Identification of UFGT-gene-associated groups of Vitis vinifera L. by the developed method of PCR-RFLP genotyping of grape

Abstract

The Vitis vinifera L. UFGT gene is one of the diagnostically significant genes for genetic testing of technical grape varieties as well as wine materials and wines produced from them. The strategy for genetic identification of grape varieties and DNA authentication of wine materials that was previously developed by us and is based on direct sequencing of the specific PCR product with a length of 99 bp gave an impulse to prognostic assessment of feasibility of PCR-RFLP analysis for detection of five diagnostically significant polymorphic positions and the following identification of 13 UFGT gene-associated groups of Vitis vinifera L. The aim of this work consisted in identification of UFGT gene-associated groups of Vitis vinifera L. by detection of diagnostically significant polymorphic positions using the developed PCR-RFLP method for genotyping of grape. Objects of research were 24 samples of technical grape varieties. Their sample preparation was carried out by extracting 50–100 mg of mature grape pulp or stone with its mechanical comminution in a mortar and transfer to an Eppendorf-type tube. Then, nucleic acids were extracted using a commercial innuPREP Plant DNA Kit or DiamondDNA Plant kit. PCR-RFLP with the extracted grape DNA was performed with Phire Plant Direct PCR Master Mix and four selected restrictases (PstI, BsaXI, BtsIMutI and HinfI) according to the protocols presented in the paper. The detection of the PCR-RFLP fragments was performed by visualization of electropherograms in a UV transilluminator after horizontal electrophoresis in 2.5% agarose gel with stained TAE buffer. The method for PCR-RFLP genotyping of grapes developed specially for identification of UFGT gene-associated groups of Vitis vinifera L. by detecting diagnostically significant polymorphic positions demonstrated its feasibility when testing 24 samples of technical grape varieties. With that, the positive result was achieved due to the practical ability of each of four selected restrictases to discriminate the strictly specified polymorphic position generating characteristic PCR-RFLP profiles of 13 UFGT gene-associated groups of Vitis vinifera L., seven of which were revealed during this study. Therefore, as a result of the performed study, the genotypic affiliation of several tested grape varieties was established: six samples were identified as representatives of the UFGT gene-associated group No.1; one sample was assigned to gene-associated group No.2; two samples were characterized by the trait of associated group No.3; four samples belonged to group No. 4; one sample to group No. 5; six samples to group No.13.