АннотацияWines DNA authentication is a technological process of their authenticity verification by genetic identification of the main plant ingredient by means of molecular genetic analysis of the residual amounts of Vitis vinifera L nucleic acids extracted from end product cellular debris. The main aim of the research was the analysis of scientific and methodological approaches to the extraction of residual amounts of nucleic acids in wine raw materials and DNA authentication of wines for their subsequent application in solving the problem of determining wine products authenticity and place of origin. The prior art includes various approaches to the extraction of Vitis vinifera L. nucleic acids among which the three methods by Savazzini & Martinelli, Pereira and Bigliazzi can be named basically. Analysis of the effectiveness of different methods of DNA extraction from wines indicates the superiority of the Pereira method over other traditional methods of extraction in terms of DNA yield and quality. Besides, the nucleic acid extracted from wines is characterized as residual since its concentration is significantly reduced in a multi-stage wine production process. The yield of extracted nucleic acid also decreases as the wine ages. The use of microsatellite DNA loci designed for grapes genetic identification is one of the approaches applicable for wine DNA authentication.
Ключевые словаWine , grapes , variety , Vitis vinifera L , DNA , authentication , identification , marker , SSR , SNP , PCR , HRM analysis
Considering its wide assortment variety and multicomponent chemical composition, wine production belongs to the segment of difficult-to- identify goods whose established authenticity serves as a basic element of consumers and producers’ rights protection [1, 2].
The search for objective identification criteria with a high degree of wine products authenticity and place of origin assessment reliability is a strategically important task achievable by multidisciplinary science- intensive approaches [3, 4].
The complex identification scheme based on the approved methods of analysis (documentary, visual, organoleptic, and physicochemical) [5–9] can be expanded by the molecular genetic method  applicable for wine DNA authentication [11, 12].
Wine DNA authentication is a technological process of authenticity verification by genetic identification of the main plant ingredient – wine grapes – by means of molecular genetic analysis of the residual amounts of Vitis vinifera L nucleic acids extracted from end product cellular debris [11, 12].Analysis of research and methodological approaches to the extraction of the residual amounts of nucleic acids in wine raw materials, and wine DNA authentication demonstrates the applicability of DNA technologies for the monitoring of counterfeit and adulterated wine products.
РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ
Methods of extraction of wine raw materials DNA residual amounts. The prior art includes various approaches to the extraction of Vitis vinifera L. nucleic acids [13–21], among which the three methods by Savazzini & Martinelli , Pereira  and Bigliazzi  can be named basically in both the original and modified set-ups .
Reagents used at various stages of sample preparation and extraction of Vitis vinifera L. DNA in the three namesake methods of nucleic acids extraction from wines are shown in Table 1.
The unifying feature of the methods presented is the precipitation of wine plant debris by centrifugation using precipitators, suchas sodium chloride, 2-propanol, and sodium acetate separately or in combination of the latter two.
Resuspension of the plant debris sediment and its lysis are carried out by a multicomponent lysing buffer consisting of ethylenediaminotetraacetic asid (EDTA), trisaminomethane hydrochloric acid (Tris-hcl), sodium chloride (NaCl), cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and 2-mercaptoethanol [14, 16, 17]. The buffer also includes the proteinase K in the Pereira method .
The stage of deproteinization of the lysate nucleoprotein complex is carried out by organic solvents (chloroform and isoamyl alcohol), with the inclusion of phenol in the three-component mixture phenol:chloroform:isoamyl alcohol at the ratio 25:24:1 in the Savazzini & Martinelli method .
The Pereira method  includes the stage of RNA removal by treatment with RNase A followed by untreated DNA deposition by centrifugation, further resuspension of the deposited DNA with an elution buffer and additional deproteinization with neutral phenol. In the Bigliazzi method , the additional stage of chloroform-methanol deproteinization at the ratio 24:1 is preceded by the stage of adding 1.1 volume of CTAB to the double centrifugation supernatant.
The selected aqueous phase is precipitated by 2-propanol in all the three methods. The subsequent deposited DNA washing in the Savazzini & Martinelli method  is performed with 70% ethanol, while in the Pereira method  with a washing buffer containing ethanol and ammonium acetate, and in the Bigliazzi method  this stage is unavailable.
However, after the precipitated DNA resuspension with an eluent, the Bigliazzi method  incorporates additional resuspended DNA treatment with proteinase K, and subsequent manipulations, including the use of QIAprep Spin Miniprep Kit.
It must be noted that a number of commercial kits selectively binding extractable nucleic acid on spin columns, such as “Plant Genomic DNA Miniprep Kit” (Sigma), “Dneasy Plant Mini Kit” (Qiagen), “High Pure PCR Template Preparation Kit” (Roche), “Power Plant DNA Isolation Kit” (MO-BIO), “Power Soil DNA Isolation Kit” (MO-BIO), “FastDNA Spin Kit for Soil” (MP Biomedicals), “NucleoSpin Food” (Macherey-Nagel), “Genomic DNA from Food” (Macherey-Nagel), and “Dneasy Mericon Food Kit” (Qiagen) were tested in Vitis vinifera L. DNA extraction, including the use of “DNA Purification System for Food” (Promega) based on DNA extraction with the help of magnetic particles .
At the same time, the procedure of DNA isolation by commercial sets from the 2-isopropanol precipitated plant debris had minor modifications associated with the introduction of a set of 100 µl α-amylase (incubation at 80°C for 30 min) and/or 40 µl of proteinase K (incubation at 55°C for 30 min) into the first used buffer .
Sequential application of α-amylase and proteinase K is also provided by the procedure of nucleic acid isolation from lyophilized, pre-dissolved in a buffer (0.1 M Tris-HCl (pH 8.0), and 0.1 M NaCl) wine powder whose DNA extraction protocol is described in Nakamura et al. (2007) . In this case, proteinase K is included in the hydrolysis by incubation at 55°C for 60 min together with sodium dodecyl sulfate (SDS). The resulting hydrolysate undergoes stepwise stages of precipitation by centrifugation in a mixture with 2-propanol; nucleic acid precipitate resuspension with an eluent buffer followed by the addition of 70% ethanol; re-deposition in a mixture of 2-propanol with sodium acetate; and deposited DNA resuspension in TE buffer. The extraction procedure also includes the stages of RNA removal by treatment of nucleic acid with RNase A and deproteinization first with neutral phenol and then with phenol:chloroform (1:1), with precipitation of the selected aqueous phase by addition of 0.2 M NaCl and 2 volumes of cold ethanol. The resulting DNA precipitate is washed with 70% ethanol and eluted with TE buffer.
Performance analysis of Vitis vinifera L. DNA extraction by different methods indicates the superiority of the Pereira method  over other traditional methods of extraction [13–15, 17] in terms of DNA yield and quality . Detailed information on the stages of Vitis vinifera L. sample preparation and DNA isolation by the Pereira method  is shown in Fig. 1.
The comparative quantitative and qualitative assessment of the isolated nucleic acid preparations pointed to the ineffectiveness of the Bigliazzi method . A number of commercial sets with the exception of “Dneasy Plant Mini Kit” (Qiagen), “High Pure PCR Template Preparation Kit” (Roche), and “Power Soil Isolation Kit” (MO-BIO) with their minor modifications associated with the addition of α-amylase and/or proteinase K providing test systems performance, also proved ineffective. Treatment with α-amylase is prescribed for the set produced by Roche, while for the MO-BIO set  it is α-amylase and proteinase K treatment.
Precipitators used for wine plant debris precipitation, such as NaCl, 2-propanol, sodium acetate used both individually and in a complex are the key reagents at the initial stage of sample preparation which involves concentration of the test material by centrifugation. From the point of view of the efficiency of DNA yield and quality, the method is not inferior to concentration by lyophilization though rather time-consuming. For example, the Pereira method  regulates storage of wine and precipitator mixture in the freezer for 2 weeks for maximum precipitation effect.
Extracted from wines Vitis vinifera L DNA has the status of residual nucleic acid since its concentration is significantly reduced during the multi-stage wine production process including decantation, purification, filtration and other processing methods [15, 20]. In addition, grapes DNA is degraded by the DNase of wine microbiota during fermentation .
Wine aging reduces the yield of the extracted nucleic acid . At the same time, it is experimentally proved  that the amount of Vitis vinifera L DNA isolated from wines with the completed stage of alcoholic fermentation is significantly reduced or practically absent depending on the terms of end product testing.
Methods of wines DNA authentication. The use of highly polymorphic microsatellite DNA loci designed for grapes genetic identification [23–27] is one of the approaches to wine DNA authentication as well [11, 13–17, 21, 22, 28, 29, 30].
Nine highly specific and reproducible polymorphic markers of the nuclear DNA microsatellite (SSR) loci presented in Table 2 form the basic set for identification and certification of grapes varieties and hybrids, where the specified sequences of oligonucleotide primers initiate amplification of SSR fragments of the extracted DNA for subsequent granular analysis in capillary gel electrophoresis  of the sequencer with interpretation of results by the genetic analyzer software.
SSR fragments amplification is carried out by multiplex PCR, combining several analyzed loci. This amplification strategy is based on working with DNA extracted from the components of grape plants (fruit, leaf, stem, root) but is not effective while studying extracted residual wine nucleic acid [11, 13, 16, 42]. Therefore, wine DNA authentication is usually carried out by PCR with a set of primers of a single SSR- marker to achieve an analyzable result .
While DNA testing monosort and assemblage commercial wines researchers noted identifiable results of the markers specified in Table 2: VrZAG79 [11, 16], VVS2, VVMD27 [14, 17], and VVMD25  These allow for a retrospective assessment of the wine materials varietal identity and actual wine DNA authentication by the interpretation of the fragmented data analysis.
The database of 3675 grape varieties genetic profiles in VIVC interactive catalogue (Vitis International Variety Catalogue) (Fig. 2) is successfully used when comparing the DNA-test-generated SSR markers profiles with the already hosted on the server published information.
Another type of SSR markers, spSSR [43–46], targeted to the chloroplast DNA, has several advantages over the nuclear DNA (nSSR) analysis due to the greater number of representation per cell, greater resistance to exonuclease influence and lower susceptibility to degradation due to its content in double membrane organelles [11, 13].
Despite the weak discriminatory ability of these SSR markers, incapable of wide range of grape varieties certification, the analysis of chloroplast DNA microsatellite loci remains an alternative approach to the varietal genetic identification of Vitis vinifera L, although not quite suitable for wine DNA authentication [11, 13, 16, 20, 21, 29] due to the low level of polymorphism of the analyzed loci, suitable only for a limited range of wine products differentiation (Table 3).
In the study of 21 grape varieties by fragmented analysis of 8 cpSSR-loci, whose oligonucleotide primers are listed in Table. 3, in the studied sample selection V. Catalano et al. (2016)  discovered 4 chlorotypes, whose results with grouping of the tested Vitis vinifera L varieties by their haplotype are shown in Table 4.
At the same time, half of these cpSSR-loci (NTcp12, ccmp2, ccmp4, and ccmp6) had no allele polymorphism, but two analyzed loci (ccmp10 and ccSSR14) were characterized by the presence of three alleles, and two more loci (NTcp8 and ccmp3) – by the presence of two alleles , respectively (Table 4).
Although this method shows relatively low resolution , it can be used as an additional test for counterfeit and adulterated wine products identification.
Microsatellite DNA is also used as a source of STS (Sequence Tagged Site – sites marked with a sequence) – unbroken unique sequences whose amplified profiles serve as molecular genetic markers [11, 15, 23]. Thus, S. Nakamura et al. (2007)  designed experimental sets of STS primers for certain SSR loci of mitochondrial and chloroplast DNA [25, 43, 51–54], having tested them in PCR during varietal genetic identification of Vitis vinifera L, and DNA authentication of wines produced from them.
Along with SSR markers, SNP markers [55, 56], applicable for wine DNA authentication , both thanks to tracing Vitis vinifera L. individual genotypes in monosort and assemblage wines with the potential for the quantitative assessment of plant ingredients and their performance in the analysis of the fragmented nucleic acid bear a high identification potential. Based on SNP markers, test systems can be designed for genetic identification of individual grape varieties .
Table 5 shows the sets of primers and probes for real-time PCR with fluorescent hybridization detection used in genetic identification of the Sangiovese variety, and DNA authentication of the wine produced from it by assessing single-nucleotide polymorphism (SNP) in three analytical positions (98, 222 and 244) .
Sangiovese variety genetic identification and DNA authentication of the wine produced from it, based on the assessment of single-nucleotide polymorphism is established by the presence of allele C in the homozygous state (CC genotype) in all the three SNP analytical positions  (Table 6).
Another type of SNP markers application is the use of knowledge about single nucleotide polymorphism in a number of Vitis vinifera L genes integrated into melting curves analysis with high resolution (HRM analysis) on PCR platforms in real time [12, 57, 58].
HRM analysis is an effective technology of genotyping  with combined stages of PCR and detection of a high degree of specificity and sensitivity, capable of differentiating between several genotypes within one analysis, and suitable for wine DNA authentication [12, 57].
Table 7 presents the sets of PCR primers with subsequent HRM analysis and/or sequencing by grape varieties genetic identification and DNA authentication of the wines produced from them.
Primers Vv3-Fwd and Vv3-Rev initiate amplification of the UFGT gene locus, 119 bp long, with localization of the flanked region in the range of 387–505, covering five SNP analytical positions (424, 425, 442, 459, 483)  interpreted by HRM analysis and/or sequencing (Table 8).
F3H_H1fwd and F3H-H1rev primers initiate amplification of the F3H1 gene locus, 375 bp long, with localization of the flanked region in the range of 5–379, covering two SNP analytical positions (47 and 291) [12, 58]; F3H_H2fwd and F3H_H2rev primers initiate amplification of the F3H1 gene locus, 532 bp long , with the flanked region localization in the range of 975–1506, covering eight SNP analytical positions (1039, 1040, 1065, 1157, 1318, 1381 and 1464) , respectively, also interpreted by HRM analysis and/or sequencing (Table 9).
As can be seen from the considered examples, single gene locus SNP identification does not allow the origin of the analyzed wine material, to be established unambiguously therefore, it is not universal. In order to be able to determine the effectiveness of identification methods, as well as their combinations, the concept of identification distance was introduced for the first time. The identification distance (ID) of grape variety A from grape variety B is the number of polymorphic nucleotide positions (SNP) which allow identifying the presence of grape variety B DNA material impurities in wine material A. As seen from the definition, ID is asymmetric due to the presence of mixed nucleotides. For example, F3H-gene locus SNP analysis (Table 9) allows to determine the presence of Chardonnay grape variety in Cabernet Sauvignon wine material while detecting polymorphic nucleotide positions 1157, 1381 and 1464 (ID = 3). At the same time, it is not possible to differentiate Cabernet Sauvignon grape variety in Chardonnay wine material by this approach (ID = 0).
Fig. 3 shows the diagram of identification distances according to F3H-gene locus SNP analysis (Table 9), where the varieties with identical polymorphic nucleotide positions are combined into a single group.
Thus, it is urgent to create a complex method of wine DNA authentication, allowing a robust analysis procedure in the context of identification distance (namely, uncovering within the method the ID minimum threshold value for all variety pairs that is acceptable for the purposes of standardization) to identify and differentiate grape varieties in both varietal and assembling commercial wines
The arsenal of molecular genetic markers used for wine DNA authentication can be significantly expanded by the development of genomic and postgenomic technologies, with the introduction of experimental developments in the product quality management system based on the standards developed for the wine industry, especially while identifying wines of protected geographical indications, wines of protected designations of origin, and for the purposes of counterfeit products identification [1, 2, 60].
In the meantime, the existing methods of wine DNA authentication, published in scientific literature, have a recommendatory status, are not regulated by GOST/ISO and other legal documents of the Russian Federation, the Customs Union, the International Organization of Vine and Wine, and the European Union.
Legal and regulatory replenishment of the approved wine identification complex scheme with new DNA authentication methods will increase assessment reliability of authenticity and place of origin of wine products.
Analysis of research and methodological approaches to the extraction of residual amounts of nucleic acids in wine raw materials and wine DNA authentication confirms the relevance of this research line and the prospects of its integration into the system of monitoring counterfeit and adulterated wine products. It shows the possibility of determining wine products authenticity and place of origin by DNA technologies, whose use implies ensured traceability of the product’s entire life cycle. The concept of identification distance (ID) between grape varieties A and B is introduced. ID stands for the number of polymorphous nucleotide positions (SNP), which allow the presence of grape variety B DNA material in variety A wine material to be identified. The algorithm of ID calculation and analysis of the information obtained is offered.
The authors declare no conflict of interest.
The study was supported by the section of agricultural products storage and processing of the Department of Agricultural Sciences of RAS and ‘V.M. Gorbatov FSC Foods Systems’ of RAS.
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