DNA-marker based identification of the RPV 3 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 vini­ fera, 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, Sayve 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. Sayve 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.

Plant breeding for immunity and performance D owny mildew is one of the most widespread and destructive fungal diseases in the grapevine, caused by oomycete Plasmopara viticola Berl.et de Toni.The pathogen affects only the vine developing in its every green organ: leafs, shoots, inflorescences, grapes and tendrils.In favorable conditions such as warm temperature and excessive humidity, mildew may cause harvest failure from 50 to 100 % in different grape varieties (Talash, 2010).
Cultivation of resistant varieties remains one of the most effective methods of disease control that allows one to reduce the amount of pesticide sprayings, and in this way improves the ampelocenosis, food safety and harvest of grapes.
The success of such cultivation is rooted into the genetic diversity of a culture and in many ways is determined by the level of knowledge about an accumulated genetic pool.Identification of the genotypes to serve as resistance donors has been one the topical issues in the science of selection.Being the foundation of high-quality vine growing cultivars Vitis vinifera have almost no genetic resistance to Plasmopara viticola, while downy mildew -resistant genotypes belong to vine species from North America and Asia (V.aestivalis, V. berlandieri, V. cinerea, V. riparia, V. rupestris, etc.) as well as to Muscadinia rotundifolia (Alleweldt et al., 1988;Wan et al., 2007).
Molecular genetic methods are widely applied these days for identification and mapping of valuable genes, gene-pool diversity analysis, and DNA-marker selection in different breeding programs.The methods have made it possible to determine around 20 mildew-resistant loci in a vine genome (http://www.vivc.de).Many of them have been mapped and given names with their linked DNA-markers identified including those appropriate for DNA marker selection (Eibach et al., 2007;Di Gaspero et al., 2012;Schwander et al., 2012;Venuti et al., 2013;Zini et al., 2014;Ochssner et al., 2016).
The Rpv3 gene, one of the major loci of resistance, was detected for the first and mapped at chromosome 18 in the genotype of a complex interspecies hybrid Bianca carrying the geneplasm of V. vinifera, V. labrusca, V. rupestris, V. berlandieri, V. lincecumii (Bellin et al., 2009).Later, in the course of a large-scale study into the North American species and varieties carrying Rpv3, one detected the seven conservative haplotypes of this gene responsible for mildew resistance (Di Gaspero et al., 2012).As the mentioned haplotypes were not found in V. vinifera, the authors came to the conclusion that Rpv3 could be found in the varieties, whose pedigree had several North American species.The valuable haplotypes localize in a single locus, that is why in case of traditional breeding, only two haplotypes can be combined in a single diploid cell.
The objective of the presented study was using DNAmarker analysis for identification of the allele state of the Rpv3 gene in different vine cultivars, and comparison of the obtained data against the genotype pedigree.

Materials and methods
The study covered 35 grapevine cultivars from the gene pools of the Anapa ampelographic collection (Anapa) and the collection of Ya.I.Potapenko Research Institute of Viticulture and Winemaking (Novocherkassk).Most of the studied cultivars were interspecies hybrids, whose parents were North American vine varieties and, based on analysis of their pedigree, could carry the resistant haplotypes of the Rpv3 gene.In the geneplasm of the studied cultivars -potential carriers of the studied gene -were present V. riparia, V. labrusca, V. aestivalis, V. rupestris, V. berlandieri, V. lincecumii.The study also covered a number of genotypes that should have no Rpv3 gene such as Vitis vinifera and its hybrids with V. amurensis.
The vines' DNA was extracted from leaves using the CTAB method (Rogers, Bendich, 1985).The genotypes were determined using polymerase chain reaction (PCR) and the DNAmarkers recommended for identification of the alleles of the Rpv3 gene (Di Gaspero et al., 2012).РCR was performed in a finite volume of 25 μl following the standard protocol and using the Sintol reagent kit (Moscow, Russia).DNA amplification was performed in Eppendorf MasterCycler Gradient Thermal Cycler (Germany) with the following protocol for every DNA-marker: 5 minutes at 95 ºС for initial denaturation followed by 35 cycles (10 seconds for denaturation at 95 ºС, 30 seconds for annealing the primers at 55 ºС, 30 seconds for synthesis at 72 ºС, and 3 minutes for the last cycle of synthesis at 72 ºС).The reaction products were separated using capillary electrophoresis, and the size of the amplified fragments was estimated with the ABI Prism 3130 automatic genetic analyzer using software packages GeneMapper and PeakScanner.The DNA of the Dunavski lazur, Seyve Villard 12-375 and Noah varieties with known allele sizes for the studied loci (Di Gaspero et al., 2012) were used as controls to specify the sizes of the amplified fragments.
The molecular genetic study was carried out using the equipment provided by Shared Equipment Center "Genomic and Postgenomic Technologies" of North-Caucasian Federal Scientific Center of Horticulture, Viticulture, Winemaking.
In the studied sample haplotype Rpv3 299-279 occurred more often than others: it was detected in 14 genotypes (see Table ).In 12 cultivars this haplotype was indentified for the first time.
Haplotype Rpv3 321-312 was for the first time identified in three cultivars: Armalaga, Poliuks and Podarok Magaracha.DNA-marker analysis identified hyplotype Rpv3 null-271 only in the melody species.The Noah variety that we used as one of the reference genotypes, also carried Rpv3 321-312 and Rpv3 null-271 .

Plant breeding for immunity and performance
Seyve Villard 12-375.These were cultivars Dekabrskiy, Dunavska gymza, Rusbol, Storgoziya, Kishmish 342, and Srebrostruy.The Seyve Villard series are complex interspecies hybrids that are often used in grapevine breeding as resistance donors, and Seyve Villard 12-375 is one of the most known hybrids in the series that carry the geneplasm of V. vinifera, V. labrusca, V. rupestris, V. berlandieri, V. lincecumii.
In the genotype of the Talisman cultivar, the resistant allele was inherited from the Frumoassa alba variety, whose parent is Seyve Villard 20-473.Seyve Villard 20-365 served as a gene donor in varieties Original and Kutuzovskiy.
It is considered that resistant haplotype Rpv3 null-271 initially originated from either V. labrusca or V. riparia.This is the haplotype we identified in American variety Melody.In the beginning, when analyzing its pedigree (Seyval blanc × Geneva white 5 (Pinot blanc × Ontario)) for its inclusion as a potential gene donor we assumed Seyval blanc (another name of Seyve Villard 5-276) to be the resistivity donor since its genetic formula contained V. rupestris and V. aestivalis.However, if we assume that Rpv3 null-271 is inherited either from V. labrusca or from V. riparia, the indicated resistance allele in Melody can be inherited from Ontario (25 % V. vinifera + 75 % V. labrusca).
Haplotype Rpv3 321-312 was identified in cultivars Armalaga, Poliuks and Podarok Magaracha.The published data indicate the source of this resistant haplotype to be either V. labrusca or V. riparia.In variety Armalaga, Rpv3 321-312 was inherited from V. labrusca, and in Poliuks -from V. riparia, which is confirmed by their pedigrees.Meanwhile, the pedigree of Podarok Magaracha remains an open issue.One of the parent varieties it inherited its mildew resistance from was probably Magarach 2-57-72 (Mtsvane × Sochinskiy cherniy).The Sochinsky cherniy first discovered by P.Ya.Golodriga in the outskirts of Sochi has been lost and its exact genetic origin remains unknown, but its high resistance to fungal pathogens it transfers to its descendants makes it possible to classify it as an interspecies hybrid.The data we collected during the study allow us to assume that the pedigree of this species included either V. labrusca or V. riparia.

Conclusion
The presented study analyzed 35 vine genotypes of different origin to identify the presence of downy mildew-resistant gene Rpv3 using DNA markers UDV305 and UDV737.The analysis identified the Rpv3 gene in 19 grapevine cultivars, including 3 varieties, in which the presence of the gene had been confirmed earlier.The abovementioned markers allow one to identify a certain haplotype of the Rpv3 gene.That way, for the first time, haplotype Rpv3 299-279 has been detected in 12 interspecies varieties; Rpv3 321-312 -in three varieties; and Rpv3 null-271 -in one variety.For the first time, the presence of the Rpv3 gene has been confirmed for such grapevine cultivars as Dekabrskiy, Dunavska gymza, Original, Talisman, Kutuzovskiy, Kodryanka, Rusbol, Storgoziya, R65, Kishmish 342, Srebrostruy, VIII 2 -2-48, Armalaga, Poliuks, Podarok Magaracha and Melody.All the genotypes with Rpv3 are characterized by high or increased level of downy mildew resistance, which has been confirmed by the results of perennial observations (Petrov, Talash, 2010;Troshin, Radchevsky, 2010).The performed DNA analyses has also allowed us to assume that the parent varieties of Podarok Magaracha, whose pedigree remains unclear, are interspecies varieties carrying the geneplasm of either V. labrusca or V. riparia.
The results obtained can be used for selection of initial varieties to breed cultivars resistant to downy mildew.