Genotyping and Phenotyping of the Potential Clones , Biotypes and Variants of Grapevine Cultivar Korinthiaki Staphis ( Vitis vinifera L . )

This study presents the results regarding the identification and discrimination of twenty seven possible clones of grapevine cultivar Korinthiaki staphis (Vitis vinifera L.), three biotypes of Korinthiaki lefki and the related cultivar Staphidampelo using the ampelographic description and the molecular method AFLP. The results from the statistical analysis showed that all the biotypes of cultivar Korinthiaki staphis show small distance and are grouped in the same cluster, depending on their origin, while Staphidampelo and Korinthiaki lefki are neither variants nor biotypes of the cultivar Korinthiaki staphis but different cultivars since they are very distant compared to the other biotypes and moreover, Korinthiaki lefki is in a separate cluster of the dendrogram. The ampelographic description in combination with the molecular method AFLP are effective for the study of the between and within genetic diversity of grapevine cultivars as well as for their identification and discrimination. The results of this study can constitute the base for the implementation of the clonal selection for grapevine cultivar Korinthiaki staphis and the seclusion of the desired clones.


Introduction
The study of the between and within genetic diversity of grapevine cultivars as well as their discrimination and identification are very difficult due to the large number of grapevine cultivars, biotypes and synonyms.It has been estimated that there are more than 8,000 grape cultivars, under 24 000 different names (Viala & Vermorel, 1909).In Greece more than 300 grapevine cultivars (Vitis vinifera L.) are grown and classic ampelographic (Krimbas, 1943;Davidis, 1967;Vlachos, 1986;Stavrakakis, 2010), biochemical (Stavrakakis & Loukas, 1983) and molecular methods (Stavrakakis at al., 1997;Stavrakakis & Biniari, 1998;Stavrakaki, 2008) have been used for their discrimination and classification.Among the various polymerase chain reaction (PCR)-based DNA marker techniques available, the Amplified Fragment Length Polymorphism (AFLP) is often used, because it is ideal when the goal is the definition of identity among different clones of the same variety or among genetically close related cultivars, with positive results in differentiating grapevine cultivars and clones (Vignani et al., 2002;Blaich et al., 2007;Stenkamp et al., 2009;Alba et al., 2011).
Korinthiaki staphis, one of the most important Greek grapevine cultivars, is considered as the oldest cultivar of the Greek vineyard and maybe its first cultivation goes back to Greek antiquity.The viticultural areas of Corinth and Egio constituted the first cultivation centers of the variety, from where it was transferred initially (in 1516) to Zante and later (in 1560) to Kefallinia (Logothetis, 1975).Today, in Greece, it is estimated that Korinthiaki staphis is cultivated in approximately 15.000 ha (ranking first in the classification of Greek grape varieties) while its production exceeds 20.000 tons of raisins.
As far as its origin is concerned, the ampelographic characters classify Korinthiaki staphis as well as Korinthiaki lefki to proles pontica, to which most Greek grapevine varieties belong and to sub-proles balcanica (Negrul, 1938(Negrul, , 1946;;Levadoux, 1956).This point of view is verified by the results of a study using molecular markers (Aradhya et al. 2003).Because of the exclusive cultivation of Korinthiaki staphis in Greece for many centuries, it has been suggested that it is an indigenous cultivar of the Greek habitat and that it has originated from a local wild grapevine population (Logothetis, 1970).This view, of the existence of one or more secondary genetic centers of grapevine varieties, is enhanced by recent research studies (Grassi et al., 2003;De Andrés et al., 2012).However, there are not sufficient data neither on the time of creation and apparition of Korinthiaki staphis nor on how it was created, if it is a product of natural crossing or the result of mutations.
It is also supported that Greek grapevine cultivar Liatiko constitutes the parent cultivar from which Korinthiaki staphis derived either through mutation (Krimbas, 1930) or through crossing with another cultivar, with grapevine cultivar Staphidampelo being a potential parent cultivar (Myles et al., 2011).In a recent study, biotypes of cultivar Liatiko from Crete and biotypes of cultivar Korinthiaki staphis from Peloponnese where studied with the use of RAPD markers (Stavrakaki & Biniari, 2014).The results showed a low degree of genetic similarity (I = 0.774 -0.791) and therefore, Liatiko and Korinthiaki staphis are different grapevine cultivars and it seems that Korinthiaki staphis did not derive from Liatiko through mutations.
During the search and selection of the biotypes of Korinthiaki staphis to be studied, there were vines bearing bunches with seeded berries on one arm and normal bunches with seedless berries on the other.Also in the same bunch, there were secondary branches with large seeded berries or with smaller seedless berries.These vines are referenced in the present study as 'Metallagmeni' (which means mutant in Greek).
The aim of this study was to identify and to discriminate twenty seven possible clones of Korinthiaki staphis (including two biotypes of Metallagmeni, which is characterized as mentioned by seeded and seedless bunches, and four biotypes of Sxistofilli, which is characterized by different leaves from the typical cultivar), the related cultivar Staphidampelo and three biotypes of Korinthiaki lefki (which is characterized by white skin color in berries) and to determine their phenotypic and genetic similarities using the ampelographic description and the molecular method of AFLP analysis.

Grapevine Material
Twenty seven possible clones of Greek grapevine cultivar Korinthiaki staphis, three biotypes of Korinthiaki lefki and Staphidampelo were chosen for identification (Table 1).Leaf material of these vines was obtained from the experimental vineyard in Korakohori, Ilia, where all the possible clones and biotypes from the various cultivation centers of the country were collected and planted by the Laboratory of Viticulture of the Agricultural University of Athens.

Ampelographic and Molecular Methods
For the ampelographic description, 66 ampelographic characters were used and measured on each grapevine biotype during the years 2011, 2012, 2013 following a list of descriptors developed by the International Organization of Vine and Wine (OIV, 2009) including the preliminary minimal traits relative to shoot, mature leave, bunch etc. (Table 2).

DNA Extraction
Grapevine DNA was extracted from young and fully expanded leaves according to Thomas et al. (1993) with minor modifications (Biniari, 2000).One g of leaves from individual vines were frozen in liquid nitrogen and ground to a fine powder, thawed and resuspended in 12.5 mL of buffer A [0.25 M NaCl, 0.2 M TRIS-Cl (pH 8.0), 50 mM EDTA, 0.1 v/v 2-mercaptoethanol, 2.5% w/v polyvinyl-pyrrolidone (MW 40.000)].A crude nuclei pellet was obtained by centrifugation at 7 000 rpm for 10 min at 4 °C.The pellet was resuspended in 2.5 mL of extraction buffer B [0.5 M NaCl, 0.2 M TRIS-Cl (pH 8.0), 50 mM EDTA, 1% v/v 2-mercaptoethanol, 2.5% w/v polyvinyl-pyrrolidone, 3% sarkosyl, 20% ethanol] and incubated at 37 °C for 45 min.An equal volume of chloroform/isoamyl alcohol (24:1) was then mixed in and the phases were separated by centrifugation at 14 000 rpm for 15 min.The aqueous layer was collected and 0.54 volume of frozen isopropanol (-20 °C) was added to precipitate the DNA.The DNA was fished and resuspended in 300 μL TE (10 mM Tris -HCl, pH 7.4, 1 mM EDTA) containing 15 μg mL -1 RNase A and incubated for 15 min at 37 °C.Protein was removed by the addition of a half volume of 7.5 M ammonium acetate, followed by centrifugation and the DNA in the supernatant was precipitated with a 0.25 of cold isopropanol; ca.120 μg DNA per g FW was obtained.

Amplification Conditions
AFLP analysis was conducted as reported by Vos et al. (1995), following the AFLP Plant Mapping Protocol by Applied Biosystems (2007), with several modifications.
For the Restriction -Ligation stage, genomic DNA (500 ng) was incubated for 14-16 h (overnight) at 20 °C with the presence of 3 U (units) of enzyme EcoRI (5'..GAATTC..3') and 1 U of enzyme MseI (5'..TTAA..3').In each sample there were 4 U T4 DNA Ligase, 1μL of EcoRI adaptor and 1 μL MseI adaptor, T4 DNA Ligase buffer, NaCl and BSA, in a final volume of 11 μL.After the incubation, 189 μL of TE buffer were added in each sample, and the products were stored at -20 °C.All pre-amplification and amplification reactions were performed in a Perkin Elmer DNA Thermal Cycler 9600.
For the Preselective Amplification (Preselective PCR), 4 μL of the diluted products of the restriction-ligation stage were used.For the reaction, 15 μL of AFLP Core Mix (Applied Biosystems, USA) and 1.0 μL of AFLP preselective primer pairs (Applied Biosystems, USA) were added in a final volume of 20 μL.The preselective PCR thermal conditions were: 2 min at 72 °C, 20 cycles of 22 s at 94 °C, 33 s at 56 °C and 2 min at 72 °C, with a final step of 30 min at 60 °C.The samples were then stored at 4 °C.After the end of the amplification, 10 μL of the pre-amplified products were checked on 1.5% (w/v) agarose gels, in TAE buffer (40 mM Tris-acetate and 1mM EDTA, pH 8), and stained in ethidium bromide (1 μg mL -1 ).The gels were photographed on a Gel Doc 1000 (Biorad).The remaining 10 μL of the pre-amplified products were diluted with 190 μL of TE buffer and stored at 4 °C.
For the Selective Amplification (Selective PCR), 1.5 μL of the diluted preselective products were used.For the reaction, 7.5 μL of AFLP Core Mix (Applied Biosystems, USA) and 0.5 μL of each selective primer (Applied Biosystems.USA) were added in a final volume of 10 μL (as mentioned, the primers were in the form of EcoRI[Primer-Axx-Dye] and MseI[Primer-Cxx]) (Table 3).Selective EcoRI primers were labeled with fluorescent dyes to enable detection using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA).The selective PCR thermal conditions were: 2 min at 94 °C, 10 cycles of 20 s at 94 °C, 30 s at 66 °C (the annealing temperature was reduced in every cycle by 1 °C) and 2 min at 72 °C, 20 additional cycles of 20 s at 94 °C, 30 s at 56 °C and 2 min at 72 °C, with a final step of 30 min at 60 °C.The samples were then stored at 4 °C.
For the loading buffer, 13 μL of deionized formamide with 0.5 μL of GeneScan-500 (LIZ) size standard (Applied Biosystems, USA) were added to 1.0 μL of each amplification sample.The samples were first denaturated at 94 °C for 5 min and then separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA).AFLP electrophoregrams were acquired and analysed using the GeneMapper v4.0 software (Applied Biosystems, USA), using the Local Southern Method.

Statistical Analysis
For the statistical analysis, the method UPGMA was used with one distance coefficient and one similarity coefficient.In order to present the morphological relationships between the cultivars, the Euclidean Distances Squared coefficient was used, as implemented in the NTSYS-pc package 2.1 developed by Rohlf (Exeter Software, New York, USA, 1993).The bigger the value of the coefficient for 2 samples, the bigger the distance between them.For the molecular analysis, the degree of genetic similarity (I) detected between each pair of cultivar studied was calculated using the DICE coefficient (Nei & Li, 1979) as implemented in the NTSYS-pc package 2.1.
Relationships among the OIV descriptors (parameters) were studied using the statistical program Jump 8.0 (SAS Institute Inc).Principal Component (PC) analysis was used to evaluate the most important parameters that contributed to the biotype separation into different groups according to their morphological traits (OIV descriptors).

OIV Ampelographic Descriptor Evaluation
According to the PC analysis, which transforms the original data set (OIV descriptors) into a smaller set of uncorrelated new variables (Principal Components, where eigenvalues was bigger than 1), 10 components have been produced in a decline series of their importance, explaining 89.72% of the total variability among the different biotypes.All descriptors that are grouped in the same principal component have strong correlation between them.Each component is strongly correlated with a set of the initial OIV descriptors so it could be estimated their contribution to variability.The OIV descriptors strongly correlated with the first 10 components are presented in Table 4 and Figure 1.For example, the OIV descriptors 004 (young shoot: density of prostrate hair on tip), 013 (shoot: density of prostrate hair on nodes), 051 (young leaf: color of the upper side of blade (4th leaf)), 083-1 (mature leaf: shape of base of upper lateral sinuses) contributed better to variability compared to OIV descriptors 068 (mature leaf: number of lobes) or 090 (mature leaf: density of prostrate hairs on petiole).In general, and with a few exceptions, the biotypes of Korinthiaki staphis of the same cultivation center show small to very small distance between them, while between biotypes of different cultivation centers, the distance is bigger.The biotypes originating from the cultivation center of Egio show the smallest distances between them but also between most of the biotypes studied, and especially with those of Kefallinia.Also, their distance with biotypes C28, C29, C30, C31 (Sxistofilli) is relatively small.Moreover, no significant differences were observed between the biotypes C26 and C27.Lastly, the biotypes of Sxistofilli (C28, C29, C30, C31) show identity between them, while relatively small is their distance with the biotypes from Egio.The ampelographic differences between these biotypes are found on the descriptors of the leaves and particularly on the number of lobes, on the bunch size and on the color of the berry skin.
The biotypes of Korinthiaki staphis and Staphidampelo are different cultivars and they are found on a totally different cluster of the dendrogram compared to the biotypes of Korinthiaki lefki.Therefore, and despite the similarities in specific ampelographic descriptors and in the nature of seedlessness, Korinthiaki lefki constitutes a different cultivar and not a color mutation of Korinthiaki staphis.
From the analysis of the above results, (Table 3, Figure 1), it is shown that there is phenotypic fluctuation between the three biotypes of Korinthiaki Lefki studied.In particular, the biotypes coming from Zante (C9, C10) do not differ significantly between them but they show a relatively high distance compared to the biotype coming from Egio (C11).

Molecular Analysis
For the molecular analysis and the identification of the biotypes and cultivars studied, five primer combinations were used to amplify genomic DNA from the twenty seven possible clones of Greek grapevine cultivar Korinthiaki staphis, cultivar Staphidampelo and the three biotypes of Korinthiaki lefki.They proved to be highly polymorphic and produced a total of more than 210 amplified fragments (Table 5), discriminating all of the samples studied.The molecular analysis data were then used to obtain a genetic similarity dendrogram (Figure 3).Since each similarity and distance coefficient has a different scale, there may be difference in their degree.The ampelographic description, especially when it takes place for several years and when a great number of descriptors is used, in combination with molecular methods can be extremely effective for the study of the between and within genetic diversity of grapevine cultivars and they can constitute reliable tools for the discrimination of grapevine cultivars, clones and biotypes.
The above mentioned results, aside from the scientific and research interest, can have an immediate implementation in the viticultural practice and in the implementation of programs of clonal selection for the emergence of the most valuable clones of the various biotypes of Korinthiaki staphis.

Figure
Figure

Table 3 .
Primers combination used for AFLP analysis

Table 4 .
Evaluation of the OIV descriptors and their contribution to the variability of the biotypes studied

Table 5 .
Primers used and number of amplified fragments