molecular characterization of vitis vinifera l. local

Molecular characterization of Vitis vinifera L. local cultivars from volcanic areas (Canary Islands and Madeira) using SSR markers

Gemma Marsal1, Juan Jesús Méndez2, Josep Maria Mateo3, Sergi Ferrer4, Juan Miguel Canals1, Fernando Zamora1 and Francesca Fort*1

1Grup de Tecnologia Enològica (TECNENOL), Departament de Bioquímica i Biotecnologia, Facultat d’Enologia,Universitat Rovira i Virgili, Campus Sescelades, C/ Marcel·lí Domingo, 1. E-43007-Tarragona, Spain2Bodegas Viñátigo, Travesía Juandana, E-38441-La Guancha Santa Cruz de Tenerife, Islas Canarias, Spain3Grup CRISES, Departament d’Enginyeria Informàtica i Matemàtiques, Universitat Rovira i Virgili, Av. Països Catalans, 26. E-43007-Tarragona, Spain4Grup Enolab, Departament de Microbiologia, Facultat de Biologia, Universitat de València, C/ Doctor Moliner, 50.E-46100 Burjassot-València, Spain

*Corresponding author: [email protected]

Aim: This study characterises and identifies 79 grapevine accessions from the Canary Islands and 14 from Madeirausing simple sequence repeat (SSR) analysis.Methods and Results: A kit of 20 microsatellites or simple sequence repeats (SSRs) were used to obtain themolecular profiles of the 93 accessions in this study. The results allowed us to identify four new cultivars(Bienmesabe tinto, Burra volcanica, Vallera, Verijadiego negro), two new colour mutations (Listan rosa, Mollarcano rosado) and two unknown molecular profiles from Madeira. Furthermore, we propose that eight names ofvarieties be included in the Vitis International Variety Catalogue (VIVC) as prime names, and 38 accession names assynonyms, 19 of which are regarded as new synonyms of the 12 varieties. Finally, we also reported eight cases ofmislabelling. The study of genetic structure shows that the cultivars from the Canary Islands and Madeira arestrongly influenced by the Iberian Peninsula. We propose that 14 varieties and three sports (mutations) at are local tothe Canary Islands: Albillo criollo, Bermejuela, Bienmesabe tinto, Burra volcanica, Albillo forastero, Huevo degallo, Listan negro, Listan rosa, Malvasia di Sardegna rosada, Malvasia volcanica, Mollar cano rosado, Torrontesvolcanico, Sabro, Uva de año, Vallera, Verijadiego, and Verijadiego negro.Conclusions: It has been reliably shown that these cultivars have a characteristic genome: phylloxera never reachedthe Canary Islands so mutations, hybridations and human selection have been able to accumulate over 500 years. Itis of great importance that this local plant material be conserved, given that it is part of our vine heritage. In the caseof Madeira, it has not been possible to propose any local varieties because phylloxera did reach the island; thisdevastated the vineyards and there was a drastic reduction in local varieties. However, one unknown cultivar was themost characteristic genotype from this region.Significance and impact of the study: This study shows the existence of non well-known varieties of Vitisvinifera L. that may be used to elaborate original wines, offering therefore new organoleptic sensations for theconsumers. Furthermore, these results suggest that this volcanic area could be considered as one centre of origin ofnew cultivars of Vitis vinifera L. (Biodiversity Centre).

grapevine, microsatellite, identification, volcanic islands, DNA, molecular profile

AB S TRACT

KEYWORD S

Received: 22 February 2019 y Accepted: 14 Juillet 2019 y Published: 21 October 2019DOI:10.20870/oeno-one.2019.53.4.2404

V I N E A N D W I N EOPEN ACCESS JOURNAL

667OENO One 2019, 4, 667-680 © 2019 International Viticulture and Enology Society - IVES

INTRODUCTION

Vitis vinifera L., the grapevine, is one of the mostimportant fruit species in the modern world. It isindigenous to southern Europe and western Asia,and is today cultivated worldwide. Wine andtable grape sales contribute significantly to theeconomy of the major wine-producing countries.Vitis vinifera L. has a diploid genome with38 haploid chromosomes and an estimatedgenome size of ~500 Mbp. Grape genotypes arehighly heterozygous and nearly all moderncultivated varieties (cultivars) are hermaphro-ditic, self-fertile and out-cross easily (This et al.,2006).

Macaronesia is a group of four North Atlanticarchipelagos that extends outwards from thesouthwest (SW) of Europe to the southwest(NW) of Africa. This group consists of theAzores, Madeira, the Canary Islands and CapeVerde (Santamarta and Naranjo Borges, 2015).The present study focuses on the volcanicarchipelagos of the Canary Islands and, to alesser extent, on Madeira.

The Madeira archipelago is an autonomousregion of the Republic of Portugal, which is520 km from the African coast and 1,000 kmfrom the European continent. It includes theislands of Madeira, Porto Santo, and the DesertasIslands, which are administered together with theseparate archipelago of the Savage Islands. Thegeneral climate of the Madeira archipelago ismild, oceanic and humid, with moderate rainfall.It is greatly influenced by the subtropicalanticyclone of the Azores and is mainlygoverned by the trade winds from the north andnortheast (GEVIC (Gran Enciclopedia VirtualIslas Canarias), 2007).

The Canary archipelago is one of theautonomous communities of Spain. The islandssit just off the northwest coast of mainlandAfrica, 100 km west of the border betweenMorocco and Western Sahara. The main islands,from largest to smallest, are Tenerife,Fuerteventura, Gran Canaria, Lanzarote, LaPalma, La Gomera, El Hierro and La Graciosa.The archipelago also has other islands and islets:Alegranza, Isla de Lobos, Montaña Clara, Roquedel Oeste and Roque del Este. The climate istropical and desert, moderated by the sea and inthe summer by the trade winds. However, thatcan vary considerably as a function of altitude,orientation and orography, and different areas

(coasts, mid-lying areas and mountain areas)have quite different precipitation, humidity,winds, etc. Generally, the precipitation patternsshow little and highly varied rainfall. The varietyof ecosystems and microclimates is favourable tothe existence and development of many plantspecies, such as Vitis vinifera L. (GEVIC (GranEnciclopedia Virtual Islas Canarias), 2007).

Several hypotheses have been put forward toexplain the colonization of Vitis vinifera L. in theCanary and Madeira Islands. One hypothesisdates back to a half century before Christ, whenQuinto Horacio Flaco claimed that “unprunedvine flowered continuously in the fortunate”(Moralejo Alvarez, 2011). However, for sometime, it has been widely accepted that the vinewas not part of the original flora of the CanaryIslands and Madeira. Nevertheless, since seedsof the Vitaceae family have been found inseveral archaeological sites in the Canary Islands(Arco et al., 2000), the theory that the vine is notpart of the autochthonous flora has beenreconsidered. The vine seeds found also seem tobe wild and very similar to the North Africanbiotypes of Vitis vinifera L. Thus, populations ofVitaceae may have existed in the Islands beforethe arrival of the first human group, a thesis thatmay seem obvious because, like all the nativeflora of the Archipelago, these plants have atertiary origin; however, also like otherpopulations of this flora, wild Vitaceaedisappeared for reasons that are still unknown(Macías, 2005). Consequently, the first cultivatedvine varieties were introduced by Europeancolonization. Europeans first visited theFortunate Islands (Macaronesia) until thefourteenth century, but it was not until thefifteenth century that monks, explorers,conquerors and traders introduced the firstvarieties of cultivated vine (domesticated).

The 19th century was marked by the entry of twomajor pests: powdery mildew (1852) and mildew(1878), but surprisingly the Macaronesiaarchipelagos (except Madeira) were not attackedby the phylloxera plague that devastatedEuropean vineyards and caused a drasticreduction in local varieties. It is for this reasonthat the Canary vineyards are considered to bethe last stronghold of some of these varieties(Hidalgo, 2011). Today many viticulturist andgrowers in these islands can see new phenotypicfeatures appearing in their vines. These newphenotypes may be due to hybridizations and/orgenetic mutations accumulated over five

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© 2019 International Viticulture and Enology Society - IVES OENO One 2019, 4, 667-680668

centuries. Therefore, selection (natural oranthropological) together with vegetativepropagation (natural or anthropological) couldhave made significant changes to the first vinephenotypes over time, and have given rise tonew varieties or different clones within the samevariety (López et al., 1993).

SSRs have been the most widely used markersfor genotyping plants over the past 20 yearsbecause they are highly informative, codominantand multi-allele genetic markers (Ibáñez et al.,2003). Moreover, SSRs are experimentallyreproducible and transferable among relatedspecies. They are enormously useful in studies ofpopulation structure, genetic mapping andevolutionary processes and also forcharacterizing and identifying individuals(Emanuelli et al., 2013).

The aim of this study was the molecularcharacterization of 79 accessions from theCanary Islands (El Hierro, La Gomera, LaPalma, Lanzarote and Tenerife) and 14 acces-sions from Madeira (Madeira archipelago) usingthe simple sequence repeats (SSR) technique toplan germplasm conservation and to find geneticsources for breeding. More specifically, wecarried out the detection of different errors(synonyms, mislabelling, etc.), evaluated geneticrelationships among different varieties (possiblepedigrees), and studied the genetic structure ofthis gene pool. The phylogeny and geneticrelationships among grapevine cultivars is ofgreat importance in genetic improvement, thepreservation of biodiversity and the exploitationof traditional wines (Emanuelli et al., 2013).Furthermore, this study may help to detect newgenotypes and minimize the homogenization ofwines from the Canary Islands and Madeira, andit would provide new material for breeding.Other equally important aspects to consider arethe updating of the Vitis International VarietyCatalogue Database (VIVC) and the possibilityof discovering and demonstrating the uniquenessof the varieties of the Canary archipelago.

MATERIAL AND METHODS

1. Plant material

Ninety-three mature grapevine leaves (Vitisvinifera L.) were collected from the CanaryIslands and Madeira (79 and 14 accessions,respectively), and conserved at -20°C until theywere processed. Four well-characterizedcultivars were also included as control samples

(Marsal et al., 2011): Chardonnay blanc,Garnacha blanca, Tempranillo tinto andCabernet sauvignon cultivar plants from theRovira i Virgili University experimentalvineyard in Constantí (AOC Tarragona, Spain;41°9′16.04″ (N) and 1°11′1.28″ (E)). SupportingInformation 1 provides detailed information onall the accessions and all other necessaryinformation.

2. Microsatellite genotyping

DNA was extracted using the method describedby Marsal et al. (2013) (based on Fort et al.(2008) and Marsal et al. (2011) protocols). Thegrapevine genepool was genotyped at 20 SSRmarkers, which were selected for their capacityfor discrimination and polymorphism inagreement with previous studies: VVS2, VVS3,VVS29 (Thomas and Scott, 1993); VVMD5,VVMD6, VVMD7 (Bowers et al., 1996);VVMD27, VVMD28, VVMD36 (Bowers et al.,1999b); VrZAG21, VrZAG47, VrZAG62,VrZAG64, VrZAG79, VrZAG83 (Sefc et al.,1999); scu06vv (Scott et al., 2000); VvUCH11,VvUCH12, VvUCH19 (Lefort et al., 2002);VChr19a (Cipriani et al., 2010). Theinternational scientific community (This et al.,2004; Maul and Röckel, 2015) uses seven ofthese as reference genetic markers.

Microsatellite amplifications were performedusing polymerase chain reaction (PCR) analysisand a MyCycler thermocycler (BioRadLaboratories, Hercules, CA, USA). PCR wascarried out with 50 ng of DNA and 1 µM of eachprimer, with an attached fluorescent dye in theupper primer (6-FAM: VVS3, VVMD7,VVMD28, VVMD36, VrZAG47, VrZAG62,VrZAG83, VvUCH11 and VvUCH19; HEX:VVS2, VVS29, VVMD6, VVMD27, VrZAG21,VrZAG79 and VChr19a; NED: VVMD5,VrZAG64, scu06vv, VvUCH12) using theAmpliTaq DNA Polymerase kit (AppliedBiosystems, Foster City, CA). The SSRs weredivided into three groups according to Marsal etal. (2011). The amplification products weremixed with 20 μL of deionized formamide and0.5 μL of DNA size standard (GeneScan 500-ROX, Applied Biosystems), and denatured at95 °C for 5 min. The fragments were separatedby capillary electrophoresis with an ABI PRISM3730® Genetic Analyzer (Applied Biosystems).Peak Scanner Software (Applied Biosystems)was used to size the amplified fragments. Each

© 2019 International Viticulture and Enology Society - IVESOENO One 2019, 4, 667-680 669

cultivar was analysed twice to prevent possibleerrors.

3. Data analysis

GenAlEx 6.5 software (Peakall and Smouse,2012) was used to estimate the four geneticparameters: the number of different alleles (Na),the number of effective alleles (Ne), observedheterozygosity (Ho), and expectedheterozygosity (He). The probability of identity(PI) and the estimated frequency of null alleles(r) were calculated using th software Identity 1.0(Wagner and Sefc, 1999). To distinguishhomozygotes and heterozygotes for each locus,the data were considered codominant for thepurposes of data analysis.

Population structure and identification ofadmixed individuals was performed using themodel-based software program Structure 2.3(Pritchard et al., 2000; Falush et al., 2003),which is a model-based Bayesian clusteringmethod. In this model, a number of populations(K) are assumed to be present, and they are eachcharacterized by a set of allele frequencies atevery locus. Individuals in the sample areassigned to populations (clusters), or jointly tomore populations if their genotypes indicate thatthey are admixed. All loci are assumedindependent, and each K population is assumedto follow the Hardy–Weinberg equilibrium. Thesubsequent probabilities were estimated usingthe Markov Chain Monte Carlo (MCMC)method. The MCMC chains were run with a100,000 burn-in period, followed by 1,000,000iterations using a model allowing for admixtureand correlated allele frequencies. The structurewas run at least ten times by setting K from 1 to15, and an average likelihood value, L (K), wascalculated across all runs for each K. The meanlog probability of the data for each K wascalculated to determine the most appropriatenumber of clusters, and the value of K for whichthis probability was highest was selected. TheΔK was then calculated using the methoddescribed by Evanno et al. (2005). ΔK is aquantity based on the rate of change in the logprobability of the data between successive Kvalues.

In addition, principal coordinate analysis (PCoA)in GenAlEx 6.5 was used to further examine thegenetic relationships between subpopulations onthe basis of the same SSR data. PCoA was based

on the standardized covariance of the geneticdistances calculated for codominant markers.

The frequency-based assignment test (Paetkau etal., 1995; Paetkau et al., 2004), also available inGenAlEx 6.5, was first used to assign theaccessions to each subpopulation generated byStructure. For each accession, a log likelihoodvalue was calculated for each subpopulationusing the allele frequencies of the respectivesubpopulations. An individual was assigned tothe subpopulation with the highest log likelihoodvalue.

Identity 1.0 software was also used to identifyputative parentage relationships (Wagner andSefc, 1999). This software prepares a list of theprobable parent-progeny relationships on thebasis of codominant inheritance (i.e. when theprogeny receives one allele from one parent andthe other allele from the other).

RESULTS

1. SSR polymorphism

The characterization of the efficiency of the20 SSR markers studied is shown in SupportingInformation 2 for a population of 44 varieties,which correspond to 41 molecular profile-SSR(MP-SSR) that are unique (without sports). Intotal, 49 out of the 93 initial accessions were notincluded because the results obtained indicatedthat they were synonyms of other accessions.Our population displayed between 5 and29 alleles per locus, with a total of 257 allelesover the 20 loci, an unbiased expectedheterozygosity (He) of 78.7 %, and 14 markershad an “r” value (the value of null alleles) lowerthan or equal to 0.01. The probability ofobtaining identical genotypes using all20 markers is 2.4 x 10–26 (cumulative PI*).

Seven SSRs for all 44 cultivars identified areshown in Supporting Information 3, togetherwith the four control samples. In this case, the14 varieties proposed as local next to the threecolour mutations from Canary Islands werehighlighted (14 varieties in blue and three casesof sports in green).

2. Cultivar analysis

2.1. Confirmation of accession name

The objective of this analysis was, first, to carryout an exhaustive bibliographic study to find outwhether the name of the accession is

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internationally known or not, and, second, tocompare the MP-SSR obtained throughmicrosatellite analysis with the MP-SSR foundin the bibliographic sources (VIVC, Maul andRöckel, 2015; Rodríguez-Torres, 2018; VitisCanarias, 2015).

For this reason, the 93 accession names werereviewed in the ampelographic section of theVIVC (Maul and Röckel, 2015), and in otherbibliographical resources specialized in varietiescultivated in the Canary Islands (Zerolo et al.,2006; Rodríguez-Torres, 2018; Vitis Canarias,2015). All the information obtained from thisbibliographic study is summarised in SupportingInformation 4.

A total of 41 grapevine varieties and three colourmutations from the 93 accessions analysed wereidentified by the bibliographic study and themicrosatellite analysis (Supporting Information1, 3, and 4). Moreover, 49 accessions weresynonyms or repetitions of other accessions. Ofthe 93 accessions, 91 were identified, of whichthere were 41 different varieties and three sports.Four of these genetic profiles coincide withothers that do not have a described name. Forthat reason, we considered it necessary to givethem a specific name after discussing with thescientist who identified them (I. Rodríguez-Torres). The selection of these new names tookinto account their history, their morphology ortheir organoleptic characteristics and thefollowing prime names are proposed:Bienmesabe tinto, Burra volcanica, Torrontesvolcanico and Vallera. Two other accessionswere from two different cultivars that did notmatch any known genotype in the databasesconsulted (VIVC, Maul and Röckel, 2015;Rodríguez-Torres, 2018; Vitis Canarias, 2015).These genotypes were referred to as unknowns 2and 3. Curiously, they were from Madeira. InSupporting Information 1 and 4 there is oneprime name with number 1 not registered byVIVC (Maul and Röckel, 2015). This primename (Uva de año) was used by other authors(Rodríguez-Torres, 2018). There are also sevennames with number 2 (Bienmesabe tinto, Burravolcanica, Listan rosa, Mollar cano rosado,Torrontes volcanico, Vallera and Verijadiegonegro) not registered by VIVC and the authors ofthis article and other authors specialized inCanarian cultivars (Rodríguez-Torres, 2018)propose should be included in VIVC. The primenames with number 3 are six varieties(Bienmesabe tinto, Burra volcanica, Torrontes

volcanico, Uva de año, Vallera and Verijadiegonegro) with their MP-SSR not included in theVIVC (Maul and Röckel, 2015). It should bepointed out that there are 19 new accessionnames (highlighted in bold) and 19 existingaccession names (highlighted in purple) that arenot recorded as a synonyms of theircorresponding cultivars nor have anybibliographic support in the VIVC (Maul andRöckel, 2015). Finally, eight accessions werecases of mislabelling (highlighted in red and bythe symbol #).

It should be emphasized that the names proposedas “new prime name” or “new synonym” areonly the names of the samples analysed, whichwere provided by the viticulturists (originalinformation in Supporting Information 1).Therefore, other names used in the CanaryIslands, and which may be valid candidates,have not been considered. In addition,unfortunately, the Madeira viticulturists did notprovided us with any information about thesamples.

2.2 Proposal of local varieties: bibliographystudy

After all cultivars had been identified andcharacterized, the next aim was to find which ofthem might be considered to be local varietiesfrom the Canary Islands and Madeira. The twounknown varieties (from Madeira) were notincluded in the study because there was noavailable information about them. Consequently,our exhaustive bibliographical study (based onhistorical and lexical terms) led us to select 14 ofthe 41 identified varieties and three sports, giventhat most of them are mentioned in thespecialised bibliography in cultivars grown inthe Canary Islands and Madeira (Zerolo et al.,2006; Rodríguez-Torres, 2018; Vitis Canarias,2015). Specifically the following genetic profilesare proposed as local: Albillo criollo,Bermejuela, Bienmesabe tinto, Burra volcanica,Albillo forastero, Huevo de gallo, Listan negro,Listan rosa, Malvasia di Sardegna rosada,Malvasia volcanica, Mollar cano rosado, Sabro,Torrontes volcanico, Uva de año, Vallera,Verijadiego and Verijadiego negro. All arehighlighted in Supporting Information 1 and 4(14 varieties in blue, and the three sports ingreen). Curiously, none of the varieties proposedas local were from Madeira. The remaining27 varieties were regarded as foreign.


3. Putative parentage relationships

Parentage was assessed using cultivars from theCanary-Madeira population used in this study. Atotal of three complete pedigrees and oneincomplete pedigree were found in thispopulation: 1) Malvasia di Sardegna xBermejuela: Malvasia volcanica (Zerolo et al.,2006; Rodríguez-Torres, 2018); 2) Palomino finox Mollar cano: Listan negro (Zerolo et al., 2006;Rodríguez-Torres, 2018); 3) Palomino fino xVerdelho branco: Albillo Forastero and Albillocriollo (Rodríguez-Torres, 2018); and 4)Alfrocheiro x Heben: Malvasia fina (Rodríguez-Torres, 2018). This last lineage was onlyobtained with data from Alfrocheiro as Hebenwas not present in this collection.

4. Genetic structure

4.1 Genetic structure of the Canary-Madeirapopulation

This section focuses on the genetic structure ofthe Canary-Madeira population, and inparticular, on the 14 cultivars and three sportsproposed as local varieties from the CanaryIslands. Although the Canary-Madeirapopulation consisted of 44 varieties, three ofthem were not taken into account because theywere closely related material; specifically, onesport included in the local population (Listanrosa) and two cultivars not included in the localpopulation (Malvasia di Sardegna and Mollarcano). Three other cultivars were also excludedbecause they are interspecific crossing (Isabellaand Flot rouge) or an author crossing (Malvasiabranca de Sao Jorge). These genetic profileswere therefore regarded as redundant (Cabezas etal., 2011; Marsal et al., 2016; Marsan et al.,2017) or as an artefact to Vitis vinifera L.population, or as an artificial crossing. Theseresting 38 genetic profiles were chosen forgenetic structure analysis, 16 of which wereproposed as local from the Canary Islands. Theanalysis was firstly made using the Structuresoftware, which provided several geneticdistributions for the population. SupportingInformation 5 shows the distributions given bythe Structure program, with K calculated usingthe method described by Evanno et al. (2005). Inaccordance with Supporting Information 5 (thegraphical representation of DK), the bestdistribution was K=4 (4K). Therefore, theCanary-Madeira population was studied in detailwhen it was divided into four groups. The

distribution of the varieties made by theStructure program is showed in Figure 1. In thiscase, the varieties in each group have beenordered from highest to lowest according to theirq value (the percentage of their inferred genomebelonging to the cluster (Bacilieri et al., 2013;Marsal et al., 2017)). Therefore, when thepopulation of 38 genotypes is divided into foursubpopulations, nine cultivars are assigned tosubPOP1 (six members with q ≥ 85 %), six tosubPOP2 (four with q ≥ 85 %), 13 to subPOP3(seven with q ≥ 85 %), and the remaining 10 tosubPOP4 (seven with q ≥ 85 %). A total of 56 %of the varieties of the subPOP1 are fromPortugal, 22 % are from Spain, 11 % are fromFrance and the origin of the rest is uncertain.This subpopulation includes both white and redgrape varieties (55 % white cultivars and 33 %red cultivars), and they are mainly used forwinemaking (67 %). It is interesting to note thattwo (Albillo criollo and Vallera) of the 16proposed local varieties placed in thissubpopulation were considered as purespecimens. All members of subPOP2 are fromSpain. This subpopulation also includes both red(33 %) and white grape varieties (67 %), winecultivars (67 %) and “double-use” cultivars(table and wine) (34 %). In this subpopulation,there are three pure local variety from the CanaryIslands (Burra volcanica, Verijadiego negro andHuevo de gallo), and one admixed cultivar(Sabro). SubPOP3, the third cluster, consists of13 cultivars, and 77 % of these are from Spain.In this cluster, 69 % are white grape varieties and54 % are used for winemaking. The rest are tableor “double-use” cultivars. Nine of them werelocal varieties (Malvasia di Sardegna rosada,Malvasia volcanica, Mollar cano rosado,Bermejuela, Torrontes volcanico, Uva de año,Listan negro, Verijadiego y Albillo forastero).However, the Listan negro, Verijadiego y Albilloforastero varieties were considered to be anadmixed genotype (q < 85 %). Finally, subPOP4is more heterogeneous in terms of the origin ofthe varieties, given that 40 % are from Spain,20 % from France, 20 % from Portugal, 10 %from Switzerland, and the remaining 10 % are ofunknown origin. In this group, the red grapevarieties are predominant, but 40 % are winecultivars and the rest are table or “double-use”cultivars. In this group, there is one pure localcultivar (Bienmesabe tinto). PCoA (Figure 2)was carried out only on the 24 varieties forwhich at least 85 % of their inferred genomebelonged to the cluster (q ≥ 85 %). Subsequently,

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the GenAlEx 6.5 program carried out theassignment test, and assignment was deemedgood in 96 % of cases (data not shown). Figure 2shows the distribution of the 24 individuals whenthe population is divided into four groups (4K).Coordinate 1 explained 12.9 % of the overallvariance, and it separated most of the individualsin the subPOP1, subPOP2 and subPOP4 (mainlylocated in the right-hand quadrants) from thesubPOP3 subpopulation (located in the left-handquadrants). Coordinate 2 explained 9.3 % of thetotal variance, and it separated the local varieties(highlighted in blue or purple, and mainlysituated in the lower quadrants) from the foreignvarieties.

4.2 Genetic structure of the local Canaryvarieties in a world population

The first step of this section was analysing thebehaviour of a group of 17 varieties incomparison with a world population consistingof 290 varieties from 21 countries. We used aworld population characterized by Marsal et al.(2017), without mutations (and sports), authorcrossing and interspecific crossing. Specifically,this group of 17 varieties includes the 14 localcultivars without sports (Albillo criollo,Bermejuela, Bienmesabe tinto, Burra volcanica,Albillo forastero, Huevo de gallo, Listan negro,Malvasia volcanica, Sabro, Torrontes volcanico,Uva de año, Vallera, Verijadiego and Verijadiegonegro), two unknown cultivars (Unknown-2 andUnkown-3) and another Canarian local cultivar

(Albillo monte Lentiscal) that was not present inthe samples provide for this study but wasanalysed for our group in a previous study. Boththe world population and the Canary-Madeirapopulation were identified using the same20 SSRs. As in the previous section, theStructure program was used to obtain the mostprobable subpopulations (no. K). SupportingInformation 6 shows the distribution of the290 varieties given by the Structure program,with K being calculated by the method describedin Evanno et al. (2005). Concerning thegraphical representation of DK, the bestdistribution for this world population was for“K=2” (2K). Supporting Information 7 showsthe detailed distribution of all the individualswhen the population is divided into two groupsalongside other information. Within each group,the varieties were ordered according to their qvalue. The cultivars are highlighted in differentcolour tones according to their q value. Theadmixed genotypes (q < 85 %) were notincluded in the genetic structure analysis.Therefore, the world population decreased from290 to 251 genomes after the varieties with q ≥ 85 % had been selected. Then, assignmentswere made by the GenAlEx 6.5 program, andwere successful in all cases (data not show). Inthis case, the subPOP1 consisted of 181 cultivarsand the subPOP2 consisted of 109 varieties. Ofthe cultivars located in subPOP1, none isconsidered to be local to the Canary Islands:29.8 % are from Italy, 27.6 % originate fromFrance and Central Europe, 26 % are from the


FIGURE 1. Representation of 38 individuals from Canary-Madeira Collection (IC-M) by Structuresoftware, when the population is divided in 4 groups (4K). The green arrows include the cultivars thathave a q ≥ 85 % and that are therefore selected for the study of the population structure. In this way, thepopulation of 38 individuals will have 24 non admixed individuals (pure).

Iberian Peninsula and 13.8 % are from BalkanPeninsula. The resting varieties are from theNear East of the Mediterranean Basin, theCaucasus or North Africa. Another characteristicof this group is that 54.7 % of its varieties arered grapes, 43 % are white, and the rest are roséor rouge. In this subpopulation, 77 % of thevarieties are used for winemaking, 21 % are“double-use” (table grapes and winemaking),and only the remaining 2 % are table varieties ortriple use (raisin, table or wine). The subPOP2 ischaracterized by having 66 % of its membersfrom Spain (14.6 % from Canary-Madeiragroup), and 17.4 % are from Italy, and the restare from the Balkan Peninsula, the Near East,France and Central Europe. Anothercharacteristic of the cultivars belonging to thesubPOP2 is that 49.5 % are red grape varieties,45.8 % are white, and the remaining 4.7 % arerosé or rouge grape varieties. Most are used forwinemaking (66.7 %). Curiously, the 16 varietiesproposed as local from the Canary and MadeiraIslands can only be found here (although Albillocriollo and Unknown-2 are regarded asadmixed). In the second step, we treated themore characteristic varieties of the Canary-Madeira Collection (13 local varieties and

Unknown-3IC) as a single subpopulation(subPOP IC-M) to determine how it was relatedto the subPOP1 and subPOP2. In this case, theassignment test had a success rate of 95 % (datanot shown). Figure 3-a shows the distribution ofthe subPOP1, subPOP2 and subPOP IC-M.Coordinate 1 explained 75.85 % of the overallvariance, whereas Coordinate 2 explained24.15 %. This representation clearly shows asplit between the subPOP IC-M and thesubPOP2, given that they are in oppositequadrants (at the ends). Although subPOP1 is inthe lower quadrants with subPOP IC-M, the twosubpopulations are very distant from each other,with subPOP1 in the right quadrant and subPOPIC-M in the left quadrant. In general, it can beobserved that the subPOP IC-M is very distantfrom the other subpopulations, which isconfirmed by the Fst values (data not show), asthe subPOP IC-M has higher Fst values than theother subpopulations. The parameter of Fst is thecorrelation of randomly chosen alleles within thesame subpopulation relative to the entirepopulation; equivalently, the proportion ofgenetic diversity due to allele frequency differsamong populations. The fixation index can rangefrom 0 to 1, where 0 means complete sharing of

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FIGURE 2. Representation of 24 non admixed individuals (q ≥ 85 %) from Canary-Madeira Collectionby Principal Coordinates Analysis when the population is divided in 4 groups (4K).

genetic material and 1 means no sharing(Holsinger and Weir, 2009). However, thesubPOP IC-M is much closer to that of subPOP2(as expected, given that the varieties of subPOPIC-M initially belonged to the subPOP2). Themain characteristic of the subPOP2 is that mostof its cultivars are from the Iberian Peninsula andItaly. The subPOP1 is mainly made up ofcultivars from Italy, France and Central Europe,and the Iberian Peninsula.

We also studied the relationship between thesubPOP IC-M and six other subpopulationscreated from the geographical origin of thecultivars. The varieties were grouped in areas(clusters of nearby countries) because some ofthe countries have few varieties (SupportingInformation 7). According to the literature(Imazio et al., 2006; Arroyo-García et al., 2006;Bacilieri et al., 2013; Marsal et al., 2017), thefive subpopulations are: subPOP EASTMED-CAU (Algeria, Cyprus, Georgia, Israel, Lebanon,Tunisia and Turkey); subPOP BP (Bosnia andHerzegovina, Bulgaria, Croatia, Greece, Serbia,Slovenia and Montenegro); subPOP ITA (Italy);subPOP FRA-CEU (Austria, France, Germany,Hungary and Switzerland); and subPOP IP

(Spain and Portugal). Each variety was assignedto the country specified in VIVC (Maul andRöckel, 2015). Figure 3-b shows the distributionof these six areas described above (thegeographical origin of the cultivar). In this case,coordinate 1 explains 44.27 % of the overallvariance, and clearly shows a split betweensubPOP IC-M and subPOP IP (only located inthe right-hand quadrant) and the othersubpopulations. Coordinate 2 explains 19.30 %of the overall variance, and divides the subPOPITA, subPOP FRA-CEU and subPOP IC-M(upper quadrant) from the rest ofsubpopulations, which are principally located inthe lower left quadrant (except subPOP IP, whichis in the lower right quadrant). This distributionshows that the subPOP IC-M is at some distancefrom all the subpopulations, but it is much closerto subPOP IP than to the others.

DISCUSSION

1. SSR polymorphism

The average expected heterozygosity was 0.787(Supporting Information 2), which is consideredrelatively high, and is very similar to that


FIGURE 3. Representation of the subpopulations from world population by Principal CoordinatesAnalysis: a) 2K + IC-M subpopulation; b) 5 area-countries + IC subpopulation.To do a better characterization of each group, only it was considered the varieties with q ≥ 85 % (non admixed individuals).

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described by other authors (Costantini et al.,2005 (0.79); Martinez et al., 2006 (0.807);Stajner et al., 2014 (0.79)). Consequently, theCanary-Madeira population shows considerablegenetic diversity. The cumulative PI* was verylow (2.4E-26) so the 20 SSRs used were efficientat distinguishing between close varieties. Thisvalue was small enough to ensure that two plantswith the same molecular profile in all loci werethe same cultivar, with sports being the soleexception. In fact, in nine cases our PI* valueswere lower than the threshold (0.05) at which amicrosatellite is considered hyperpolymorphic ingrapes (Costantini et al., 2005), and only VVS3,VVS29, and VVMD6 had values higher than0.1. In addition, 13 markers had a probability ofnull alleles (r) lower than or equal to 0.01,meaning that there was a very small proportionof excess heterozygosity. This confirms that thehomozygote profiles are true, so they would nothave lost any alleles. All this data confirms thatthe 20 SSRs are appropriate for this study.

2. Cultivar analysis: Confirmation ofaccession name and proposal of local varieties

Eight of the prime names in this collection arenot recorded in the VIVC: Bienmesabe tinto,Burra volcanica, Listan rosa, Mollar canorosado, Torrontes volcanico, Vallera, Verijadiegonegro and Uva de año (SupportingInformation 1). All are the prime names of sixcultivars and two sports that we propose shouldbe inclused in the VIVC database. Uva de Año isa name that has been used by other authors(Rodríguez-Torres, 2018) but it does not appearin this database. Moreover, the MP-SSR of theseeight cultivars are not described in VIVC. Thissuggests, therefore, that these names and theirsMP-SSR could be included in the VIVCdatabase. The MP-SSR of Torrontes volcanicocorresponds to MP-SSR of the cultivar namedPedro Ximenez/Torrontes by Rodríguez-Torres(2018). Interestingly, this molecular profilecorresponds neither to MP-SSR Pedro Ximeneznor to the MP-SSR Torrontes, which indicatesthat it is a different cultivar.

We consider that 38 names from this collectionare synonyms (either new synonyms orsynonymous names for a particular cultivar). Ofthese, there are 19 accessions names that are notrecorded or have no bibliographic support in theVIVC. These accession names are proposed asnew synonyms given that they are very commonin the Canary Islands. Furthermore,

19 accessions names are recommended assynonyms of 16 varieties. These names arealready accepted synonyms for other varietiesaccording to VIVC, but in the Canary Islandsthey are also commonly used to refer to thecultivars of this collection. Therefore, thisinformation should also be included in theampelographic section of VIVC (Maul andRöckel 2015). Finally, eight accessions havebeen identified as cases of mislabelling inasmuchas they all coincide with the prime name of othercultivars.

The studied Canary-Madeira population is madeup of 41 varieties and three sports. However,according to several bibliographic resourcesspecialized in the grapevines of the CanaryIslands (Zerolo et al., 2006; Rodríguez-Torres,2018; Vitis Canarias, 2015), only 26 have beentraditionally cultivated in these islands: Albillocriollo, Baboso blanco/Bastardo blanco, Babosonegro, Bastardo negro, Breval negro, Burrablanca, Castellana negra (Tintilla castellana),Forastera blanca (Albillo forastero), Gual, Huevode gallo, Listan blanco de Canarias, Listannegro/Almuñeco, Listan prieto, Malvasiaaromatica, Malvasia rosada, Malvasia volcanica,Marmajuelo, Moscatel de Alejandría, Negramoll,Torrontes volcanico (Pedro Ximenez/Torrontesby Rodríguez-Torres (2018)), Sabro, Tintilla,Verdello, Verijadiego, Vijariego blanco andVijariego negro. For this reason, these26 varieties have been selected and studied indetail to identify which can be considered aslocal varieties of the Canary Islands (SupportingInformation 4). A particular case to highlight isthat of three unknown varieties, which coincidedwith other unknown ones identified by otherauthors specializing in Canarian varieties(Rodríguez-Torres, 2018). In this case and byconsensus of all the scientists who analysed theirgenetic profiles, the names of Bienmesabe tinto,Burra volcanica and Vallera were chosen, takinginto account different aspects such as historicalinformation (when available), phenologicalbehaviour and plant morphology.

After an exhaustive bibliographic revision andmolecular analyses, we proposed that14 varieties and three sports are local to theCanary Islands. We found that their names andtheir MP-SSR are unique (except sports) in theworld and to our knowledge they are only grownin the Canary Islands. These cultivars are Albillocriollo, Bermejuela, Bienmesabe tinto, Burravolcanica, Albillo forastero, Huevo de gallo,

Gemma Marsal et al.


Listan negro, Listan rosa, Malvasia di Sardegnarosada, Malvasia volcanica, Mollar cano rosado,Sabro, Torrontes volcanico, Uva de año, Vallera,Verijadiego and Verijadiego negro. Curiously,none of these come from Madeira. This suggeststhat, nowadays, the Canary Islands (unlikeMadeira) keep genuine cultivars, which havebeen brought from different regions of the worldover the years, because phylloxera never reachedthere. Unfortunately, Madeira suffered thedisaster of phylloxera and probably lost many ofits original varieties. However, there are twounknown varieties from Madeira (SupportingInformation 1 and 4), but we cannot put forwarda hypothesis about these since we have found noinformation.

3. Putative parentage relationships

In the Canarian and Madeiran population, wefound four pedigrees. Two pedigrees (lineages 1and 2) were reported in a study with nine SSRs(Zerolo et al., 2006) and four pedigrees (lineages1, 2, 3 and 4) were described in another studywith 48 SNPs (Rodríguez-Torres, 2018). Cabezaset al. (2011) reported that 48 SNPs had adiscrimination power similar to a set of 15 SSRmarkers. Therefore, the present study confirmsthese lineages through molecular characteri-zation using 20 SSRs. It should be pointed outthat lineage number 3 is not included or has nobibliography to support its inclusion in theVIVC.

4. Genetic structure

The Canary-Madeira population (Figures 1and 2), can be divided into two large groups.One contains mainly foreign varieties (located insubPOP1 and subPOP4), and the other containsmainly local varieties (located in subPOP2 andsubPOP3, these local varieties are highlighted inblue whereas their sports in purple). Curiously, itcan be seen (Figure 2) that coordinate 2 separatesthese populations by placing the population offoreigners (almost totally) in the upper rightquadrant and most of the local Canary-Madeirapopulation in the lower quadrants. Conversely,coordinate 1 separates subPOP3 (isolating it inthe lower left quadrant) from the rest of thesubpopulations that disperse, occupying thequadrants on the right. SubPOP3 is characterizedby having the highest proportion of localvarieties at 69 %, which increases to 86 % ifonly pure local varieties are considered. Most ofthese cultivars come from Lanzarote Island. This

subpopulation groups some cultivars that have astrong influence of varieties from the east of theMediterranean Basin (Malvasia di Sardegna orMuscat of Alexandria). The other group thatincludes a significant amount of local varieties(66.6 %) is subPOP2, which increases to 75 %when we consider only the pure individuals thatcome entirely from the Island of El Hierro(Burra volcanica, Huevo de gallo andVerijadiego negro). This subpopulation ischaracterized because three of its members havethe ancestral Spanish cultivar Heben as theparent (Sumoll, Vijariego blanco (admixed) andSabro (admixed)). The three local pureindividuals together with the variety Bienmesabetinto, from the island of La Palma and belongingto subPOP4, are the furthest local group.subPOP4 is the most disparate subpopulation:apparently its members are disconnected,although there is a certain Central Europeaninfluence. Quite the opposite occurs in subPOP1in which all its members, except the Vallera andUnknown-2IC varieties, are directly or indirectlyrelated to the Central European ancestral varietyknown as Savagnin blanc. This last cultivar isthe one of the parents of the varietiesSamarrinho (PRT), Molar (PRT), Trousseau noir(a French cultivar widely implanted in Portugaland known by the name synonym of Bastardonegro), Verdelho branco (PRT) and Alfrocheiro(PRT), and the grandfather of the cultivarsAlbillo criollo (local cultivar from La Palmaisland) and Malvasia fina (PRT). Many authorssupport the theory that from the Middle Agesthere was a large influx of Central Europeanvarieties through the Route of Santiago(Casanova et al., 2011). This is possibly why theSavaging blanc cultivar was introduced in thisarea, since it is one of the varieties with morecrossings in the northern Iberian Peninsula.Therefore, the Central European influencemeans that subPOP1 and subPOP4 are muchrelated, between them occupying the upper rightquadrant of the PCoA representation in Figure 2.These subpopulations only contain three localvarieties: Albillo criollo from La Palma andVallera from Tenerife (subPOP1) andBienmesabe tinto from La Palma (subPOP4).

Consequently, the study of the genetic structureof the local Canary varieties in a worldpopulation must be based on the subPOP IC-M.This subpopulation is assumed to consist of15 Canary cultivars (14 from this work and theAlbillo monte Lentiscal from the world


population) and two varieties from Madeira(Unknown-2IC and Unknown-3IC), all of whichbelong to the area of subPOP IC-M (SupportingInformation 7). The subPOP IC-M cultivars wereremoved from subPOP2, where most of thepeninsular cultivars are found. The results showthat these varieties have a direct relationshipwith the Iberian Peninsula. The assignment testsalso show that the varieties in the subPOP IC-Mare highly characteristic and constitute their owngroup (data not shown). The distribution of thesubpopulations (Figure 3) shows that thesubPOP IC-M is very distant from all othersubpopulations. This proves the singularity ofthe molecular profiles of the subPOP IC-M.However, it is always nearer to the subPOP2(Figure 3-a) and to the subPOP IP (Figure 3-b)than the others (Myles et al., 2011). This may bebecause the main colonizers of these islandscame from Spain and Portugal, so most of thefirst vines in the Canary Islands and Madeiracome from these countries.

These results reinforce the idea that subPOP IC-M has characteristics that differentiates itsvarieties from the rest, although is clearlyinfluenced by the peninsular population.

The Canary Islands never experienced thephylloxera plague. For 500 years the cultivarsintroduced by the first colonizers and thesuccessive migrations have adapted to the newedaphoclimatic conditions (mutations) andhybridized spontaneously. In addition,grapegrowers have selected the most interestingspecimens. All these actions have led to cultivarsthat are quite different from the initial ones, withcharacteristic and unique genetic profiles.Consequently, the Canary Islands and Madeiraseem to be a biodiversity centre or a centre oforigin of new cultivars of Vitis vinifera L.

CONCLUSIONS

This study proposes two new mutations, Listanrosa and Mollar cano rosado, which are sports oftwo well-known varieties (Listan negro andMollar cano, respectively). The study has alsodetected four new cultivars for which there areno previous references (Bienmesabe tinto, Burravolcanica, Vallera and Verijadiego negro). Eightnames of varieties have also been proposed forinclusion in the VIVC as new prime names(Bienmesabe tinto, Burra volcanica, Listan rosa,Mollar cano rosado, Torrontes volcanico, Uva deaño, Vallera and Verijadiego negro).

Furthermore, the MP-SSR of five varieties isgiven (Bienmesabe tinto, Burra volcanica,Torrontes volcanico, Uva de año, Vallera andVerijadiego negro). We also suggest that38accession names commonly used in theCanary Islands be added to the list of synonymsin the VIVC. Nineteen of these must beconsidered new synonyms of the 12 varieties(PN): Abillo grano chico (PN: Albillo criollo);Uvallón (PN: Breval negro), Huevo gallo (PN:Huevo de gallo); Uva olor (PN: Isabella);Muñeco negro, Negra gruesa, and Negra mulata(PN: Listan negro); Marmajuelo rosada (PN:Listan rosa); Bermejuelo, Vermejuela, andMarmajuelo blanco (PN: Bermejuela);Negramoll negra (PN: Mollar cano); Negramollmulato, and Negramoll rosada (PN: Mollar canorosado); Albillo grano pintado (PN: Muscat apetits grains rouges); Blanca peluda, and ViñaJavier (PN: Palomino fino); Tintilla castellana(PN: Tinto cao). The rest, 19 synonymous namesof 38, corresponding to 16 cultivars, are existingsynonyms for other varieties. In this case, wesuggest that they be incorporated as synonymsfor the varieties of this collection. Two varietiesare labelled as unknown and their MP-SSRs areprovided. Eight cases of mislabelling weredetected and identified.

The study of genetic structure reveals that thecultivars from the Canary and Madeira Islandshave been strongly influenced by the IberianPeninsula. Furthermore, 14 local varieties andthree local sports from the Canary Islands havebeen proposed: Albillo criollo, Bermejuela,Bienmesabe tinto, Burra volcanica, Albilloforastero, Huevo de gallo, Listan negro, Listanrosa, Malvasia di Sardegna rosada, Malvasiavolcanica, Mollar cano rosado, Torrontesvolcanico, Sabro, Uva de año, Vallera,Verijadiego, and Verijadiego negro. They are allpart of a single group with a genomecharacteristic of the Canary Islands. In Madeira,however, we were unable to find a local variety:the molecular profile of Unknown 3IC seems tobe the most characteristic, but did not manage toidentify this variety.

It can be concluded, therefore, that this volcanicarea could be considered as a biodiversity centreor centre of origin of new cultivars of Vitisvinifera L.

Acknowledgements: The authors are grateful toProfessors Josep Cano, Javier Ibáñez, AgustíVillarroya and Isabel Pardo for their valuable

Gemma Marsal et al.


support. We would also like to thank Rosa Pastorand Santiago Moreno for their support in thelaboratory.

Supplementary data are available from thewebsite: https://oeno-one.eu/article/view/2404.

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