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doi: 10.15389/agrobiology.2020.3.523eng

UDC: 634.8:577.22

Acknowledgements:
The authors thank the JSC Evrogen company for the help in sequencing.
Supported financially by Russian Foundation for Basic Research and the Administration of the Krasnodar Territory, grant No. № 19-44-233003

 

COMPARATIVE ANALYSIS OF THE VvMybA1 LOCUS ALLEAL STATE IN SOME INDIGENOUS AND INTRODUCENT GRAPEVINE VARIETIES

A.V. Milovanov1, E.T. Ilnitskaya2, V.V. Radchenko1, A.V. Garkovenko1, A.S. Zvyagin1, L.P. Troshin1, A.G. Koshchaev1

1Trubilin Kuban State Agrarian University, 13, ul. Kalinina, Krasnodar, 350044 Russia, e-mail milovanov1991@mail.ru (✉ corresponding author), vitalyradchenko@yandex.ru, chief@gene-expert.ru, ziag@mail.ru, lptroshin@mail.ru, kagbio@mail.ru;
2North Caucasian Federal Scientific Center of Horticulture, Viticulture, Wine-making, 39, ul. im. 40-letiya Pobedy, Krasnodar, 350901 Russia, e-mail ilnitskaya79@mail.ru

ORCID:
Milovanov A.V. orcid.org/0000-0002-6312-1147
Zvyagin A.S. orcid.org/0000-0002-2684-5875
Ilnitskaya E.T. orcid.org/0000-0002-2446-0971
Troshin L.P. orcid.org/0000-0001-5177-352X
Radchenko V.V. orcid.org/0000-0002-4449-3105
Koshchaev A.G. orcid.org/0000-0002-3904-2860
Garkovenko A.V. orcid.org/0000-0001-6809-5999

Received October 10, 2019

 

European cultivated grape Vitis vinifira L. is one of the most common agricultural crops grown by man since ancient times. Of course, the usual companion of the cultivation of this culture is a traditional hybridization and clonal selection, which allows you to select spontaneous mutants. Nevertheless, the study of such an important agrobiological trait as anthocyanin coloration and, in particular, the genes that determine it, is one of the most important problems both in modern grape genetics and in studies of plant metabolic pathways in general. The study of this problem can not only provide the basis for subsequent fundamental research on the functioning of both individual genes and the genome as a whole, but also create a basis for the selection of varieties for economically valuable traits. The VvMybA family of grape transcription factors is responsible for anthocyanin accumulation in berries of cultivated and wild grapes. In the present work, alleles of the VvMybA1 gene were first identified in native Russian grape varieties. Theses alleles in the colored and uncolored grape varieties were found out to be the same in size. Sequence alignment showed the characteristic features of alleles for each of the studied genotypes. The purpose of our study, we set a description of the four alleles of the VvMybA1 gene in the Chardonnay, Sibirkovskii, Cabernet Cortis and Sypun cherny varieties. Two introduced and well-known varieties and two indigenous varieties also cultivated in the Krasnodar Territory were investigated. The leaves for DNA extraction was collected at the Anapa zonal experimental station. DNA was isolated by CTAB method with mercaptoethanol. PCR was performed using primers and amplification parameters published in the literature. For PCR amplification and isolation of VvMybA1 gene alleles we used markers which allow simultaneous identification of two alleles, VvMybA1b and VvMybA1c. We sequenced and compared with each other and with the GenBank NCBI database the sequences of the VvMybA1 gene alleles of these varieties. Alignment of sequences in the ClustalO program revealed structural features of the allele nucleotide sequences. In particular, a single nucleotide insert was found in white-berry varieties and nucleotide substitutions in different places in varieties with colored and uncolored berries. Further, comparing with the GenBank NCBI database found that the alleles of colored varieties have a structure characteristic for varieties with a pronounced color of berries, while uncolored varieties apparently have a specific reason for the loss of color. Thus, it was found that the Chardonnay variety has an allele with the insertion of Gret-1 transposon which blocks the normal expression of VvMybA1 gene. It was also revealed that the Sibirkovky variety also has the alleleof VvMybA1 gene which is not functional due to the blocking by Gret-1 transposon. As shown in previous studies by other authors, this allele is also present in other uncolored varieties, and, therefore, this is the reason for blocking gene expression. A study of the amino acid sequence also revealed differences between the groups of colored and uncolored varieties. These differences can be obviously divided into those characteristic of colored and uncolored varieties. However, a mutation was detected in Sypun cherny variety, which affected the replacement of the amino acid isoleucine with valine, but did not affect the overall color of the berries. When searching for the amino acid sequence in the GenBank NCBI database, it was revealed that this mutation is not unique in nature, as it was found in the Alphonse Lavallée variety, as well as in interspecific hybrids.

Keywords: Vitis vinifera L., indigenous varieties, introduced varieties, allele, gene, VvMybA1, sequencing, anthocyanin, mutations, amino acids, transposons.

 

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