Application of DNA markers in molecular breeding and genetic studies of grapevine

DNA technology has increasingly become more and more important over last years. They can be used to improve the breeding of agricultural plants. Grapevine is one of the oldest and most important cultured plants. This article presents a review of the world’s main achievements in grapevine genetics and markerassisted selection after the Vitis genome sequence. The process of creating new forms of grapes, as well as all crops, is based on the use of existing genetic diversity. For this reason, the problem of a detailed study of the gene pool of the genus Vitis, wild populations and varieties created by men during the long cultivation history of this crop becomes more and more important every year. The genome of Vitis vinifera L. is the fourth sequenced nuclear genome of higher plants. A number of genes and quantitative trait loci were identified and mapped by various research groups, and so were sets of DNA markers for the genes of economically valuable traits. The genes for resistance to the most harmful fungal pathogens Plasmopara vitikola, Erysiphe necator are the most studied. Advancements in genetic mapping and the use of DNA markers in traditional breeding made it possible to refine the genetic mechanism of seedlessness, an important trait in the breeding of table grapes. The study of the genetic control of the content of substances that determine the organoleptic properties of wines has also progressed. Marker-assisted selection (MAS) is used in practice increasingly. Markers associated with disease resistance genes currently used for elimination of susceptible seedlings at the initial stage of large-scale breeding programs carried out in Germany, Italy and the United States. Thus, advances in molecular biology of grapevines creates a conducive situation for active use of DNA-marker technology in this culture

DNA marker identification of Rpv3 downy mildew resistance gene in seedless grape varieties

Table grapes are a valuable dietary product. Seedless grapes are in high demand among consumers. For this reason, the breeding of seedless varieties is one of the popular trends in modern viticulture, along with the production of environmentally friendly products. Downy mildew (Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni) is one of the most common fungal diseases of the grapevine. Most downy mildew resistant grape accessions belong to North American species like Vitis aestivalis Michx., V. berlandieri Planch., V. cinerea (Engelm. ex A. Gray) Engelm. ex Millard, V. riparia Michx., V. rupestris  Scheele, etc. The search for donors of resistance genes is an urgent task. Rpv3 is one of the most significant resistance genes from a number of North American grape varieties. The aim of this work is to identify the downy mildew resistance gene Rpv3 in seedless grape varieties by means of DNA-marker analysis. The grape varieties with rudimentary development of seed in berries and with North American species in the pedigree were chosen as the object of the study. The varieties “Dunavski lazur” and “Seyve Villard 12-375” with reference alleles were used as the positive control, while V. vinifera L. was used as the negative control. UDV305 and UDV737 DNA-markers were used in this study to identify the allelic type of the Rpv3 gene. The work was performed using the polymerase chain reaction. The reaction products were separated by capillary electrophoresis using the ABI Prism 3130 automatic genetic analyzer. Evaluation of the results was done using the GeneMapper and PeakScanner software. Functional alleles of the downy mildew resistance gene Rpv3 were revealed in grape  varieties “Kishmish zaporozhskiy”, “Lady Patricia”, “Remaily seedless”, “Pamyati Smirnova” and “Shayan”. Rpv3299-279, one of the seven known haplotypes, was identified in all the varieties. The pedigree analysis of the studied varieties indicated that the parental forms – “Seyve Villard” and “Seibel” hybrids – are presumably the donors of the gene. Grape accessions with the identified Rpv3 gene can be used in seedless varieties breeding as donors of resistance to downy mildew

A study of genetic profiles of grape plants preserved under the name of Dagestan variety ‘Khatmi’

Background. Traditionally, the description of grape varieties is a task of ampelographic studies. However, several different grape cultivars have similar phenotypic traits. Molecular genetic characterization is the most accurate tool for cultivar identification. The development of DNA fingerprinting of varieties is the first step in this direction. An extensive database of DNA profiles of grape genotypes makes it possible to determine the varietal affiliation of unknown forms, confirm or refute the varietal correspondence of planting material. ‘Khatmi’ is an autochthonous grape variety from Dagestan. The profile of ‘Khatmi’ is presented in the VIVC international database of DNA fingerprints for grape varieties. However, an application of DNA analysis methods in grape variety studies has determined that several ancient varieties were cultivated under one name, while for others a certain variability of genotypes was observed. The objectives of the work were to study samples of ‘Khatmi’ plants from different places of growth in Dagestan by standard microsatellite loci used for grape genotyping, to assess the level of genetic similarity of the samples, and to refine the DNA profile of ‘Khatmi’. Materials and methods. Molecular genetic study was carried out on 10 samples from different ‘Khatmi’ populations. The material was picked from the collection sites of Dagestan breeding experimental station of viticulture and vegetable growing and the Dagestan Experiment Station of VIR, as well as from production plantations. DNA was extracted from herbarium specimens of young grape shoot tips by the CTAB method. The samples were genotyped by polymerase chain reaction using a standard set of primers for 9 microsatellite markers VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79 recommended by the International Organization of Grapes and Wine (OIV) for grapevine DNA fingerprinting. Amplification products were separated, and their sizes were assessed using capillary electrophoresis on an ABI Prism 3130 genetic analyzer. Results. Genotyping was done for 10 samples of grapes growing in Dagestan under the name ‘Khatmi’, including samples from different collections and places of industrial cultivation, as well as clonal variations of this variety and putative clonal variations. The two base pair differences within one of the loci distinguished the DNA profiles of the analyzed samples from that of ‘Khatmi’ presented in the international grape varieties database VIVC. It was determined that the sample under the name ‘Khatmi krupnoyagodnyi’ is closely related to ‘Khatmi’ variety by its genotype, but probably represents a clonal variation of ‘Koz uzyum’, another local variety of Dagestan. Conclusion. The DNA profile of the local Dagestan grape variety ‘Khatmi’ has been refined

Allelic diversity of the SSR locus VVIB23 linked to the flower sex in wild-growing grapevine genotypes of Krasnodar Territory

Background. Studying wild-growing grapevines is of interest because it expands the knowledge on the genetic diversity within the gene pool and helps to search for new resistance sources. One of the main differences between wild vines and cultivars is the type of their reproductive system: wild grapes are represented by cross-pollinated dioecious vines, while cultivated ones are, with rare exceptions, hermaphrodites capable of self-pollination. A DNA marker applicable to determine the grape flower sex has been identified.Materials and methods. The VVIB23 DNA marker to the Sex gene determining the flower sex was used to study 36 genotypes of wild-growing grapes selected in 2019–2021 within the Utrish Nature Reserve near Gelendzhik and the Krasny Les State Nature Reserve on the right bank of the Kuban River. PCR technique was applied, and the results were evaluated on a Nanofor 05 genetic analyzer.Results. The studied grapevine accessions were found to be variable for the VVIB23 SSR locus: 7 types of alleles were identified. Alleles corresponding to the male flower type were present in 14 genotypes, and those corresponding to the female type in 21. A PCR fragment whose size, according to the published data, correlated with hermaphroditism was not found, which is typical for wild genotypes. The DNA marker analysis failed to determine the flower sex of one accession.Conclusion. The results of the DNA marker analysis showed that the studied wild-growing grapevine genotypes were dioecious form

DNA-marker based identification of the RPV3 gene determining downy mildew resistance in grapevines

Downy mildew is one of the most common fungal diseases of the vine, caused by Plasmopara viticola. An effective way to control the spread of the pathogen is to cultivate resistant varieties. Cultivars of Vitis vinifera, being the basis of high-quality viticulture, practically do not possess genetic resistance to P. viticola, so screening for resistance donors is an important stage in breeding. One of the major resistance loci to downy mildew, the Rpv3 gene, was identified in the genotype of a complex interspecific hybrid of grapes Bianca. Later, it was found that this gene had seven haplotypes of resistance inherited from North American grape species, and that it was possible to identify the allelic status of the gene using DNA-markers UDV305, UDV737. However, only two haplotypes can be combined in one diploid form. To determine the Rpv3 gene in the grape gene pool we, using these markers, studied 35 different genotypes of grapevines, most of which are interspecies cultivars. Three varieties with known allelic status of the Rpv3 gene (Dunavski lazur, Noah, Seyve Villard 12-375) were included in the study as reference genotypes. The genotypes were studied through polymerase chain reaction with separation of amplification products by capillary electrophoresis in automatic genetic analyzer ABI Prism 3130. In the studied grape cultivars DNA marker analysis indentified the Rpv3 gene in sixteen genotypes of interspecific origin, including haplotype Rpv3299-279 found in twelve varieties, Rpv3321-312 – in three, and haplotype Rpv3null-271 – in one variety. Seyve Villard 12-375 turned out to be the donor of resistance gene in the most of the genotypes carrying Rpv3 in this study. The obtained data can be useful in selection of mildew resistant grape varieties and screening for hybridization pairs

DNA marker identification of downy mildew resistance locus <i>Rpv10</i> in grapevine genotypes

One of the most common and harmful diseases of grapevine is downy mildew, caused by Plasmopara viticola. Cultivars of Vitis vinifera, the basis of high-quality viticulture, are mainly not resistant to downy mildew. Varieties with natural resistance to downy mildew belong to the vine species of North America and Asia (V. aestivalis, V. berlandieri, V. cinerea, V. labrusca, V. amurensis, etc.), as well as Muscadinia rotundifolia. The breeding of resistant cultivars is based on interspecific crossing. Currently, molecular genetic methods are increasingly used in pre-selection work and directly in breeding. One of the major loci of downy mildew resistance, Rpv10, was first identified in the variety Solaris and was originally inherited from wild V. amurensis. DNA markers that allow detecting Rpv10 in grapevine genotypes are known. We used PCR analysis to search for donors of resistance locus among 30 grape cultivars that, according to their pedigrees, could carry Rpv10. The work was performed using an automatic genetic analyzer, which allows obtaining high-precision data. Rpv10 locus allele, which determines resistance to the downy mildew pathogen, has been detected in 10 genotypes. Fingerprinting of grape cultivars with detected Rpv10 was performed at 6 reference SSR loci. DNA marker analysis revealed the presence of a resistance allele in the cultivar Korinka russkaya, which, according to publicly available data, is the offspring of the cultivar Zarya Severa and cannot carry Rpv10. Using the microsatellite loci polymorphism analysis and the data from VIVC database, it was found that Korinka russkaya is the progeny of the cultivar Severnyi, which is the donor of the resistance locus Rpv10. The pedigree of the grapevine cultivar Korinka russkaya was also clarified

Genetic structure of the population of wild-growing vines of the Utrish Nature Reserve

Grapes are one of the most common agricultural crops in the world. Currently, the analysis of genotypes directly at the DNA level is considered to be the most accurate method for studying the plant gene pool. The study of wild vines and ancient varieties in various regions of viticulture is an important direction of research in this field. The purpose of this work was to study the population of wild grapes growing on the territory of the Utrish Nature Reserve on the Black Sea coast of Krasnodar Region. The territory of the reserve is of interest as it is a site of ancient settlements, and the environmental conditions are suitable for the growth of wild grapes. During the survey of the territory, 24 samples of wild grapes were found, which were described according to the main morphological characteristics and analyzed by the molecular genetic method. The found vines were genotyped using 15 DNA markers, including nine commonly used for DNA fingerprinting (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) and VVIb23, which allows determining hermaphrodite and dioecious vines. Statistical processing of microsatellite loci polymorphism data was carried out using the GenAlEx 6.5 program. The genetic relationships of the studied vines were evaluated using the PAST 2.17c program. The samples were found to be morphologically and genetically polymorphic. The number of alleles identified in the sample varied from 5 to 18 and averaged 8 alleles per locus. Statistical processing of DNA analysis data made it possible to identify two genetically different populations among the wild discovered vines. An assessment of genetic similarity of the found vines with some local varieties of geographically close viticulture regions, rootstocks and representatives of Vitis sylvestris from other territories was made. One of the populations found in the Utrish Nature Reserve is close to a number of V. sylvestris genotypes, the DNA profiles of which are presented in the Vitis International Variety Catalogue

Characterisation of native Dagestan grape cultivars using SSR-analysis and the main ampelographic features of the leaves

Local, ancient grape cultivars of different cultivation regions are important part of grapevine genetic resources. Dagestan is one of the oldest regions of viticulture in the Russian Federation. Some Dagestan aboriginal grape varieties are cultivated on an industrial scale, while others are found in single numbers. The study of the native gene pool is given special attention in all grapes producing countries of the world. Currently, the most informative method of plant genotypes analysis is the study at the DNA level. The main features of the leaves of grape varieties are a key ampelographical characteristic. We studied cultivars Agadai, Alyi terskyi, Bor kara, Buday shuli, Gok ala, Gok izyum, Mahbor cibil, Yai izyum beliy, Yai izyum rozovyi by using these approaches. DNA profiles of 9 local Dagestan grape cultivars were obtained on microsatellite loci VVMD5, VVMD7, VVMD27, VVS2, VrZAG62 and VrZAG79 using an automated genetic analyzer ABI Prism 3130. The SSR-markers are recommended as the main for Vitis vinifera L. genotyping. The cultivars analyzed have different sets of allele combination by the loci studied. Evaluation of the genetic similarity of cultivars according to the results of microsatellite analysis showed that the genotypes of Mahbоr cibil and Aliy terskiy are closer to the Western European gene pool of V. vinifera L. than any other native varieties in the sample studied. In addition, the grapevine cultivars studied were described for the main features of the formed leaves according to the method of the international organization of vine and wine. The similarity of cultivars Gok Ala and Agadai was shown by the results of analysis of the leaves characteristics and according to SSR-profiling

THE АNAPA AMPELOGRAPHIC COLLECTION IS THE LARGEST CENTER OF VINE GENE POOL ACCUMULATION AND RESEARCH IN RUSSIA

Nowadays there is much tension in the Russian modern vine growing industry around the issue of enhancing the range of grape varieties, which is aimed at production of highly adaptive grape plants and sustainable production of competitive grape varieties in the unstable stressing weather conditions of the moderate continental climate in the south of Russia. In this view, we believe it important to carry out research into the origin, generation and preservation of genetic resources so that they can be involved in the selection process and reach most important objectives of the national economy. The role of a variety and ampelographic collections becomes more important. The Anapa ampelographic collection (http://azosviv.info/category/osnovnye_razdely/anapskaya_ampelograficheskaya_kollekciya) is Russia’s largest gene pool depository of grape varieties brought from various countries of Europe, Asia and America, and Russia’s regions as well. It contains 4921 grape varieties, including Vitis vinifera L. (2975), V. amurensis Rupr. (40), V. labrusca L. (50), the interspecies varieties V. vinifera L.×V. amurensis Rupr. (210), V. vinifera L.×V. labrusca L. (168), blended interspecies hybrids V. vinifera L.× hybrids SV (220), V. vinifera L.×V. amurensis Rupr.×hybrids SV (70), and other samples. We carry out extensive research into collection varieties for their production and selection. We select varieties and forms showing good agronomic characters in productivity, quality of grapes and wine products, winter hardiness, drought resistance, and resistance to pests. We have identified, preserved and now use in selection, as sources of good agronomic characters of resistance to low temperatures in winter, such varieties as Riesling of the Rhine, Rkatsiteli, Aligoté, Riesling of Italy, Traminer Pink, Pinot Noir, Rara Neagra, Cabernet Sauvignon, Coarna Neagra, Pinot Gris, Gamay Freaux, Saperavi, Muscat Ottonel, Madeleine Angevine, Cabernet Franc, Khikhvi, Pearl of Csaba, Chardonnay, Krasnostop of Anapa, Dostoiniy, Krasnostop AZOS, etc. The collection has given birth to twenty seven varieties and elite forms, of which twelve varieties have been sent to the State Committee for the Testing of New Varieties of Agricultural Plants (Pluto, Muzhestvenniy (Courageous), Gordiy (Proud), Maran, Varvarovsky, Harmony, Progress, Gorniy (Mountaneous), Saturn, Dimitry, Vladimir, Kurchansky). We carry out a molecular genetic testing of the accumulated gene pool and DNA-classification of varieties. We have studied the microsatellite polymorphism in the genotypes of autochthonous grape varieties of the Don River and the Republic of Dagestan. We are performing the DNA-classification of varieties selected by the North Caucasian Regional Research Institute of Horticulture and Viticulture (Krasnodar), which is advantageous for the identification of pure varieties in planting material and vineyards and specifying of the parent forms of grape varieties, as well as when any disputes arise as to the variety authorship

Genetic polymorphism of local Abkhazian grape cultivars

Local grape cultivars from different countries of the world are an important part of the gene pool of this culture. Of particular interest are the genotypes of the most ancient regions of viticulture. The territories of the subtropical zone of Georgia and the central part of Abkhazia belong to one of the centers of origin of the cultural grapevine. The purpose of the work was to genotype native Abkhazian grape cultivars, to study their genetic diversity based on DNA profiling data and to compare them with the genotypes of local varieties of other viticultural regions. Samples of plants were taken on the territory of the Republic of Abkhazia in private farmsteads and in the collection of the agricultural firm “Vina i Vody Abkhazii“ (“Wines and Waters of Abkhazia”). The genotyping of the Abkhazian cultivars Avasirhva, Agbizh, Azhapsh, Azhizhkvakva, Azhikvaca, Atvizh, Atyrkuazh, Achkykazh, Kachich was carried out using 14 DNA markers, 9 of which are standard microsatellite markers recommended for the identification of grape varieties. To improve our knowledge about the sizes of the identified alleles, we used the DNA of grape cultivars with a known allelic composition at the analyzed loci. Statistical analysis of the data showed that the observed heterozygosity for the analyzed loci exceeded expected values, which indicates a genetic polymorphism of the studied sample of varieties. Evaluation of genetic similarity within the analyzed group based on the results of genotyping at 14 loci showed that the cultivars Kachich and Azhapsh differed from the other Abkhazian varieties. The obtained DNA profiles of the Abkhazian cultivars were checked for compliance with DNA-fingerprints of grape varieties in the Vitis International Variety Catalogue. The Georgian varieties Azhizhkvakva and Tsitska turned out to be synonyms according to DNA profiles, two varieties from the Database (Italian Albana bianca and Georgian Ojaleshi) have differences in DNA-fingerprints from the varieties Atyrkuazh and Azhikvatsa only in one allele, respectively. When comparing the identified Abkhazian grape genotypes, their difference from the sample of Dagestan, Don, Greek, Turkish, Italian, Spanish, and French varieties and genetic similarity with the genotypes of Georgian grapes were shown