SSR and SNP genetic profiling of armenian grape cultivars gives insights into their identity and pedigree relationships

OENO One 2021, 4, 101-114 101 © 2021 International Viticulture and Enology Society - IVES SSR and SNP genetic profiling of Armenian grape cultivars gives insights into their identity and pedigree relationships Anna Nebish1,2, Javier Tello1, Yolanda Ferradás1, Rouben Aroutiounian2, José Miguel Martínez-Zapater1 and Javier Ibáñez1 1 Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de la Rioja), Departamento de Viticultura, Logroño, 26007 La Rioja, Spain 2 Yerevan State University, Department of Genetics and Cytology, 0025 Yerevan, Armenia *corresponding author: [email protected] Associate editor: Thierry Lacombe a b s t r a c t The South Caucasus is recognised as the primary Vitis vinifera L. (grapevine) domestication centre and has a high diversity of wild and cultivated grapevines. Archaeological findings indicate that winemaking activities have existed in Armenia for more than 6,000 years, viticulture being one of the most important activities of the modern Armenian agricultural sector. Despite this relevance, some grapevines in local collections have not yet been properly identified, thus hindering the efficient conservation, characterisation and eventual use of autochthonous genetic resources. In the present study, a combined SNP and SSR profiling strategy was used for the genetic identification of a series of grapevine accessions from the Grape Collection of the International Academy of Viticulture and Winemaking in Nalbandyan, presumed to be autochthonous Armenian varieties. The results provided useful information for the correct identification of these genetic resources, revealing multiple cases of synonyms, homonyms and misnames. The genetic data made it possible to confirm the pedigree proposed for some of the cultivars identified in this study and to clarify the origin of others. In addition, we propose, for the first time, a series of new trios and duos involving autochthonous Armenian grapevines. The singularity of this genetic pool compared to other Western and Central European varieties, as well as the potential novel sources of variability in traits of interest (e.g., seedlessness) that were found, highlight the importance of improving knowledge of the Armenian grapevine genetic pool. k e y w o r d s Grapevine, homonyms, genetic identification, misnames, Simple Sequence Repeat (SSR), Single Nucleotide Polymorphism (SNP), synonyms Supplementary data can be downloaded through: https://oeno-one.eu/article/view/4815 Received: 16 July 2021 y Accepted: 8 October 2021 y Published: 10 November 2021 DOI:10.20870/oeno-one.2021.55.4.4815 © 2021 International Viticulture and Enology Society - IVES 102 OENO One 2021, 4, 101-114 Anna Nebish et al. INTRODUCTION The South Caucasus is acknowledged as the primary grapevine (Vitis vinifera L.) domestication center (This et al., 2006). The genetic characterisation of grapevines from this region indicates the uniqueness and singularity of this germplasm when compared to Western genetic pools (De Lorenzis et al., 2015; Imazio et al., 2013; Maul et al., 2015; Riaz et al., 2018), representing an important source of diversity for future breeding programmes and sustainable agriculture (Dallakyan et al., 2020). Analyses of South Caucasus grapevines by molecular markers show a moderate genetic differentiation between V. vinifera L. sativa and sylvestris subspecies (De Lorenzis et al., 2015; Ekhvaia et al., 2014; Salayeva et al., 2010), high levels of genetic diversity and heterozygosity (Arroyo-García et al., 2006; Imazio et al., 2013), and some particular alleles, scarcely represented in other genetic pools (Riaz et al., 2018). All these features are commonly found in crop domestication centers. Consistent with this evidence, archaeological and archeobotanical findings indicate that viticultural activities first began in this region during the early Neolithic Period (This et al., 2006), the earliest evidence of winemaking activities in Armenia being traced back to around 4,000 BCE (Barnard et al., 2011). Nowadays, viticulture is one of the leading branches of the Armenian agricultural sector, accounting for more than 16,500 hectares of vineyards, which mainly produce grapes for wine production. According to the Vine and Wine Foundation of Armenia (https://vwfa.am/), the Armenian grape and wine sector produced 264,000 tonnes of table and wine grapes in 2020, and more than 11 M litres of wine. More than 80 % of grape production took place in the Armavir, Ararat and Aragatsotn provinces. The presence of abundant wild grapevine populations and the Armenian edafoclimatic conditions, altitudinal variation, isolated valleys and soil types likely prompted the generation of highly diverse grape varieties that are adapted to the local conditions (Dallakyan et al., 2020), which have been exploited for grape cultivation and winemaking in Armenia until today (Barnard et al., 2011). Nowadays, some of these varieties are exclusively found in private vineyards or gardens, where owners preserve old vines for their own consumption. Other cultivars can still be found in old viticultural areas and are at risk of extinction (Dallakyan et al., 2020; Margaryan et al., 2019; Nebish et al., 2017). Attempts have been made to minimise the loss of these local genetic resources, including the establishment of a series of grapevine collections. The first Armenian grapevine collection was established in 1950 by the Armenian Scientific Research Institute of Viticulture, Winemaking and Fruit growing in Yerevan (FAO Institute Code: ARM 02); it held 850 accessions of local cultivars, interspecific bred hybrids, international cultivars and wild forms. After its closure in the early 1990s, many of these cultivars were irreversibly lost, and only around 140 varieties were stored in the three minor grapevine collections created at (i) the Institute of Botany of the National Academy of Sciences of Armenia in Yerevan (ARM005), (ii) the Scientific Center of Viticulture, Winemaking and Fruit growing in Yerevan (ARM 06), and (iii) the Armenian Academy of Viticulture, Wine-Making and Fruit-Growing in Nalbandyan (ARM011). In 2016, most of the genetic resources stored in the Nalbandyan grapevine collection were transferred to Echmiadzin to be part of the new Armenian Grape Collection of the Scientific Center of Agriculture (Margaryan et al., 2019). During this process, grape varieties were named under different appellations, and while new names were established in some areas, the old ones remained in others (Dallakyan et al., 2020). Molecular genetic studies are useful for identifying possible synonyms (accessions with identical genetic profiles, but with different names), homonyms (accessions with identical names, but with different genetic profiles) and misnames (accessions of one cultivar registered under the name of another cultivar) in grapevine collections. Such information contributes to the true-to-type identification of local varieties, making it easier to register them in national databases and to study their genetic relationships with local and international cultivars, as has been proved in numerous studies (Carka et al., 2015; Maul et al., 2015; Popescu et al., 2017). Nowadays, the combined use of nuclear Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers has proved efficient in the genetic identification of grapevine varieties, as well as in the description of first degree genetic relationships between local and international cultivars (Cunha et al., 2020; Maraš et al., 2020; Zinelabidine et al., 2012). When this information is combined with chloroplast genotype profiling, the maternal lineage of any cultivar can be determined, thus enabling its historical origin to be tracked down (Arroyo-García et al., 2006). OENO One 2021, 4, 101-114 103 © 2021 International Viticulture and Enology Society - IVES TABLE 1. List of the 37 Armenian grapevine samples (AM) included in this work. When available, we indicate accession name and code, as well as berry colour (B: Black; P: Pink; R: Red; W: White), flower sex (F: Female; H: Hermaphrodite) and formation of seeds (C: Complete; NC: No Complete). Sample ID Accession name Holding Institute Accession code Described origin Berry colour Flower sex Seeds AM01 Ararati ARM011 V-56 Autochthonous cultivar R F C AM02 Areni ARM011 IV-20 Autochthonous cultivar BH C AM03 Areni Clone ARM011 IV-68 Autochthonous cultivar BH C AM04 Avagi 1 ARM011 IV-66 Unknown B H C AM05 Avagi 2 ARM011 IV-26 Unknown B H C AM06 Avagi 3 ARM011 IV-27 Unknown B H C AM07 Avagi X ARM011 IV-28 Unknown R H C AM08 Charentsi ARM011 VI-9 Bred cultivar B H C AM09 Eraskheni ARM011 V-79-81 Autochthonous cultivar BH C AM10 Gervaghahas Karmir ARM011 IV-36 Autochthonous cultivar BF C AM11 Hakobi Vordi ARM011 IV-25 Autochthonous cultivar R H C AM12 Hastamashk ARM011 V-47-48 Autochthonous cultivar W H NC AM13 Hayreniq ARM011 IV-41 Bred cultivar B H C AM14 Kakhet ARM011 V-27-28 Autochthonous cultivar BH C AM15 Kaqavik ARM011 V-22 Autochthonous cultivar W H NC AM16 Karmir Khach - - Autochthonous cultivar R H C AM17 Karmir Muscat - - Autochthonous cultivar R F C AM18 Khach Kharji ARM011 V-23 Autochthonous cultivar W H C AM19 Kharji ARM011 V-23 Autochthonous cultivar W H C AM20 Lyustra-1 ARM011 IV-43 Unknown B H C AM21 Lyustra-2 ARM011 IV-44 Unknown B H C AM22 Mskhali ARM011 5/27/2006 Autochthonous cultivar W H C AM23 Muscat Spitak ARM011 9/27/2010 Autochthonous cultivar W H C AM24 Nazeli ARM011 IV-1-25 Bred cultivar W H NC AM25 Nerkeni ARM011 XI-8-9 Bred cultivar B H C AM26 Parvana ARM011 28 Autochthonous cultivar W H NC AM27 Qrdi Khaghogh ARM011 IV-70-71 Autochthonous cultivar W H C AM28 Sali ARM011 V-32 Autochthonous cultivar BH C AM29 Sev Araqseni ARM011 5 Autochthonous cultivar BH C AM30 Shahumyani ARM011 V-75-76 Autochthonous cultivar W H C AM31 Tokun ARM011 IV-30 Autochthonous cultivar W H C AM32 Tozot ARM011 V-31 Autochthonous cultivar BH C AM33 Vaghahas Areni ARM011 V-36 Autochthonous cultivar BH C AM34 Vani ARM011 IV-23 Bred cultivar W H C AM35 Vanki ARM011 IV-27 Autochthonous cultivar P H C AM36 X4 ARM011 IV-26 Unknown W H C AM37 Wild, unknown - - - - - - © 2021 International Viticulture and Enology Society - IVES 104 OENO One 2021, 4, 101-114 Anna Nebish et al. Despite recent efforts to characterise the genetic diversity of Armenian cultivars (Dallakyan et al., 2020; Margaryan et al., 2019; Nebish et al., 2017), local grapevine collections still hold a high number of synonyms, homonyms and misnames (Dallakyan et al., 2020), which hinders their efficient conservation, characterisation, evaluation and eventual utilisation. In addition, the genetic relationships between the Armenian cultivars have been little explored, and information regarding their pedigree and likely origin is scarce. Therefore, the aim of this study was to use SSR and SNP markers in order to carry out a genetic characterisation of a series of traditional cultivars, most of them stored in the International Academy of Viticulture and Winemaking in Nalbandyan and presumed to be autochthonous Armenian varieties. From the results, it was possible to identify almost all the analysed accessions, as well as provide pedigree information about some of them, thereby improving knowledge of the Armenian grapevine genetic pool. MATERIALS AND METHODS 1. Plant material Thirty-seven grapevine accessions from the Grape Collection of the Armenian Academy of Viticulture, Wine-Making and Fruit-Growing in Nalbandyan, Armavir (FAO Institute Code: ARM011) and from a local private orchard (two samples: AM16 and AM17) were analysed (Table 1). Accessions in the Nalbandyan grape collection correspond to local cultivars traditionally grown in Armenia or bred cultivars from recent breeding programmes, except for one sample (AM37) which was originally collected as a feral vine. Young leaves from all accessions were collected in situ, dried in silica gel and stored at room temperature until DNA extraction. 2. DNA extraction and genotyping Whole genomic DNA was extracted from approximately 100 mg of ground leaf tissue according to the methods described by Zinelabidine et al. (2010). DNA quality and quantity was evaluated using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA) and by visual inspection with lambda DNA on an ethidium bromide-stained agarose gel (0.8 %). All samples were then genotyped at seven SSR markers in a single multiplex polymerase chain reaction (PCR): VVMD5, VVMD7, VVMD27, VVMD32, VVS2, VrZAG62, and VrZAG79, including the reference set of six SSR markers recommended for grapevine identification (Maul et al., 2012; This et al., 2004). Multiplex PCR reaction was carried out with 5 ng of DNA, 0.07 μM of VVMD32, 0.10 μM of VrZAG62, 0.12 μM of VVS2, 0.15 μM of VVMD7 and VrZAG79, 0.35 μM of VVMD27 and 0.50 μM of VVMD5 primers using QIAGEN multiplex PCR kit (Qiagen, Hilden, Germany). The forward primer of each pair was fluorescently labelled with different dyes: 6-FAM for VVMD27 and VVMD5, VIC for VVMD32 and VrZAG62, NED for VVS2 and VVMD7, and PET for VrZAG79. Amplification reactions were performed in a Thermal Cycler T100 (Bio-Rad, Hercules, USA) using the following PCR cycle: initial denaturation at 95 °C for 15 min, followed by 30 cycles of 95 °C for 45 s, 55 °C for 60 s, and 72 °C for 30 s, and a final extension step at 72 °C for 1 h. Sample AM23 was genotyped for one additional SSR marker (VVMD28), and samples AM08 and AM25 for four additional SSRs (VrZAG112, VrZAG29, VrZAG67 and VrZAG83), following the methods detailed in Ibáñez et al. (2009). DNA amplification products were mixed with 20 μl of highly deionized (Hi-Di) formamide, and 0.2 μl of GeneScan-500 LIZ size standards (both from Applied Biosystems, Foster City, CA, USA) and denaturalised at 95 °C for 5 min prior to capillary electrophoresis, which was performed in the genotyping platform of the Centro de Investigación Biomédica de La Rioja (CIBIR). Fragment sizes were determined by means of GeneMapper v.4.1 (Applied Biosystems, Darmstadt, Germany). In each analysis, one Tempranillo Tinto DNA was included as a positive control. In parallel, DNAs were profiled for a set of 240 nuclear SNP markers applying Fluidigm technology, through the genotyping services provided by the Sequencing and Genotyping Unit of the Universidad del País Vasco (UPV/EHU). This set includes a core set of 48 SNPs used for cultivar identification and an extended set of 192 SNPs for parentage and diversity analyses (Cabezas et al., 2011; Cunha et al., 2020; Lijavetzky et al., 2007). The core set of 48 SNPs contained three chloroplast SNPs for the identification of the four most common grapevine chloroplast haplotypes (A, B, C and D) (Arroyo-García et al., 2006). 3. Variety identification Non-redundant 48-SNP genetic profiles were compared pair-wise with those stored in the ICVV-SNP database for cultivar identification. OENO One 2021, 4, 101-114 105 © 2021 International Viticulture and Enology Society - IVES To date, this database contains more than 3,000 unique 48-SNP genotypes. In parallel, non-redundant SSR genetic profiles were compared with those stored in the VIVC (Maul, 2020), the European Vitis Database (Maul et al., 2012) and in the Armenian Vitis Database (Margaryan et al., 2019) for cultivar identification within a broader context. 4. Phylogenetic and parentage analyses An Unweighted Neighbor Joining (UwNJ) distance tree was calculated to explore the relationship between the Armenian cultivars considered in this work and a group of 27 traditional grapevine cultivars from ten European countries: Austria (2 cultivars), France (5), Germany (2), Italy (5), Moldova (1), Montenegro (2), Portugal (2), Serbia (1), Slovenia (2) and Spain (5) (Supplementary Table 1). The 240-SNPs genetic profiles of these 27 cultivars were retrieved from the ICVV-SNP database. For this analysis, a dissimilarity matrix with 10,000 bootstrap steps was calculated using the DARwin software package v. 6.0.21 (Perrier and Jacquemond-Collet, 2006), discarding those SNPs with any missing data (90 SNPs were removed). This matrix was used to construct an UwNJ distance tree based on 1,000 bootstrap replicates. Similarly, another UwNJ distance tree was constructed for the Armenian cultivars analysed in this study following the same procedure (81 SNPs were removed), using VIVC information of cultivar main use (wine/table/ multi-purpose) for graphical representation. For parentage analyses, the non-redundant 240-SNPs genotypes obtained in this study were merged with those stored in the ICVV-SNP database to detect possible first-order kinship relationships (trios and duos or parent–offspring pairs), using the likelihood-based method implemented in Cervus v.3.0 (Kalinowski et al., 2007) as previously described (Cunha et al., 2020). The likelihood of each detected trio and duo was evaluated taking into account the natural logarithm of the overall likelihood ratio (LOD) score. The maximum number of mismatching loci for trios and duos was set to 2 and 1 respectively, and only duos with LOD > 25 were considered. For each trio, chloroplast haplotype information was used to determine which of the putative parents acted as mother, according to the maternal transmission of chloroplasts in grapevine (ArroyoGarcía et al., 2006). RESULTS The combination of SNP and SSR genotyping of the 37 accessions explored in this work revealed 27 different genetic profiles (Table 2 and Supplementary Table 2). Twenty-four grapevine cultivars were identified via the parallel comparison of the 48-SNP genetic profiles with those stored in the ICVV-SNP database, and the 7-SSR genetic profiles with international databases; the genetic profile of three accessions (AM16, AM17 and AM37), however, did not match the genetic profile of any previously registered grapevine varieties (Table 2). The set of 7-SSRs markers was informative enough for the identification of most of the accessions analysed here, except for AM23 (‘Muscat Spitak’), whose genetic profile for these seven loci matched with those of ‘Muscat Ottonel’ (VIVC variety number 8243) and ‘Muscat St. Laurent’ (VIVC variety number 8252). According to the VIVC information, these two cultivars are full siblings from ‘Chasselas Blanc’ × ‘Ingram’s Muscat’, and they differ for VVMD28 (‘Muscat Ottonel’: 258:268; ‘Muscat St. Laurent’: 218:246). After genotyping AM23 for VVMD28 by means of a simplex PCR, this accession was confidently identified as being ‘Muscat Ottonel’ (Supplementary Table 2), confirming the results obtained when comparing its 48-SNP genetic profile with those stored in the ICVV-SNP database, for which ‘Muscat Ottonel’ and ‘Muscat St. Laurent’ differ in 23 SNPs. Moreover, accessions AM08 (‘Charentsi’) and AM25 (‘Nerkeni’) showed the same genetic profile for the seven SSRs initially screened and for the four SSRs genotyped in an additional multiplex PCR (VrZAG112, VrZAG29, VrZAG67 and VrZAG83), but they differed in 14 of the 240 SNPs used for genetic profiling (Supplementary Table 2). For these 14 SNPs, AM25 was found to be homozygous for one of the two different alleles that were present in a heterozygous manner in AM08. Given their different SNP genetic profiles, these two accessions can be considered as two different cultivars. As can be seen in Table 2, the vast majority of the 24 identified cultivars (19) are already registered in the VIVC as Armenian cultivars, whilst three are from neighbouring or Near East countries like Afghanistan (‘Kandahari Siah’), Turkmenistan (‘Eskeri’) or Uzbekistan (‘Kishmish Khishrau’), and one is from France (‘Muscat Ottonel’). The most commonly found variety was ‘Areni Sev’, which was found six times, followed by ‘Areni Spitak’, ‘Eskeri’, ‘Hakobi Vordi’, ‘Khusaine © 2021 International Viticulture and Enology Society - IVES 106 OENO One 2021, 4, 101-114 Anna Nebish et al. TABLE 2. Results of the genetic identification of 37 grapevine accessions obtained by SNP and SSR genetic profiling. Variety Name* Short Name N (Sample/s ID) Variety number* SNP-ICVV genotype code Chlorotype Use* Origin* Arakseni Chernyi ACH 1 (AM29) 530 1417 D T Armenia Areni Sev ASE 6 (AM02, AM03, AM04, AM06, AM14, AM21) 576 3834 BT/W Armenia Areni Spitak ASP 2 (AM27, AM36) 577 3933 C W Armenia Charentsi CHA 1 (AM08) 2459 4009 C W Armenia Eghegnadzori Sev EGH 1 (AM05) 26229 4008 C W Armenia Eraskheni ERA 1 (AM09) 3924 4010 D T/W Armenia Eskeri ESK 2 (AM12, AM24) 3960 204 C T/R Turkmenistan Hakobi Vordi HVO 2 (AM07, AM11) 24880 3932 C W Armenia Hayastan HAY 1 (AM34) 22017 4022 C T Armenia Hayreniq HYR 1 (AM13) 16453 4012 C T Armenia Kandahari Siah KAS 1 (AM10) 5956 305 D T Afghanistan Karmir Kakhani KKA 1 (AM01) 6000 879 C T Armenia Karmir Khach KKH 1 (AM16) - 4014 C - - Karmir Muscat KMU 1 (AM17) - 4015 C - - Khardji Sev KHS 1 (AM20) 25976 4017 C W Armenia Khatun Khardzhi KHK 1 (AM19) 6168 4016 D W Armenia Khusaine Belyi KHB 2 (AM26, AM30) 6203 315 C T Armenia Kishmish Khishrau KIK 1 (AM15) 6258 4021 C T Uzbekistan Masis MAS 1 (AM22) 7470 4024 D T Armenia Muscat Ottonel MUO 1 (AM23) 8243 755 C T/W France Nerkeni NER 1 (AM25) - 4020 C - - Tokun TOK 1 (AM31) 12558 4023 D T/R/W Armenia Tozot TOZ 2 (AM28, AM32) 12600 3823 C W Armenia Unknown UNK 1 (AM37) - 3949 C - - Vaghahas Areni VAR 1 (AM33) 25978 3838 C W Armenia Vanki VAN 1 (AM35) 25977 4013 D W Armenia Voskeat VOS 1 (AM18) 13165 1450 D T/W Armenia *According to VIVC database. For Use, T: Table grape; R: Raisin grape; W: Wine grape. OENO One 2021, 4, 101-114 107 © 2021 International Viticulture and Enology Society - IVES Belyi’, and ‘Tozot’, all of them found twice. Regarding their main use, eight of the identified genotypes are registered in the VIVC database as table grape cultivars, nine as wine grape cultivars and six as cultivars with multiple uses. Chlorotype C was found to be the most abundant within the different genotypes analysed (18 times, 66.6 %), followed by chlorotype D (8 times, 29.6 %) and chlorotype B (once, 3.7 %) (Table 2). The UwNJ distance tree constructed from the 27 unique genetic profiles identified in this work and 27 profiles from autochthonous grapevine cultivars from diverse European countries grouped the genotypes into two clusters that clearly reflected their origin (Figure 1A). The UwNJ tree constructed exclusively with the 27 unique genetic profiles identified in this study generated two main clusters (Figure 1B), which mostly reflected the main use of the cultivar, as recorded in the VIVC database. Thus, one cluster grouped most of the varieties with a clear aptitude for winemaking (such as the white-berried cultivar ‘Areni Spitak’, or the black-berried cultivars ‘Khardji Sev’ and ‘Tozot’), whilst the other grouped most of those suitable for table grape production (such as the cultivars ‘Hayastan’, ‘Hayreniq’ and ‘Masis’). Parentage analysis based on SNP data was useful for revealing the full pedigree (trio) of eight of the genotypes identified in this work (Table 3), as well as eight first-order genetic relationships (Table 4). The full pedigrees identified for ‘Kishmish Khishrau’, and ‘Muscat Ottonel’ had been already reported and confirmed in the literature (Lacombe et al., 2013), but it is the first time that the remaining six pedigrees (either previously reported in the literature or not) are FIGURE 1. Unweighted neighbor-joining (UwNJ) unrooted trees obtained from SNP data for (a) 27 unique genotypes analysed in this work (in orange; ACH (Arakseni Chernyi), ASE (Areni Sev), ASP (Areni Spitak), CHA (Charentsi), EGH (Eghegnadzori Sev), ERA (Eraskheni), ESK (Eskeri), HVO (Hakobi Vordi), HAY (Hayastan), HYR (Hayreniq), KAS (Kandahari Siah), KKA (Karmir Kakhani), KKH (Karmir Khach), KMU (Karmir Muscat), KHS (Khardji Sev), KHK (Khatun Khardzhi), KHB (Khusaine Belyi), KIK (Kishmish Khishrau), MAS (Masis), MUO (Muscat Ottonel), NER (Nerkeni), TOK (Tokun), TOZ (Tozot), UNK (Unknown), VAR (Vaghahas Areni), VAN (Vanki), VOS (Voskeat)) and 27 genotypes for Western and Central Europe countries (in blue; ALE (Aledo), ALV (Alvarelhao), ARA (Aramon), BLA (Blaufraenkisch), BOB (Bobal), CAS (Castelao), COA (Coarna Alba), ELW (Elbling Weiss), GRA (Graciano), KRA (Kratošija), KRS (Krstac), LIP (Listán Prieto), MER (Merlot), MDT (Moscato di Terracina), PIN (Pinot), POB (Portugieser Blau), PRO (Prokupac), RIW (Riesling Weiss), SCG (Schiava Grossa), SEM (Semillon), SIG (Silvaner Gruen), SYR (Syrah), TEM (Tempranillo), TRT (Trebbiano Toscano), VER (Vermentino), VUL (Vulpea), WEL (Welschriesling)); and (b) 27 unique genotypes analysed in this work coloured according to their main use description available in the VIVC database (green: table grape; red: wine grape; purple: multi-purpose grape; grey: unknown). A B © 2021 International Viticulture and Enology Society - IVES 108 OENO One 2021, 4, 101-114 Anna Nebish et al. Offspring* Chlorotype Parent 1 (mother) Chlorotype Parent 2 (father) Chlorotype SNPs compared Mismatching SNPs LOD score Reference Areni Spitak (VIVC: 577) CTozot (VIVC: 12600) CAreni Sev (VIVC: 576) B223 0 100.3 - Hayreniq (VIVC: 16453) CAngur Kalan (VIVC: 8561) CAlphonse Lavallee (VIVC: 349) B220 0 88.9 - Nerkeni (ICVV: 4020) CCharentsi (VIVC: 2459) CCharentsi (VIVC: 2459) C 201 0 83.8 - Kishmish Khishrau (VIVC: 6258) CAngur Kalan (VIVC: 8561) CKishmish Chernyi (VIVC: 6256) D 215 0 81.6 Lacombe et al. (2013) Hayastan (VIVC: 22017) CAngur Kalan (VIVC: 8561) CItalia (VIVC: 5582) C 220 0 80.5 - Muscat Ottonel (VIVC: 8243) CIngram’s Muscat (VIVC: 5531) CChasselas Blanc (VIVC: 2473) D 229 1 73.9 Laucou et al. (2018) Khusaine Belyi (VIVC: 6203) CDzhandzhal Kara (VIVC: 3760) CUnknown (ICVV: 3949) C 223 2 70.2 - Karmir Muscat (ICVV: 4015) CAngur Kalan (VIVC: 8561) CMathiasz Janosne (VIVC: 7503) B220 0 68.2 - TABLE 3. Summary of the full trios detected by SNP analyses involving (at least) one of the genotypes identified in this work. If available, the VIVC variety number is given between brackets. If not, the SNP-ICVV genotype code is provided. *For Khusaine Belyi (VIVC: 6203), both parents could have acted as parent 1 (mother) or parent 2 (father). OENO One 2021, 4, 101-114 109 © 2021 International Viticulture and Enology Society - IVES Variety 1 Variety 2 SNPs compared Mismatching SNPs LOD score Reference Muscat Ottonel (VIVC: 8243) Muscat St. Laurent (VIVC: 8252) 222 0 62.9 Lacombe et al. (2013) Areni Sev (VIVC: 576) Khardji Sev (VIVC: 25976) 221 0 48.1 - Vanki (VIVC: 25977) Khatun Khardzhi (VIVC: 6168) 223 0 46.3 - Masis (VIVC: 7470) Mskhali (VIVC: 8109) 211 0 44.4 - Vaghahas Areni (VIVC: 25978) Madeleine Angevine (VIVC: 7062) 218 0 37.7 - Eskeri (VIVC: 3960) Sultanina (VIVC: 12051) 211 0 37.6 Lacombe et al. (2013) Karmir Khach (ICVV: 4014) Karmir Kakhani (VIVC: 6000) 223 0 35.0 - Areni Spitak (VIVC: 577) Khardji Sev (VIVC: 25976) 221 1 28.2 - TABLE 4. Summary of the first-order genetic relationships (duos) detected by SNP analyses involving (at least) one of the genotypes of the Armenian samples studied in this work. If available, the VIVC variety number is given between brackets. If not, the SNP-ICVV genetic code is provided. supported by molecular analyses. All pedigrees were fully confirmed by means of the SSR data available, except the one proposed for ‘Khusaine Belyi’, which has one allele for VVS2 (147) absent in its suggested two genitors (‘Khusaine Belyi’ is 147:155, whereas ‘Dzhandzhal Kara’ and ‘Unknown’ (AM37) are 125:155 and 143:155 respectively) (Supplementary Table 2) (Maul, 2020). Moreover, our results indicate a relevant role for the female cultivar ‘Angur Kalan’ in the genotypes identified here, being a genitor in four of the eight full trios identified in this study. In addition, the genotype obtained for ‘Nerkeni’ was found to be compatible with a self-cross of the genotype obtained for ‘Charentsi’. Lastly, six new duos were identified within the genotypes explored in this study; as shown in Table 4, in many of them the cultivars involved were Armenian, indicating the close pedigree relationships that exist among local grapevines. DISCUSSION Ancient and almost neglected cultivars in the South Caucasus have been found to have unique traits for facing current viticulture challenges (Bitsadze et al., 2015; Sargolzaei et al., 2021); this highlights the relevance of exploring local grapevine germplasm as alternative genetic sources of variability for future breeding programmes. The identification of local Armenian grapevines is the first step in their study and eventual use; however, recent reports have shown that many have not been properly identified (Dallakyan et al., 2020; Margaryan et al., 2019; Nebish et al., 2017). In the present study, the analysis of 37 grapevine local accessions by means of SNP and SSR markers resulted in the identification of 27 unique genotypes, of which 24 were already recorded in the VIVC database, most of them as Armenian cultivars (Table 1). Therefore, three new genetic profiles, which were not registered in the three international databases used in the study, were identified: ‘Karmir Khach’, ‘Karmir Muscat’ and ‘Unknown’. These three samples were obtained from a private orchard (‘Karmir Khach’ and ‘Karmir Muscat’, both locally appreciated for fresh fruit production), and from the wild (‘Unknown’). Although the latter sample was collected in the wild, it is unlikely that it is a sylvestris plant, since in a preliminary genetic structure analysis between European sativa and sylvestris genotypes it clustered with the sativa individuals (data not shown). In addition, it was identified as one of the potential genitors of the cultivar ‘Khusaine Belyi’ (Table 3), so it is more likely that this ‘Unknown’ genotype corresponds to an ancient cultivar in traditional Armenian viticulture. The parent-offspring relationship