A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr


  • Jana Žiarovská Slovak University of Agriculture, Faculty of Agrobiology and Food Resources, Department of Genetics and Plant Breeding, Tr. A. Hlinku 2, 949 76 Nitra
  • Lucia Zeleňáková Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety, Tr. A. Hlinku 2, 949 76 Nitra
  • Miroslava Kačániová Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
  • Eloy Fernández Cusimamani Czech University of Life Sciences Prague, Faculty of Tropical AgriSciences, Kamýcká 961/129, 165 00, Prague-Suchdol




direct PCR, Vitis vinifera L., thaumatin-like sequence, stormy wine, must


Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs) of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too. 


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Aubert, Ch., Chalot, G. 2018. Chemical composition, bioactive compounds, and volatiles of six table grape varieties (Vitis vinifera L.). Food Chemistry, vol. 240, p. 524-533. https://doi.org/10.1016/j.foodchem.2017.07.152 PMid:28946307

Baleiras-Couto, M. M., Eiras-Dias, J. E. 2006. Detection and identification of grape varieties in must and wine using nuclear and chloroplast microsatellite markers. Analytica Chimica Acta, vol. 563, no. 1-2, p. 283-291. https://doi.org/10.1016/j.aca.2005.09.076

Bellstedt, D. U., Pirie, M. D., Visser, J. Ch., de Villiers, M. J., Gehrke, B. 2010. A Rapid and Inexpensive Method for the Direct PCR Amplification of DNA from Plants. American Journal of Botany, vol. 97, no. 7, p. 65-68. https://doi.org/10.3732/ajb.1000181

Bošeľová, D., Žiarovská, J. 2016. Direct PCR as the platform of Hedera helix, L. genotypying without the extraction of DNA. Journal of Central European Agriculture. vol. 17, no. 4, p. 941-949. https://jcea.agr.hr/en/issues/article/1795

Bošeľová, D., Žiarovská, J., Hlavačková, L., Ražná, K., Bežo, M. 2016. Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR. Acta fytotechnica et zootechnica, vol. 19, no. 4, p. 144-149.

Briciu, D., Pamfil, D., Briciu, A., Curticiu, D., Balazs, E., Taoutaou, A., Pop, I., and Coia, L. 2010. Development of methods for DNA extraction from leaves and must grapes. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Animal Science and Biotechnologies. vol. 67, no. 1-2, p. 1843-1849.

Drábek, J., Stávek, J., Jalůvková, M., Jurček, T., Frébort, I. 2008. Quantification of DNA during winemaking by fluorimetry and Vitis vinifera L. specific quantitative PCR. European Food Research and Technology, vol. 226, no. 3, p. 491-497. https://doi.org/10.1007/s00217-007-0561-8

D'Angeli, S., Altamura, M. M. 2007. Osmotin induces cold protection in olive trees by affecting programmed cell death and cytoskeleton organization. Planta, vol. 225, no. 5, p. 1147-1163. https://doi.org/10.1007/s00425-006-0426-6 PMid:17086398

Faria, M. A., Magalhães, R., Ferreira, M. A., Meredith, C. P., Monteiro, F. F. 2000. Vitis vinifera must varietal authentication using microsatellite DNA analysis (SSR). Journal Agricultural and Food Chemistry, vol. 48, no. 4, p. 1096-1100. https://doi.org/10.1021/jf990837h PMid:10775355

Faria, M. A., Nunes, E., Oliveira, M. 2008. Relative quantification of Vitis vinifera L. varieties in musts by microsatellite DNA analysis. European Food Research and Technology , vol. 227, p. 845-850. https://doi.org/10.1007/s00217-007-0795-5

Ferreira, R. B., Piçarra-Pereira, M. A., Monteiro, S., Loureiro, V. B., Teixeira, A. R. 2001. The wine proteins. Trends in Food Science and Technology, vol. 12, no. 7, p. 230-239. https://doi.org/10.1016/S0924-2244(01)00080-2

Garcia-Beneytez, E., Moreno-Arribas, M. V., Borrego, J., Polo, M. C., Ibanez, J. 2002. Application of a DNA analysis method for the cultivar identification of grape musts and experimental and commercial wines of Vitis vinifera L. using microsatellite markers. Journal of Agricultural and Food Chemistry, vol. 50, no. 21, p. 6090-6096. https://doi.org/10.1021/jf0202077 PMid:12358485

Garcia-Casado, G., Collada, C., Allona, I., Soto, A., Casado, R., Rodriguez-Cerezo, E., Gomez, L., Aragoncillo, C. 2000. Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chestnut seeds. Physiologia Plantarum, vol. 110, no. 2, p. 172-180. https://doi.org/10.1034/j.1399-3054.2000.110205.x

Ho., V. S., Wong, J. H., Ng, T. B. 2007. A thaumatin-like antifungal protein from the emperor banana. Peptides, vol. 28, no. 4, p. 760-766. https://doi.org/10.1016/j.peptides.2007.01.005

Husaini, A. M., Abdin, M. Z. 2008. Development of transgenic strawberry (Fragaria x ananassa Duch.) plants tolerant to salt stress. Plant Science, vol. 174, no. 4, p. 446-455. https://doi.org/10.1016/j.plantsci.2008.01.007

Chu, K. T., Ng, T. B. 2003. Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis. Biochemical and Biophysical. Research Communication, vol. 301, no. 2, p. 364-370. https://doi.org/10.1016/S0006-291X(02)02998-4

Chum, P. Y., Haimes, J. D., André, Ch. P., Kuusisto P. K., Kelley, M. L. 2012. Genotyping of plant and animal samples without prior DNA purification. Journal of Visualized Experiments, vol. 67, p. e3844. https://doi.org/10.3791/3844

Işçi, B., Yildirim, H. K., Altindisli, A. 2014. Evaluation of methods for DNA extraction from must and wine. Journal of Institute Brewing, vol. 120, no. 3, p. 238-243. https://doi.org/10.1002/jib.129

Jayasankar, S., Li, Z., Gray, D. J. 2000. In-vitro selection of Vitis vinifera 'Chardonnay' with Elsinoe ampelina culture filtrate is accompaniedby fungal resistance and enhanced secretion of chitinase. Planta, vol. 211, no. 2, p. 200-208. https://doi.org/10.1007/s004250000285 PMid:10945214

Liu, J., Sturrock, R., Ekramoddoullah, A. K. 2010. The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function. Plant Cell Reporter, vol. 29, no. 5, p. 419-436. https://doi.org/10.1007/s00299-010-0826-8

Marangon, M., Van Sluyter, S. C., Waters, E. J., Menz, R. I. 2014. Structure of Haze Forming Proteins in White Wines: Vitis vinifera thaumatin-Like Proteins. PLOS ONE, vol. 9, no. 12, e113757 https://doi.org/10.1371/journal.pone.0113757 PMid:25463627

Medo, J., Maková, J., Kovácsová, S., Majerčíková, K., Javoreková, S. 2015. Effect of Dursban 480 EC (chlorpyrifos) and Talstar 10 EC (bifenthrin) on the physiological and genetic diversity of microorganisms in soil. Journal of environmental science and health, vol. 50, no. 12, p. 871-883. https://doi.org/10.1080/03601234.2015.1062659

Milella, L., Martelli, G., Salava, J., Fernández, C. E., Ovesná, J., Greco, I. 2011. Total phenolic content, RAPDs, AFLPs and morphological traits for the analysis of variability in Smallanthus sonchifolius. Genetic Resources and Crop Evolution, vol. 58, no. 4, p. 545-551. https://doi.org/10.1007/s10722-010-9597-x

Oslovičová, V., Simmonds, J. R., Snape, J. W., Gálová, Z., Balážová, Ž., Matušíková, I. 2014. Molecular marker-based characterization of a set of wheat genotypes adapted to Central Europe. Cereal research communications, vol. 42, no. 2, p. 189-198. https://doi.org/10.1556/CRC.42.2014.2.2

Parkhi, V., Kumar, V., Sunilkumar, G., Campbell, L. M., Singh, N. K., Rathore, K. S. 2009. Expression of apoplastically secreted tobacco osmotinin cotton confers drought tolerance. Molecular Breeding, vol. 23, no. 4, p. 625-639. https://doi.org/10.1007/s11032-009-9261-3

Revák, O., Golian, J., Židek, R., Čapla, J., Zajác, P. 2014. Detection of lupine (Lupinus spp. L.) as a food allergen using three methods: end-point PCR, real-time PCR and ELISA. Potravinarstvo, vol. 8, no. 1, p. 207-215. https://doi.org/10.5219/384

Siret, R., Gigaud, O., Rosec, J. P., This, P. 2002. Analysis of grape Vitisvinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers. Journal Agricultural and Food Chemistry, vol. 50, p. 3822-3827. https://doi.org/10.1021/jf011462e

Trebichalský, A., Kalendar, R., Schulman, A., Stratula, O., Gálová, Z., Balážová, Ž., Chňapek, M. 2013. Detection of genetic relationships among spring and winter triticale (× Triticosecale Witt.) and rye cultivars (Secale cereale L.) by using retrotransposon-based markers. Czech journal of genetics and plant breeding, vol. 49, no. 4, p. 171-174. https://doi.org/10.17221/56/2013-CJGPB

Vincze, T., Posfai, J., Roberts, R. J. 2003. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Research, vol. 31, no. 13, p. 3688-3691. https://doi.org/10.1093/nar/gkg526 PMid:12824395

Wang, Q., Li, F., Zhang, X., Zhang, Y., Hou, Y., Zhang, S., Wu, Z. 2011. Purification and characterization of a CkTLPprotein from Cynanchum komarovii seeds that confers antifungal activity. PloS one. vol. 6, no. 2, e16930. https://doi.org/10.1371/journal.pone.0016930

Waters, E. J., Alexander, G., Muhlack, R., Pocock, K. F., Colby, C., et al. 2005. Preventing protein haze inbottled white wine. Austrian Journal Grape Wine Research, vol. 11, no. 2, p. 215-225. https://doi.org/10.1111/j.1755-0238.2005.tb00289.x

Van der Wel, H., Loeve, K. 1972. Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. European Journal of Biochemistry, vol. 31, no. 2, p. 221-225. https://doi.org/10.1111/j.1432-1033.1972.tb02522.x PMid:4647176

Yan, X., Qiao, H., Zhang, X., Guo, Ch., Wang, M., Wang, Y., Wang, X. 2017. Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance. Scientific Reports, vol. 7, no. 1, p. 4269. https://doi.org/10.1038/s41598-017-04105-w

Yang, G. Y., Kim, J. Y., Moon-Soo, S., Doo-Sik, K. 2007. A Simple and Rapid Gene Amplification from Arabidopsis Leaves Using AnyDirect System. Journal of Biochemistry and Molecular Biology, vol. 40, no. 3, p. 444-447. https://doi.org/10.5483/BMBRep.2007.40.3.444

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T. L. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, vol. 13, p.134. https://doi.org/10.1186/1471-2105-13-134 PMid:22708584

Zhang, Z., Schwartz, S., Wagner, L. and Miller, W. A. 2000. A greedy algorithm for aligning DNA sequences. Journal of Computational Biology, vol. 7, no. 1-2, p. 203-214. https://doi.org/10.1089/10665270050081478 PMid:10890397

Žiarovská, J., Hricová, A., Gálová, Z., Záhorský, M., Bošeľová, D. 2016. Utilization of DNA without isolation approaches in plant genome analyzes (Využitie DNA bez-izolačných prístupov v analýzach genómov rastlín). Chemické listy, vol. 110, p. 931-934. (In Slovak)

Žiarovská, J., Grygorieva, O., Zeleňáková, L., Bežo, M., Brindza, J. 2015. Identification of sweet chesnut pollen in bee pollen pellet using molecular analysis. Potravinarstvo. vol. 9, no. 1, p. 352-358. https://doi.org/10.5219/497

Žiarovská, J., Bošeľová, D., Zeleňáková, L., Bežo, M. 2016a. Utilization of different markers for Hedera helix, L. germplasm evaluation. Journal of Microbiology, Biotechnology and Food Sciences, vol. 5, no. 1, p. 23-26. https://doi.org/10.15414/jmbfs.2016.5.special1.23-26

Žiarovská, J., Kyseľ, M., Cimermanová, R., Knoteková, Ľ. 2017. Effect of DNA extraction method in the Rosa canina L. identification under different processing temperature. Potravinarstvo Slovak Journal of Food Sciences, vol. 11, p. 190-196. https://dx.doi.org/10.5219/695

Židek, R., Bajzík, P., Maršálková, L, Golian, J. 2012. Detection of the beef meat adulteration with Real-Time PCR. Maso, vol. 23, no. 1, p. 15-17.




How to Cite

Žiarovská, J. ., Zeleňáková, L. ., Kačániová, M. ., & Cusimamani, E. F. (2018). A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr. Potravinarstvo Slovak Journal of Food Sciences, 12(1), 226–232. https://doi.org/10.5219/892

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