8% to the differences among
individuals within populations, and 81% to the differences within
individuals, with levels of significance estimated over 999
permutations lower than 0.001. F
ST
,F
IS
, and F
IT
parameters over
all the loci and populations were 0.108, 0.092 and 0.190sss,
respectively (p ≥0.001).
3.5 Genetic polymorphism at the Ren1
locus of Armenian wild genotypes
A total of 107 V. sylvestris individuals from in situ habitats were
analyzed at three SSR markers linked to the Ren1 locus on
chromosome 13 (Supplementary Table S8). A total of 28 wild
genotypes carried three R-alleles, and 34 wild genotypes carried
two R-alleles associated with PM resistance. Based on the obtained
results, the Artsakh population had the most individuals carrying
the three R-alleles (16), followed by Syunik (9), Lori (2), and Tavush
(1). R-allele 242 at marker SC47-18 was the most frequent.
4 Discussion
The Armenian Highlands lies on the northern edge of Western
Asia and stretches up to the Caucasus from the north. The recent
story about dual domestication origin and evolution of grapevine
comes to state that in Armenian Highland, human and grapevine
stories are intertwined through millennia and roots of grapevine
domestication are found deep in the Pleistocene, ending 11.5
thousand years ago (Dong et al., 2023). According to authors’
findings, glacial episodes split V. sylvestris into eastern and
western ecotypes approximately 200–400 ka. The last glacial
advance saw the split of the eastern ecotype into two groups that
separately and parallel gave rise to a domestication process. The
limited migration from the area under study is the key factor for the
uniqueness of grapevine diversity preserved in Armenia. The in-
depth study of this gene pool can be used to shed light on the
complex grapevine history in the region.
The genus Vitis contains approximately 60–70 inter-fertile
species, and among them was Vitis vinifera L., which is the
species with the greatest agronomic importance. V. vinifera ssp.
sylvestris is the only extant wild Vitis taxon native to Eurasia, and it
is considered the progenitor for almost 10,000 domesticated
grapevine cultivars nowadays (This et al., 2006). Wild grapes are
endangered in all their distribution areas, and preservation
measures are needed to maintain the genetic integrity and
survival of the remnant diversity. Within this context, in the
proposed study, our goal was to characterize genetic diversity via
population genetics methods to decode the population signature
and decipher the potential of wild grapes growing in Armenia.
Despite the importance of grapevine cultivation in human
history, religion, culture, and the role of economic values of
cultivar improvement, comprehensive genetic and genomic data
for wild grapes in Armenia are lacking. The present work is the first
study evaluating the genetic diversity and population structure of
wild grapes originating from Armenia with DNA-based markers.
Until now, no inventory or systematic genetic and morphological
characterization of V. sylvestris has been carried out in Armenia.
The surveys started in 2018, and thousands of plants have been
registered, and this activity is an ongoing process. One of the tasks
of the research group is to know the current distribution and main
habitats of the fragmented populations of wild plants in the country,
to carry out morphological description of V. sylvestris along their
phenological development, to analyze its sanitary status, and to
estimate genetic richness of wild plants.
4.1 Genetic diversity and population
structure of wild grapevine in Armenia
Accurate detection and precise quantification of genetic
variation is pivotal for cost-effective management and successful
conservation of grapevine genetic resources. Morphological
characteristics are used to distinguish wild grapes from cultivated
grapes; however, in some cases, these tools do not correctly identify
genotypes escaped from vineyards or hybrids between wild and
cultivated plants. The application of DNA markers such as simple
sequence repeats (nSSR) provides the possibility of characterizing
wild accessions on the basis of their genomic signature. To
characterize Vitis genetic resources, we have adopted a symbiotic
approach as the precise strategy combining ampelography and
molecular fingerprinting used in our previous studies.
Ampelographic characterization of wild accessions involved in this
study is ongoing. The genotyping results of the present study
TABLE 2 Results of the AMOVA analysis carried out among and within five populations of wild grapevine.
Source of variation Degree of
freedom
Sum of
squares Variance components Percentage of
variation (%) F
st
F
is
F
it
Among populations 4 350.235 1.066 *** 11
Among individuals within populations 191 1,844.622 0.812 *** 8
Within individuals 196 1,574.500 8.033 *** 81
Total 391 3,769.357 9.911 100 0.108 *** 0.092 *** 0.190 ***
a. The inbreeding coefficient within individuals relative to the subpopulation. b. The inbreeding coefficient within individuals relative to the total. c. The inbreeding coefficient within
subpopulations relative to the total. *** p ≥0.001 estimated over 999 permutations.
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provides the basis to identify duplicate accessions among the
sampling set and to maintain only unique genotypes and to
establish a SSR marker-based database of distinct wild plants of
Armenia that in the future will facilitate germplasm collection efforts.
Obtained results ensure essential information that advances the
understanding of the allelic diversity, population structure of
Armenian wild grapes, and hybridization patterns shown only in
particular locations and in few cases. The most admix samples were
observed within the Syunik population. The presence of cross-
hybridization between V. sylvestris and V. sativa has been
demonstrated to be a well-known phenomenon in V. vinifera L.
(Di Vecchi-Staraz et al., 2009;De Andres et al., 2012). The detection
of naturalized hybrids in V. sylvestris populations is in accordance
with described cases of pollen flow between vineyards and wild
grapes reported by authors (Di Vecchi-Staraz et al., 2009;Riaz et al.,
2018). This level of gene flow between two subspecies, occurring
over many generations, could have strong effects and consequences
such as introgression, pollution of the gene pool, and loss of genetic
makeup on the evolution of small populations of wild grapevines
(Grassi et al., 2006). Furthermore, in few cases, our results show
evidence of hybridization between rootstocks and wild individuals,
and these cases were only found for the Lori populations. This could
be due to the existence of Phylloxera in the region since 1926, where
some rootstocks and their offspring have been detected across the
road and where grafted vineyards predominated. For example,
among collected accessions of Millardet et Grasset 420 A,
Couderc 3309 have been detected after molecular fingerprinting
and comparison based on VIVC database. For some of genotypes,
the hybrid alleles have been registered, like VVMD28 (252 bp),
VVIP31 (269 bp), and VVS2 (123 bp).
The 24 nSSR loci analyzed displayed a different range of
polymorphism, genetic diversity, and inbreeding level within the
Armenian wild grape populations, as shown by the variability of the
measured indices. We expected to see a high level of heterozygosity
because wild grapevine species are dioecious and obligate out-
crossers. According to the obtained results, the observed and
expected heterozygosity in Armenian wild populations were
higher than reported before for other wild populations that
originated from Europe and from the Mediterranean basin (Barth
et al., 2009;Lopes et al., 2009;De Andres et al., 2012;Biagini et al.,
2014), but were comparable to the results registered for the
Georgian wild grapes (Imazio et al., 2013). In another manuscript
by Doulati-Baneh et al. (2015), the genetic diversity of wild
grapevine originated from Zagros mountains was studied. Wild
grapes have been collected from five different forest locations, and
based on the data, 182 alleles were detected with an average 7.9
allele per loci. The lower genetic diversity observed in their study
according to the authors may be due to the small population size
and the effect of random drift (Doulati-Baneh et al., 2015).
The first large-scale study performed by Margaryan et al. (2021)
focused on genetic diversity and parentage analysis of 492 grapevine
genotypes collected across country. The obtained high number of
alleles (347 alleles, 14,485 allele/per loci), high level of observed and
effective heterozygosity, and presence of female APT3-allele 366,
which is absent in western European cultivars, illustrate the huge
diversity of the Armenian germplasm. Authors concluded that these
findings are related to recurrent introgression of V. sylvestris into the
cultivated compartment during domestication events. Another study
(data are not published) demonstrated clear distinction between
cultivars and wild grapes of Armenia and showed high assignment
to the sativa and sylvestris clusters. Furthermore, data also showed
mixed clades or overlaps between two clusters falling in the transition
zone and representing as admixed genotypes, indicating a possible
common gene pool for the two subspecies. The gap related to the
introgression, gene flow, and hybridization pattern between these two
subspecies are still open and could be resolved after analysis of whole
genome data, which is our ongoing activity. However, the obtained
results indicate the high genetic diversity still preserved in the wild
populations in Armenia. Previous studies based on molecular
markers showed a higher wild grapevine haplotype diversity in
Caucasus, which expressed the highest value, suggesting the area as
a possible center of origin of the species (Grassi et al., 2006).
Evaluation of population genetic structure becomes an
increasing focus providing essential insight into patterns of
migration, gene flow, and demography among “populations”
(Janes et al., 2017). The Structure is the most cited among several
clustering-based methods, which was created to provide precise
estimates without the need for populations to be determined a
priori. However, the study by Janes et al. (2017) evidenced some
facts, when the application of Structure and DK method introduced
the problem to select “true”number of clusters, and authors
concluded that many studies may have been over- or
underestimating population genetic structure.
In our study, Structure analysis provided two possibilities of
population clustering, namely, K = 2 and K = 8, as the second-order
rate of change of likelihood distribution, which characterize more
precisely the structure of Armenian wild grape populations
distributed within different biogeographic regions of Armenia.
The interpretation of K should be done having in mind that it
provides ad hoc approximation and there is probability to over- or
underestimate population structure. K=8 value was selected because
it is biologically sensible.
The approaches used to analyze the genetic structure of V.
sylvestris in Armenia showed that the wild population have distinct
groupings and differentiations based on geographic location; at the
same time, a tendency to split between the northern and southern
genotypes and values of apparently dissimilar genetic indexes was
recorded. Admixture analyses showed that hybridization is not high
and that wild and domesticated grapevines have essentially
remained reproductively isolated, most likely due to diverse relief
and the geographical distance frequently observed among vineyards
and mountains, forests, and riverbanks, where wild populations
were mainly found.
The ampelographic characterization of wild grapes is the first
important step in the conservation of V. sylvestris populations as an
essential source of genetic diversity. Traits such as dioeciousness, color
of internodes and young leaves, anthocyanin coloration on tips and
buds, opening of petiole sinus, presence of teeth on sinuses, bunch and
berry size, and seed shape are key traits to find and preserve wild
populations in Armenia. The in situ and ex situ conservation of
valuable source of wild grape diversity is essential to prevent the
extinction of these unique accessions that are potentially valuable for
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breeding programs. Nevertheless, it would not be advisable to use only
ampelography to establish relationships among wild Armenian
genotypes by geographical location or gender. Some differences may
be attributed to genetic adaption strategies to environmental
conditions rather than genetic relationships.
Until now, no systematic genetic and morphological
characterization of Armenian wild grapevine accessions has been
carried out to determine whether they correspond to bona fide V.
vinifera ssp. sylvestris individuals, naturalized cultivars, or
spontaneous hybrids. There are only a few records, and the only
detailed characterization of Armenian wild grape populations
originated from Lori province, Debed River canyon, done by D.I.
Sosnovskiy in 1947 in the book “Dikorastushchaya vinogradnaya
loza Pambakskogo ushchelya”in Russian, translated as “Wild grape
of Pambak canyon.”He described the five most important
subgroups of wild plants, which show extensive morphological
diversity. According to our observations the phenotypic
characteristics of the leaves in Lori province are varying widely,
from circular to pentagonal and wedge-shaped with three or five
lobes, with petiole sinus open or just open and blistering light or
moderate (Figure 6). Quite differently, even the pilosity of the lower
side of the leaf, ranging from absent to high, was characterized by
prostrate hairs, while erect hairs were detected only in a few cases in
the Lori population. The variable is detected also for the color of the
veins and the petiole, from light colored to the totally absent of
anthocyanin coloration, which was the dominant characteristic.
Describing the wild grape populations in Caucasus, Sosnovskiy
(1947) concluded that only wild grapes growing in Lori region
shared similar morphology with Caucasian V. sylvestries, while the
majority of wild accessions have obvious heterogenic morphology
typical of this region. At the same time, it was underlined that all
these “non-canonic”wild individuals show the obvious typical
characteristics of wild grapes: dioecious small sized plants, black
and rounded berries, and loose or very loose bunches.
Since 2018, the collection and characterization of wild grapes
growing in Syunik and in Artsakh were also carried out. The large
diversity of leaf and bunch morphology of wild grapevines growing in
Syunik province are presented in Figures 7,8. In contrast to Tavush
and Lori provinces, where wild grapes are growing in forests and river
banks, in Syunik, Artsakh, and Vayots Dzor provinces, the wild plants
are mainly growing on cliffs, climbing the rocks and in gorges.
The population of V. sylvestris that originated from Syunik has
shown the highest genetic and also phenotypic diversity in terms of
leaf, berry, and bunch morphology. The plants of Syunik province
showed weaker anthocyanin coloration on shoot tips, higher
frequency of bronze-colored young leaves, from wedge-shaped to
five-lobed and circular mature leaves, and no teeth on the petiole
sinus or in the lower and upper lateral sinus, compared to the Lori
population, where teeth are presented. Other characters found to be
discriminant traits, such as open petiole sinus of wild vines, loose
and small bunches, small dark-blue berries with a globose
(dominated shape) or ellipsoid shape, and rounded seeds.
The ellipsoid shape of berries was found only for the Syunik
population. The same phenomenon is described also for Spanish
wild populations, where an ellipsoid shape of berries was typical for
populations from the southern part of Spain compared to the
northern populations, in which spherical shaped berries are
predominant (Benito et al., 2016). All these characteristics are in
agreement with the results reported by other authors (Olmo, 1995;
Martınez de Toda and Sancha, 1999;Barth et al., 2009;Di Vecchi-
Staraz et al., 2009;Maghradze, 2022).
Nowadays, advances in both genetics and breeding programs
made use of wild grapevine plants for the introgression of disease
resistance genes and tolerance to diverse abiotic and biotic stresses
FIGURE 6
Leaf morphology of wild grapevines growing in Lori province.
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and climate environments. Mapping genes of interest associated
with agronomic and/or fruit quality traits opens up the possibility
for using molecular markers for assisted selection (MAS) (De
Lorenzis et al., 2022). The generated information on the
domestication process and genetic resources helps to understand
the gene pool available for the development of cultivars that
respond to producer and consumer requirements.
In the proposed research, for the first time, we have analyzed a
gene pools of wild grapes from different geographic origins and
genetic backgrounds with the purpose to identify potential resistance
sources against powdery mildew using three SSR markers linked to
the Ren1 and Ren1.2 loci. According to the results obtained, 28 wild
genotypes carried three alleles test, and for 34 of them, we have
detected the presence of two R alleles providing the opportunity to
eventually expand the gene pool for powdery mildew resistance
breeding. The data of sequencing of resistance genes from these wild
accessions would be very informative to gain further insight into the
evolution of powdery mildew resistance in Armenia, where possibly
powdery mildew disease existed for thousands of years and
resistance in wild populations evolved over a longer time via
FIGURE 8
Bunch morphology of wild grapes, Syunik province.
FIGURE 7
Leaf morphology of wild grapevines growing in Syunik province.
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sexual recombination. Recent studies demonstrated that the
Caucasian-resistant individuals have an allelic profile different
from the Ren1-carrying genotypes from Central Asia. It could be
hypothesized that Eurasian V. vinifera grapes may have developed
multiple and independent resistance genes located on chromosome
13 close to Ren1 genetic region. Summarizing all the arguments, we
can conclude that PM resistance is predominant in the subsp.
sylvestris from Armenia and neighbor countries and may have
been present at the time of domestication thousands of years ago.
Future studies are planned for the assessment of the structure and
evolution of the underlying local resistance genes.
5 Conclusions
The presented study is the first extensive research forwarded on
the assessment of the genetic diversity of Armenian wild grapes
using nSSR markers for molecular fingerprinting. Prospections in
traditional viticulture regions, forests, and mountainous areas
across the country provided insights into the large diversity of V.
sylvestris existing in the country. A combination of ampelography
and nuclear microsatellite markers was argued to be valuable to
determine the identity of wild plants and distinguish them from
cultivars and feral types.
Population structure and genetic diversity analyses identified
eight genetic groups extended through different geographic regions
of the country. The genetic structure analysis of wild plants revealed
groupings of population and differentiation based on geographic
locations. The tendency to split between the northern and southern
genotypes was observed. According to the admixture data
hybridization is not high and wild and domesticated grapevines
have essentially remained reproductively isolated apparently due to
diverse relief. Obtained results indicated that high genetic and
morphological diversity as a source of novel alleles and genotypes
is still preserved in the wild populations in Armenia. Based on the
preliminary screening for Ren1 resistance loci against powdery
mildew, it is necessary that future in-depth studies need to bring
more light into the unexploited genepool of Armenia. In conclusion,
this study aims to highlight the importance of V. sylvestris germplasm
conservation in Armenia as the unique genetic resource, which can
contribute to the development of improved cultivars with enhanced
disease resistance, adaptability,and quality traits.
Data availability statement
The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding author/s.
Author contributions
KM: Conceptualization, Data curation, Formal Analysis,
Investigation, Methodology, Resources, Supervision, Writing –
original draft, Writing –review & editing. RT: Writing –review
& editing. BG: Writing –review & editing. AA: Writing –review &
editing. OT: Writing –review & editing. FR: Writing –review &
editing. EM: Conceptualization, Data curation, Formal Analysis,
Investigation, Methodology, Writing –review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This
research was funded by DAAD, grant number 57552334, Ministry
of Education, Science, Culture and Sports RA, Higher Education
and Science Committee, grant number 21T-1F076 and by ANSO-
CR-PP-2020-04-A project.
Acknowledgments
The experiments and analyses were conducted at the Institute of
Grapevine Breeding, JKI. We would like to thank laboratory
technicians for their assistance. Our special acknowledgements
are dedicated to our colleague Dr Gagik Melyan, who,
unfortunately, passed away. We acknowledge the support given
by FAST (Foundation of Armenian Science and Technology) in the
frame of the ADVANCE program. The authors also want to
acknowledge the “Areni-1 Cave”Consortium (“Areni-1 Cave”
Scientific-Research Foundation, Armenia and “Gfoeller
Renaissance”Foundation, USA) team members for their support
in collecting of samples.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1276764/
full#supplementary-material
Margaryan et al. 10.3389/fpls.2023.1276764
Frontiers in Plant Science frontiersin.org14
References
Alishan, G. (1895). Haybusak kam haykakan busabarutyun (in Armenian) (St.
Lazzaro, Venice).
Arnold, C., Gillet, F., and Gobat, J. M. (1998). Situation de la vigne sauvage Vitis
vinifera L. ssp. silvestris (Gmelin) Hegi en Europe. Vitis 37 (4), 159–170.
Barth, S., Forneck, A., Verzeletti, F., Blaich, R., and Schumann, F. (2009). Genotypes
and phenotypes of an ex situ Vitis vinifera ssp. sylvestris (Gmel) Beger germplasm
collection from the Upper Rhine Valley. Genet. Resour. Crop Evol. 56 (8), 1171–1181.
doi: 10.1007/s10722-009-9443-1
Benito, A., Muñoz-Organero, G., De Andres, M. T., Ocete, R., Garcıa-Muñoz, S.,
Lopez, M.A
., et al. (2016). Ex situ ampelographical characterisation of wild Vitis
vinifera from fifty-one Spanish populations. Aust. J. Grape Wine Res. 23, 143–152.
doi: 10.1111/ajgw.12250
Biagini, B., De Lorenzis, G., Imazio, S., Failla, O., and Scienza, A. (2014). Italian wild
grapevine (Vitis vinifera L. subsp. sylvestris) population: insights into eco-geographical
aspects and genetic structure. Tree Genet. Genomes 51369–1385. doi: 10.1007/s11295-
014-0767-4
Dallakyan, M., Esoyan, S., Gasparyan, B., Smith, A., and Hovhannisyan, N. (2020).
Genetic diversity and traditional uses of aboriginal grape (Vitis vinifera L.) varieties
from the main viticultural regions of Armenia. Genet. Resour Crop Evol. 67, 999–1024.
doi: 10.1007/s10722-020-00897-5
De Andres, M. T., Benito, A., Perez-Rivera, G., Ocete, R., Lopez, M. A., Gaforio, L.,
et al. (2012). Genetic diversity of wild grapevine populations in Spain and their genetic
relationships with cultivated grapevines. Mol. Ecol. 21, 800–816. doi: 10.1111/j.1365-
294X.2011.05395.x
De Lorenzis, G., Carbonell-Bejerano, P., Toffolatti, S. L., and Tello, J. (2022).
Editorial: Advances in grapevine genetic improvement: Towards high quality,
sustainable grape production. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.1080733
Di Vecchi-Staraz, M., Laucou, V., Bruno, G., Lacombe, T., Gerber, S., Bourse, T., et al.
(2009). Low level of pollen-mediated gene flow from cultivated to wild grapevine:
consequences for the evolution of the endangered subspecies Vitis vinifera L. subsp.
silvestris. J. Hered. 100, 66–75. doi: 10.1093/jhered/esn084
Dong, Y., Duan, S., Xia, Q., Liang, Z., Dong, X., Margaryan, K., et al. (2023). Dual
domestications and origin of traits in grapevine evolution. Science 379 (6635), 892–901.
doi: 10.1126/science.add8655
Doulati-Baneh, H., Mohammadi, S., Labra, M., De Mattia, F., Bruni, I., Mezzasalma,
V., et al. (2015). Genetic characterization of some wild grape populations (Vitis vinifera
subsp. sylvestris) of Zagros mountains (Iran) to indentify a conservation strategy. Plant
Genet. Resour. 13 (1), 27–35. doi: 10.1017/S1479262114000598
Earl, D. A., and vonHoldt, B. M. (2012). STRUCTURE HARVESTER: a website and
program for visualizing STRUCTURE output and implementing the Evanno method.
Conserv. Genet. Resour. 4, 359–361. doi: 10.1007/s12686-011-9548-7
Evanno, G., Regnaut, S., and Goudet, J. (2005). Detecting the number of clusters of
individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–
2620. doi: 10.1111/j.1365-294X.2005.02553.x
Excoffier,L.,Smouse,P.E.,andQuattro,J.M.(1992).Analysisofmolecularvariance
inferred from metric distance among DNA haplotypes: application to human
mitochondrial DNA restriction data. Genetics 131, 479–491. doi: 10.1093/genetics/131.2.479
Fechter, I., Hausmann, L., Daum, M., Sörensen, T. R., Viehöver, P., Weisshaar, B.,
et al. (2012). Candidate genes within a 143 kb region of the flower sex locus in Vitis.
Mol. Genet. Genom. 287, 247–259. doi: 10.1007/s00438-012-0674-z
Giovino, A., Guarino, C., Marchese, A., Sciarillo, R., Gianniantonio, D., Marco, T.,
et al. (2022). Genetic variability of Chamaerops humilis (Arecaceae) throughout its
native range highlights two species movement pathways from its area of origin.
Botanical J. Linn. Soc. 201 (3), 361–376. doi: 10.1093/botlinnean/boac053
Grassi, F., Labra, M., Imazio, S., Ocete, R., Failla, O., Scienza, A., et al. (2006).
Phylogeographical structure and conservation genetics of wild grapevine. Conserv.
Genet. 7, 837–845. doi: 10.1007/s10592-006-9118-9
Hobosyan, S., Gasparyan, B., Harutyunyan, H., Saratikyan, A., and Amirkhanyan, A.
(2021). Armenian Culture of Vine and Wine (Yerevan, Armenia: Publishing House of
the Institute of Archaeology and Ethnography).
Imazio, S., Maghradze, D., De Lorenzis, G., Bacilieri, R., Laucou, V., This, P., et al.
(2013). From the cradle of grapevine domestication: molecular overview and
description of Georgian grapevine (Vitis vinifera L.) germplasm. Tree Genet.
Genomes 9 (3), 641–658. doi: 10.1007/s11295-013-0597-9
Janes, J. K., Miller, J. M., Dupuis, J. R., Malenfant, R. M., Gorrell, J. C., Cullingham, C.
I., et al. (2017). The K=2 conundrum. Mol. Ecol. 26, 3594–3602. doi: 10.1111/mec.14187
Kalinowski, S. T., Taper, M. L., and Marshall, T. C. (2007). Revising how the
computer program CERVUS accommodates genotyping error increases success in
paternity assignment. Mol. Ecol. 16, 1099–1106. doi: 10.1111/j.1365-294X.2007.03089.x
Kumar, S., Stecher, G., and Tamura, K. (2016). MEGA7: molecular evolutionary
genetics analysis version 7.0 for bigger datasets. Mol. Bio l. Evol. 33, 1870–1874.
doi: 10.1093/molbev/msw054
Lala, S., Amri, A., and Maxted, N. (2018). Towards the conservation of crop wild
relative diversity in North Africa: checklist, prioritisation and inventory. Genet. Resour
Crop Evol. 65, 113–124. doi: 10.1007/s10722-017-0513-5
Laucou, V., Lacombe, T., Dechesne, F., Siret, R., Bruno, J. B., Dessup, M., et al. (2011).
High throughput analysis of grape genetic diversity as a tool for germplasm collection
management. Theor. Appl. Genet. 122, 1233–1245. doi: 10.1007/s00122-010-1527-y
Lopes, M. S., Mendonca, D., Rodrigues dos Santos, M., Eiras-Dias, J. E., and da
Camara MaChado, A. (2009). New insights on the genetic basis of Portuguese
grapevine and on grapevine domestication. Genome 52 (9), 790–800. doi: 10.1139/
g09-048
Luksic, K., Zdunic, G., Hancevic, K., Mihaljevic, M. Z., Mucalo, A. D., Maul, E., et al.
(2022). Identification of powdery mildew resistance in wild grapevine (Vitis vinifera
subsp. sylvestris Gmel Hegi) from Croatia and Bosnia and Herzegovina. Sci. Rep. 12,
2128. doi: 10.1038/s41598-022-06037-6
Maghradze, D. (2022). Wild grapevine in Georgia (Georgia: Universal, Tbilisi), 1–
388.
Margaryan,K.,Maul,E.,Muradyan,Z.,Hovhannisyan,A.,Melyan,G.,and
Aroutiounian, R. (2019). Evaluation of breeding potential of wild grape originating
from Armenia. Bio Web Conf. 15, 01006. doi: 10.1051/bioconf/20191501006
Margaryan, K., Melyan, G., Röckel, F., Töpfer, R., and Maul, E. (2021). Genetic
diversity of Armenian grapevine (Vitis vinifera L.) germplasm: molecular
characterization and parentage analysis. Biology 10, 1279. doi: 10.3390/
biology10121279
Martınez de Toda, F., and Sancha, J. C. (1999). Characterization of wild vines in La
Rioja (Spain). Am. J. Enology Viticulture 50, 443–446. doi: 10.5344/ajev.1999.50.4.443
McGovern, P. E. (2003). Ancient Wine: The Search for the Origins of Viniculture
(Princeton, NJ, USA: Princeton University Press).
Myles, S., Boyko, A. R., Owens, C. L., Brown, P. J., and Grassi, F. (2011). Genetic
structure and domestication history of the grape. Proc. Natl. Acad. Sci. U.S.A. 108,
3530–3535. doi: 10.1073/pnas.1009363108
Nanagulyan, S., Zakaryan, N., Kartashyan, N., Piwowarczyk , R., and Łuczaj, L.
(2020). Wild plants and fungi sold in the markets of Yerevan (Armenia). J.
Ethnobiology Ethnomedicine 16 (26). doi: 10.1186/s13002-020-00375-3
Nebish, A., Tello, J., Ferradas, Y., Aroutiounian, R., Martınez-Zapater, J. M., and
Ibañez, J. (2021). SSR and SNP genetic profiling of Armenian grape cultivars gives
insights into their identity and pedigree relationships. OENO One 55 (4), 101–114.
doi: 10.20870/oeno-one.2021.55.4.4815
Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proc. Natl.
Acad. Sci. U.S.A. 70, 3321–3323. doi: 10.1073/pnas.70.12.3321
Ocete, R., Del Tıo, R., and Lara, M. (1995). Les parasites des populations de lavigne
sylvestre, Vitis vinifera sylvestris (Gmelin) Hegi, des Pyrene esAtlantiques (France).
Vitis 34 (3), 191–192.
Olmo, H. P. (1995). “The origin and domestication of the Vinifera grape,”in The
Origins and Ancient History of Wine. Eds. P. E. McGovern, S. J. Fleming and S. H. Katz
(Luxembourg: Gordon and Breach), 31–43.
Peakall, R., and Smouse, P. E. (2006). GenAlex 6: Ge netic analysi s in Excel.
Population genetic software for teaching and research. Mol. Ecol. Notes 6, 288–295.
doi: 10.1111/j.1471-8286.2005.01155.x
Picq, S., Santoni, S., Lacombe, T., Latreille, M., Weber, A., Ardisson, M., et al. (2014).
A small XY chromosomal region explains sex determination in wild dioecious V.
vinifera and the reversal to hermaphroditism in domesticated grapevines. BMC Plant
Biol. 14, 229. doi: 10.1186/s12870-014-0229-z
Possamai, T., Wiedemann-Merdinoglu, S., Merdinoglu, D., Migliaro, D., De Mori,
G., Cipriani, G., et al. (2021). Construction of a high-density genetic map and detection
of a major QTL of resistance to powdery mildew (Erysiphe necator Sch.) in Caucasian
grapes (Vitis vinifera L.). BMC Plant Biol. 21, 528. doi: 10.1186/s12870-021-03174-4
Pritchard, J. K., Stephens, M., and Donnelly, P. (2000). Inference of population
structure using multilocus genotype data. Genetics 155, 945–959. doi: 10.1093/genetics/
155.2.945
Pusadee, T., Jamjod, S., Chiang, Y. C., Rerkasem, B., and Schaal, B. A. (2009). Genetic
structure and isolation by distance in a landrace of Thai rice. Proc. Natl. Acad. Sci.
U.S.A. 18 106(33), 13880–13885. doi: 10.1073/pnas.0906720106
Ramasamy,R.K.,Ramasamy,S.,Bindroo,B.B.,andNaik,V.G.(2014).
STRUCTURE PLOT: a program for drawing elegant STRUCTURE bar plots in user
friendly interface. Springerplus 3, 431. doi: 10.1186/2193-1801-3-431
Riaz, S., Boursiquot, J. M., Dangl, G. S., Lacombe, T., Laucou, V., Tenscher A., C.,
et al. (2013). Identification of mildew resistance in wild and cultivated Central Asian
grape germplasm. BMC Plant Biol. 13, 149. doi: 10.1186/1471-2229-13-149
Riaz, S., De Lorenzis, G., Velasco, D., Koehmstedt, A., Maghradze, D., Bobokashvili,
Z., et al. (2018). Genetic diversity analysis of cultivated and wild grapevine (Vitis
vinifera L.) accessions around the Mediterranean basin and Central Asia. BMC Plant
Biol. 18, 137. doi: 10.1186/s12870-018-1351-0
Riaz, S., Menendez, C. M., Tenscher, A., Pap, D., and Walker, M. A. (2020). Genetic
mapping and survey of powdery mildew resistance in the wild Central Asian ancestor of
cultivated grapevines in Central Asia. Hortic Res. 7, 104. doi: 10.1038/s41438-020-0335-z
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Margaryan et al. 10.3389/fpls.2023.1276764
Frontiers in Plant Science frontiersin.org15
Sefc, K. M., Regner, F., Turetschek, E., Glössl, J., and Steinkellner, H. (1999).
Identification of microsatellite sequences in Vitis riparia and their applicability for
genotyping of different Vitis species. Genome 42, 367–373. doi: 10.1139/g98-168
Smith, A., Bagoyan, T., Gabrielyan, I., Pinhasi, R., and Gasparyan, B. (2014). “Late
Chalcolithic and Medieval Archaeobotanical Remains from Areni-1 (Birds’Cave),
ARMENIA,”in Stone Age of ARMENIA, A Guide-book to the Stone Age Archaeology in
the Republic of ARMENIA, Monograph of the JSPS-Bilateral Joint Research Project. Eds.
B. Gasparyan and M. Arimura (Japan: Kanazawa University Press), 233–260.
Sosnovskiy, D. I. (1947). Dikorastushchaya vinogradnaya loza Pambakskogo
ushchelya (in Russian) (Yerevan, Armenia: National Academy of Science of
Armenian SSR), 1–34.
This, P., Lacombe, T., and Thomas, M. R. (2006). Historical origins and genetic
diversity of wine grapes. Trends Genet. 22, 511–519. doi: 10.1016/j.tig.2006.07.008
Vavilov, N. L. (1926). Studies on the origin of cultivated plants. Bull. Appl. Bot. 26 (2),
1–248.
Vezzulli, S., Gramaje, D., Tello, J., Gambino, G., Bettinelli, P., Pirrello, C., et al.
(2022). “Genomic Designing for Biotic Stress Resistant Grapevine,”in Genomic
Designing for Biotic Stress Resistant Fruit Crops. Ed. C. Kole (Cham, Switzerland:
Springer) 11, 6330. doi: 10.1007/978-3-030-91802-6_4
Wan, Y., Schwaninger, H. R., Baldo, A. M., Labate, J. A., Zhong , G.-Y., Simon, C.,
et al. (2013). A phylogenetic analysis of the grape genus (Vitis L.) reveals broad
reticulation and concurrent diversification during neogene and quaternary climate
change. BMC Evol. Biol. 13 (141). doi: 10.1186/1471-2148-13-141
Zhou, Y., Massonnet, M., Sanjak, J. S., Cantu, D., and Gaut, B. S. (2017). Evolutionary
genomics of grape (Vitis vinifera ssp. vinifera) domestication. Proc. Natl. Acad. Sci.
U.S.A. 114, 11715–11720. doi: 10.1073/pnas.170925711
Zohary, D., Hopf, M., and Weiss, E. (2012). “Domestication of Plants in
the Old World,”in The Origin and Spread of Domesticated Plants inSouth-West
Asia, Europe and the Mediterranean Basin,4th ed (Oxford, UK: Oxford University
Press).
Margaryan et al. 10.3389/fpls.2023.1276764
Frontiers in Plant Science frontiersin.org16
