Combining Microsatellite Markers and Ampelography for Better Management of Romanian Grapevine Germplasm Collections

Aiming to investigate the correspondence of phenotypic variability and molecular markers, 50 varieties of Vitis vinifera ssp. vinifera, ancient grapevine varieties grown on Romanian territory, were selected to be analysed. All varieties were subjected to ampelographic analyses with OIV descriptors and also to molecular analyses with 13 microsatellites. The morphology description with recommended methods corresponded with the reference literature, proving the correct registration of the varieties at the moment of acquisition. Markers efficiency for assessing the genetic diversity among studied varieties was evaluated by computation of statistic parameters referring to the polymorphisms found. Among the internationally recommended SSR markers, ISV4 and VMCNG4b9 markers displayed interesting PIC and PI values. By comparing the SSR profiles of the 50 Romanian grapevine varieties with those in the EU database, the following can be concluded: confirmation of 10 synonymies mentioned in old documents, while 3 synonymies mentioned in old reference literature were not confirmed, new synonyms were found for 4 accessions, additional synonyms were found for 4 accessions, which are to be added to the previous ones already mentioned in the literature, the synonymies in three groups of accessions were documented for the first time in the specialised literature, the unicity of three Romanian accessions was proved (ʻMoroştinăʼ, ʻNegru mareʼ and ʻRomânieʼ) and the genetic profiles of 15 varieties were identical to those already recorded in the investigated databases.


Introduction
A comprehensive characterization of accessions preserved in germplasm collections entails ampelographic description with standardized descriptors and molecular analyses with an internationally shared set of SSR markers.The two approaches ensure accurate identification and guarantee the authenticity of plant material used in research work, preserved in germplasm collections, or distributed for establishing new vineyards.The first international standard for the classification of grapevine based on morphological characters was established in 1873 by the International Ampelography Committee in Vienna.Permanently improved, this system for describing and characterizing species and varieties of Vitis genus comprises today 150 descriptors, of which 48 are essential.
Since the mid-1990s, in addition to the descriptor system, microsatellite markers have been used for the genetic characterization of grapevine varieties, as a useful tool complementing ampelography.Thomas et al. (1993) first applied the SSR analysis for identifying grapevine varieties and their results showed that the microsatellite sequences are: a) abundant in the genome; b) highly informative for identifying Vitis vinifera varieties; c) transmitted to descendants following the Mendelian laws of heredity (co-dominant); d) suitable for genetic mapping and investigation of the degree of genetic relatedness among genotypes.The main advantage of microsatellite sequences is the high reproducibility, which allows the exchange of data between laboratories around the world, the final result being a unique genetic profile for each variety and, implicitly, unambiguous characterization of any variety.
These features were considered underpinned for the OIV decision to recommend in 2009 the use of six SSR loci to identify Vitis genotypes (OIV, 2009).Subsequently, after testing improved methods on a number of over 2,000 genotypes, belonging to 30 germplasm collections, nine SSR markers were finally recommended (Maul et al., 2012).Thus, identifying grapevine varieties using at least 9 SSRs following the protocol described by Migliaro et al. (2013), using fluorescent primers and an ABI3130xl genetic analyser (Applied Biosystems, Foster City, CA).

Statistical analysis
Data analysis: statistics on scored alleles were computed, such as the number of alleles (Na), the number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), the probability of identity (PI), and the probability of null alleles (F(null) using the freely available Cervus 3.0.7 and GenAlEx softwares.

Ampelographic description
From the total of fifty (50) varieties, five (5) were very well appreciated and grown in many other countries with long time viticulture tradition, seven (7) are major local cultivars, extensively grown in Romania and two (2) are minor local cultivars, fairly utilized in wine production (Table 1).Most of the studied varieties (36) are considered autochthonous, grown for centuries on the study territory, but today rarely notified in private households, or preserved in few germplasm collections.Valuable information with detailed ampelographic descriptions was found for 44 varieties in old documents, so it was possible to obtain a preliminary confirmation of the authenticity of the corresponding accessions.No information was found for ʻBraghină albăʼ, ʻGalbenă maruntă'ʼ, ʻMoroștinăʼ, ʻRomânieʼ, ʻȚâța caprei neagrăʼ and ʻȚâța vacii neagrăʼ, so it was not adequate to establish their possible authenticity only on the basis of our morphological characterization.
Ampelographic description of all varieties was performed at specific phenophases (shoot tip and young leaf, during spring; mature leaf after flowering; bunch and berry between ripening and harvesting), on target organs, in specific position on the vine and the right notifications were recorded for expression level of each character.All features and their descriptor records were compared with those in the reference literature, presenting comprehensive information for each variety.Some characters were slightly different, such as: intensity of anthocyanin coloration of the shoot tip (OIV 003), colour of upper side of the young leaf (OIV 051), shape of the mature leaf (OIV 067), degree of opening/overlapping of petiole sinus (OIV 079), shape of petiole sinus (OIV 080), density of prostrate hairs between main veins on lower side of mature leaf (OIV 084), bunch density (OIV 204), bunch shape (OIV 208), berry shape (OIV 223), sugar content of must (OIV 505) and total acidity of must (OIV 506).These differences are often explained as different response of each variety to variable cultural conditions, healthy status of the plants, observer subjectivity (Fatahi et al., 2003;Carimi et al., 2010;Tassie, 2010).In some instances, were noted varieties that were morphologically very similar varieties, so that they cannot be distinguished by visual comparison, but had different DNA profiles, and, on the contrary, clones of the same cultivar showed different phenotypes, even if their DNA profile was identical.
has become a common practice for synonyms and homonyms detection, preliminary investigation on genetic relatedness between or among varieties, and identification of plant materials of unknown origin by comparing their profiles with reference genotypes collected in the international databases.
Grapevine germplasm collections in Romania have a wide range of varieties, an overwhelming majority having a long tradition of vineyard culture.The series of seven "Ampelografia of the Romanian People's Republic" published between 1959 and 1966 with the detailed description of the old grapevine varieties and the works of Indreas and Visan (2001) and Rotaru (2009) about those considered of economic importance, are the reference literature for the field of ampelography.In the last two decades, for many of the vine varieties considered valuable and important for the production of grapes and wine, the ampelographic description was completed with molecular characterization (Bodea et al., 2009;Butiuc-Keul et al., 2010;Coste et al., 2010;Gheorghe et al., 2008;Gheţea et al., 2010 and2012;Popescu et al., 2017).
The objectives of this study were: 1) to complete the identification of some grapevine varieties, supposed to be autochthonous, on the base of 48 OIV descriptors and 13 SSR markers; 2) to evaluate the genetic diversity of the analysed Romanian grapevine varieties.

Biological material
Table 1 presents the fifty (50) varieties of grapevine used in the present study and selected because considered indigenous and cultivated for many centuries in our country.All of them are growing in the ex situ germplasm collection held by NRDIBH Stefanesti.

Protocol for molecular analyses
For molecular analyses, the DNA extraction was performed from 100 mg fresh leaf with Qiagen DNeasy Plant mini-kit (Qiagen, Hilden, Germany).For genotyping were used 13 SSR markers: nine of them belonged to the set of those internationally recommended (Maul et al., 2012) as a standard for grapevine (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79), plus additional four, namely ISV2, ISV3, ISV4 and VMCNG4b9.The SSR analyses were performed

SSR profiles for identifying synonyms, homonyms, misnomers and unique genotypes
In the last 30 years, in Europe, the interest of grapevine growers and wine producers for old and autochthonous varieties has increased and therefore it became necessary to correctly identify the varieties.It is very well known that the same variety may be grown in a number of countries or regions under different names (synonyms) or different varieties may be known under the same name (homonyms).Part of this information is found in Romanian most representative literature in ampelography domain.The results obtained with SSR profiles comparison provided valuable and sometimes unexpected information for the identification of varieties and revealed new or different synonyms and homonyms.SSR profiles also allowed the detection of unique varieties and accessions incorrectly identified by means of ampelographic descriptors.
B. Some synonymies presented in reference old literature were not confirmed and new ones or different from previous statements were found: -ʻBusuioacă de Bohotinʼ had the same SSR profile with ʻMuscat à petits grains blancsʼ, but a different colour of the berries; thus, it can be concluded that ʻBusuioacă de Bohotinʼ is the red somatic variant for berry colour of ʻMuscat à petits grains blancsʼ and not a synonym with ʻMuscat rouge de Madereʼ as mentioned Constantinescu et al. (1960); -ʻGalbenă uriaşăʼ SSR profile matched that of ʻMirkovacaʼ, an endangered Croatian variety described by Maletić et al. (1999); so, the present results proved that ʻGalbenă uriaşăʼ is not a clonal variant of ʻGalbenă de Odobeştiʼ as mentioned Constantinescu et al. (1961); -ʻTămâioasă româneascăʼ accession, one of the most important and appreciated grapevine varieties in Romania, was proven not to share the same SSR profile of ʻMuscat à petits grains blancsʼ, and therefore not to be a synonym with it, as mentioned by Constantinescu et al. (1960).Because the investigated accession seemed to be similar to those from other collections, it is supposed the existence of a mixture of at least two varieties of ʻTămâioasă românescăʼ in Romanian vineyards and, accordingly, Popescu et al. (2017) recommended the study of the mix of varieties in old ʻMuscat à petits grains blancsʼ vineyards all over the country.
196 C. Synonyms were found for some accessions for which there is no information in the Romanian ampelographic literature, such as: -ʻGalbenă măruntăʼ SSR profile matched that of ʻKakotrygisʼ, a Greek variety, so it is a synonym with this one; -ʻŢâţa caprei neagrăʼ SSR profile matched that of ʻHoraʼ, a well-known Bulgarian variety; -ʻTâţa vacii neagrăʼ matched that of ʻKozi Cici cherveniʼ, also a Bulgarian variety; -ʻBraghină albăʼ and ʻBraghină rozʼ showed two different molecular profiles, so they are different varieties, not somatic variants for berry colour and with no relatedness with ʻBakatorʼ as is supposed by Constantinescu et al. (1959).D. New synonyms, which will be added to the previous mentioned in the literature, were proven for some varieties: -ʻCeauş rozʼ has the same profile with ʻChaouch rozovyiʼ from Turkey; -ʻCoada oilorʼ is synonym with ʻJuhfarkʼ from Hungary; -ʻŢâţa vacii albăʼ showed the same SSR profile as ʻHalholyagʼ from Ukraine; -ʻCârcioasăʼ shared the same genotype with ʻBalint weissʼ from Hungary.E. For the first time in Romanian literature, synonymies were documented for the following varieties: -ʻBătută neagrăʼ shares the same SSR profile of ʻNegru românescʼ, though they were considered different varieties and were described separately by Constantinescu et al. in 1959 and1966, respectively; -ʻGordanʼ, ʻIordanăʼ and ʻZemoasăʼ accessions showed identical microsatellite profiles, proving to be synonyms; -ʻZghihară de Huşiʼ and ʻGalbenă de Odobeştiʼ varieties present in many germplasm collections, grown on large areas and used in wine producing, have the same SSR profiles, and proven to be synonyms.
F. The different genetic profiles obtained for ʻMoroştinăʼ, ʻNegru mareʼ and ʻRomânieʼ varieties, proved their uniqueness in the European Vitis germplasm collections.
The results obtained and presented in the hereby paper prove the usefulness of genotyping as a precious and convenient tool for supporting ampelography in the correct identification of varieties managed in the germplasm collections.This way, there were found similarities among accessions from Romanian collections and the varieties cultivated in collections from other countries, discovered incorrect records of the accessions, and were able to rename the misnomered ones.Molecular methods provide scientific support for documenting inconsistencies and inaccuracies in ampelographic descriptions and complete the picture for description of all accessions present in germplasm collections.

SSR markers for assessing genetic diversity in Romanian grapevine varieties
Statistics about the discriminatory efficiency of the 13 SSRs markers used are presented in Table 2.The high degree of genetic variability among the 50 cultivars is proven by the high number (112) of different alleles.With analysed accessions, the number of different alleles per locus ranged from 6 for VVMD25 and ISV4 to 13 for VVMD28, with an average of 8.62, comparable to the results obtained in other laboratories (Westman et al., 1997;Salayeva et al., 2010).The highest Ne was for VVMD28 locus (8.16), while the lowest for ISV3 locus (2.74) with the mean value of 4.84.Mean observed heterozygosity (82.9%) showed to be higher than that reported for other grapevine germplasm collections, like Iran with 76% (Fatahi et al., 2003) and Spain with 70.7% (Ibanez et al., 2003), and comparable to the values obtained by Carimi et al. (2010) and Lacombe et al. (2013).
For microsatellite markers efficiency were considered observed and expected heterozygosity (Ho, He) to evaluate the genetic variability among analysed grapevine.Ho ranged from 0.64 for VVS2 to 0.93 for VVMD5; mean Ho was slightly higher (0.829) than the expected one (0.784), with great differences locus by locus.
The ability of microsatellites to identify grapevine varieties is linked to the polymorphic information content (PIC) (Weber, 1990) and the probability of identity (PI) (Paetkau et al., 1995).The correspondence between these parameters is the following: an SSR effective in discriminating among genotypes has high levels of Ho, He and PIC, and low PI values (Salayeva et al., 2010).Among the four additional SSR markers tested in the current study, were remarked the ISV4 and VMCNG4b9 markers which displayed high diversity levels regarding the expected heterozygosity (higher than mean value of 0,784), high values of PIC (higher than mean value of 0,742) "and low values of PI (in comparison with other markers).
In the present case, the PI values ranging from 0.028 to 0.200 and the combined PI value of 3.39E-15, classified all the 13 markers as effective in discriminating the analysed varieties and highly informative for proving genetic diversity among studied grapevine varieties.These values are in concordance with those presented by Lopes et al. (1999) and Sefc et al. (2000).
Null-allele frequency F (null) showed to be very interesting, because it was negative for 11 out of 13 SSRs.This parameter is valuable to reveal some problems with allele amplification during genotyping or a possible deletion in the target sequence.In the hereby study were obtained positive values for F (null) with VVS2 and VVMD28 markers indicating possibility of the presence of null alleles.With these two markers, were registered lower values of the observed heterozygosity in comparison with the expected ones, also probably due to the occurrence of null alleles at VVS2 and VVMD28 locus.Between these two markers, VVS2 was a little bit critical, despite the high number of alleles.

Conclusions
Variety identification is important to breeders, curators, grape growers, winemakers and grape and wine consumers and, also it is a proof of a good traceability system throughout producing of grapevine planting material and winemaking process.Beside ampelographic description, the DNA analyses are necessary to verify grapevine identity before acquisition for germplasm collection, distribution or exchange plant material, planting and establishing a vineyard or investing in wine production.The efficiency of the two methods used for the complete characterization of the autochthonous grapevine varieties represents overwhelming evidence for the necessity of further investigation of other accessions existing in germplasm collections.These data with entire documentation of plant material authenticity are essential for improving the knowledge on Romanian grape varieties, completing information on the existence of synonyms with other varieties in European germplasm collections, as well as identifying unique genotypes that require particular attention for preservation.The complete description and compliance with the requirements for European germplasm collections of each accession is the primary requirement for the exchange of genetic material among germplasm collections.

Table 1 .
Grapevine cultivars characterized using OIV descriptors and SSR markers Accessions under evaluation received from Research and Development Station for Viticulture and Oenology Drăgășani; (2) Accession under evaluation received from University of Agronomic Sciences and Veterinary Medicine of Bucharest *310 = local cultivar, spread all over, international cultivar; 320 = major local cultivar, of local importance, but extensively grown; 330 = minor local cultivar, of local importance, fairly utilized; 360 = local neglected cultivar, at risk of extinction **VIVC The Vitis International Variety Catalogue is a database of various species and varieties/cultivars of grapevine.

Table 2 .
Genetic markers (13 microsatellite loci) and parameters applied to analyze their effectiveness to highlight genetic variability from the grapevine germplasm collection Na: number of alleles; Ne: number of effective alleles, Ho: observed heterozygosity; He: expected heterozygosity; PIC: Polymorphism information content, PI: probability of identity, F(null): probability of null alleles