Behavior of grape breeding lines with distinct resistance alleles to downy mildew ( Plasmopara viticola )

Downy mildew (Plasmopara viticola) is the main grapevine disease in humid regions. In the present investigation, marker-assisted selection (MAS) was used to develop grapevine lines homozygous in loci Rpv1 and Rpv3 for resistance against P. viticola. The experimental populations UFSC-2013-1 (n = 420) and UFSC-2013-2 (n = 237) were obtained by self-pollination of two F1 full-sib plants, originated from a cross between two distinct breeding lines containing the downy mildew resistance loci Rpv1 and Rpv3 in heterozygosity. The two experimental populations were genotyped with four microsatellite markers flanking the two downy mildew resistance loci. Among 637 genotyped plants, 300 (48.2%) were homozygous for at least one resistance locus and 10 (1.57%) were homozygous for both Rpv1 and Rpv3 loci. These 10 plants challenged with P. viticola inoculum showed a clearly enhanced level of resistance. These plants have a great potential as resistance donors in grapevine breeding.


INTRODUCTION
The diploid obligate biotrophic oomycete Plasmopara viticola (Berk & Kurt) causes downy mildew, one of the main diseases that reduce grape (Vitis vinifera) production (Caffi et al. 2010).This yield loss is not confined to one season; early leaf death and defoliation caused by the disease weakens the vine and leads to poor harvests in subsequent seasons (Matasci et al. 2008).The pathogen was found originally in North America on wild Vitis (Olmo 1986); however, it has since spread across the globe to many countries, including Brazil, by introduction of American vines contaminated with the pathogen (Santos-Neto 1955).Currently, the pathogen is responsible for extreme damages to vineyards without effective crop protection in South Brazil, where the humid climate is especially amenable for the pathogen's life cycle and proliferation (Hamada et al. 2008, Peruch andBruna 2008).
For more than two centuries, V. vinifera and Muscadinia rotundifolia, a donor of resistance genes, have been bred with the objective of combining high-quality fruit production with effective disease resistance (Olmo 1986, Alleweldt andPossingham 1988).Nevertheless, high levels of distorted segregation in plants FD Sánchez-Mora et al. from interspecific crosses led to low survival rates in the progeny, thus hindering breeding efforts (Bouquet 1986, Lu et al. 1998); however, some successful hybridizations were obtained when M. rotundifolia was used as the male parent (Lu et al. 1998).
Grapevine improvement, similar to all perennial species, takes a considerably long time to develop new varieties because maturity often requires several growing seasons before breeding success can be evaluated.However, indirect selection with the assistance of molecular markers, which can be used at any developmental stage of the plant, can reduce this interim time considerably (Töpfer et al. 2011a).
More recently, these markers have allowed the construction of genetic maps, QTL analyses, and implementation of marker-assisted selection (MAS), particularly applied for pyramiding resistance alleles in grapevine (Alzate-Marin et al. 2005, Eibach et al. 2007, Töpfer et al. 2011b).In addition to the pyramiding of resistance genes the development of inbred lines has a crucial role in gene introgression, because it ensures that all progeny will inherit the alleles of interest.Moreover, vines homozygous for downy mildew resistance allele Rpv12 (Venuti et al. 2013) and for seedlessness (Sdl) [according to VIVC table of loci] locus responsible for apyrenic grape fruits have been reported (Doligez et al. 2002, Akkurt et al. 2012).
The locus Rpv1 is located on chromosome 12 of M. rotundifolia, and it is associated to locus Run1, which confers resistance to downy mildew (Barker et al. 2005, Merdinoglu et al. 2003).Rpv1/Run1 alleles were introduced in V. vinifera by backcrossing (Pauquet et al. 2001).The locus Rpv3 was identified on chromosome 18 in the grape variety 'Regent' (Fischer et al. 2004, Welter et al. 2007) and conferred significant resistance to downy mildew on both leaves and berries in all evaluated years (Welter et al. 2007).In grapevine, this Rpv3 locus was responsible for causing the hypersensitive response (HR) at infection sites within two days after the inoculation (dpi) (Bellin et al. 2009, Casagrande et al. 2011).
The resistance alleles Rpv1 and Rpv3 revealed complete dominance at both of their loci.Through pyramiding Rpv1 and Rpv2, Eibach et al. (2007) obtained heterozigous plants that exhibited resistance to downy mildew.The effect of either Rpv1 or Rpv3 in single plants showed significantly higher resistance to downy mildew; however, genotypes with both resistance alleles showed no infection (Eibach et al. 2007).In other studies, the combination of multiple resistance alleles, such as Rpv1 and Rpv3 (Calonnec et al. 2013); Rpv3 is equal to QTLRgD as already described in Bellin et al. (2009), Di Gaspero et al. (2012), Zyprian et al. (2016), Rpv3 and Rpv10 (Schwander et al. 2012), Rpv3 and Rpv12 (Venuti et al. 2013), likewise conferred significantly enhanced resistance to pathogen infection in comparison to single resistance alleles.Thus, the objective of this study was to develop, characterize, and select breeding lines homozygotic for the pyramided resistance alleles Rpv1 and Rpv3 for future breeding purposes.

DNA extraction
DNA was extracted from approximately 100 mg of leaf samples dissected on silica gel.The samples were macerated using four 2.8 mm ceramic beads placed in 2 ml self-standing micro-tubes (Precellys ® 24 homogenizer, Bertin Technologies) during two 30 s agitation cycles at 5000 rpm.Subsequently, the DNA was isolated using a NucleoSpin ® Plant Kit as described by the supplier (Macherey-Nagel).The DNA samples were quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific).The DNA was diluted to a concentration of 1 ng μL -1 for further use in PCR reactions.

Amplification of microsatellite markers by PCR
Microsatellite markers Sc34_8 and Sc35_2 (NCBI STS database accession: GF111545 and GF111546), flanking the Rpv1 locus located on chromosome 12, and the markers GF18-06 (Schwander et al. 2012) and GF18-08 (Zyprian et al. 2016), located on chromosome 18, which flank the Rpv3 locus, were used to genotype the plants.The four pairs of primers were added in the same polymerase chain reaction (PCR) to obtain all amplicons at once.The PCR reactions were performed in a total volume of 5 µL, using the KAPA 2G Kit (Kapa Biosystems, Inc. Boston, USA).The mix contains 1x of KAPA Fast Multiplex Mix and four pairs of primers (0.01 µM).The forward primer (5') was labeled with 6-FAM, VIC or PET fluorophore.The PCR reactions were carried out in a C-1000TM Thermal Cycler (Biorad), with the following thermal profile: 94 °C for 3 min, followed by 30 touchdown cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and a final elongation of 40 min at 72 °C.To confirm amplification, 5 uL of PCR product was used for electrophoresis in 1.5% agarose gel and visualized under UV transilluminator.

Capilar electrophoresis
Sequencing reaction was prepared to a final volume of 10 μL of diluted PCR product (1.0 µl PCR reaction in 27 µl autoclaved ultrapure water), 0.5 µl of size ladder Gene Scan 600 LIZ (Life Technologies) and 8.5 µL of Formamide (Life Technologies).The alleles were separated by capillary electrophoresis done in a 3500 XL Applied Biosystems with the polymer POP-7.Allele sizes were determined using a Gene Mapper Version 4.1 software (Applied Biosystems).

Resistance phenotyping on artificially inoculated leaf discs
To validate the genotyping data, 142 randomly-selected plants in variable proportions from the nine obtained allelic combinations were evaluated for resistance to P. viticola (Table 1).Inoculations were performed on leaf discs according to Schwander et al. (2012).All materials used during the inoculations, except the inoculum, were autoclaved at 120 °C and 1.1 atm for 19 min.
The fourth leaf from the apex to the base of all plants were collected.The leaves were sterilized with sodium hypochlorite (1% of active chlorine) for 30 s, and, subsequently, triple-rinsed in ultrapure water (Milli-Q®) for 30 s, 30 s and 60 s, respectively.In a laminar flow hood, six leaf disks 12 mm in diameter were excised from each plant and placed abaxial-side-up on moist filter paper in glass petri dishes to produce a moist chamber for pathogen proliferation.Then, a droplet of 30 µL of the sporangia suspension, at a concentration of 50,000 spores mL -1 , was applied to each leaf disk.Three replicates were performed for each plant.
The Petri dishes were sealed and incubated in a growth chamber.In the first 10 h the disks were kept in the dark at 22 °C.Subsequently, conditions were changed to a photoperiod of 14 h light and a constant temperature of 25 °C.After 15 h, the droplet was removed from each disc, and left incubated for seven days to evaluate the sporulation of downy mildew.The degree of infection was rated in each genotypic class on the basis of sporangia intensity formed by leaf disc.

Statistical analysis
Frequency distribution and chi-square tests were used to analyze the obtained segregations in genotypic data from the expected ones.When distorted segregation was statistically significant, the Lorieux et al. (1995) methodology was used to determine the type of selection acting in the analyzed populations.Confidence intervals (CIs) of 95% were used for comparison of means of phenotypic evaluations of the downy mildew.The degree of resistance of other genotypes to P. viticola was calculated based on the susceptible genotypes rpv1/rpv1 and rpv3/rpv3 that showed 100% of mildew infection.

Genotyping
Genotyping with four microsatellite markers linked to resistance alleles Rpv1 and Rpv3 (with two pairs of primers flanking each of the loci) classified plants according to the number of resistance alleles present in each plant.Plants were considered to have the resistance loci when the two microsatellite markers for each locus presented alleles originally linked to the resistance alleles.Out of 637 genotyped plants, 15 showed genetic recombination (2.3%) between the pairs of microsatellite markers and were excluded from the analysis.This recombination rate of 2.3% rate is quite acceptable to breeders in terms of indirect selection efficiency.
The segregation of the markers GF18-06 and GF18-08, linked to the Rpv3 resistance locus, was Mendelian in one population and distorted in the other (Table 2).The Mendelian segregation occurred in the UFSC-2013-2 population: 53 Rpv3/Rpv3, 104 Rpv3/rpv3 and 52 rpv3/ rpv3 [x 2 = 0.014 (P = 0.99)].In the UFSC-2013-1 population, both markers segregated to proportion of 132 Rpv3/Rpv3, 205 Rpv3/rpv3 and 76 rpv3/rpv3 [x 2 = 15.20 (P= 0.0005)], therefore, distorted.Significant deviations in the value of χ 2 in that population were partly influenced by the death of 51 sensitive plants.The deviation from the expected Mendelian segregation ratio has often been observed in the offspring of intra-and inter-species hybrids (Harushima et al. 1996).Thus, segregation distortion comprises a group of systems of genic drive found in a wide range of organisms, usually involving a small number of interacting primary loci (Lyttle 1991).
Because the parental donor VRH 3082-1-42 had been obtained from interspecific cross between Muscadinia rotundifolia G52 x Malaga seedling No.1 (V.vinifera), followed by four backcrosses to V. vinifera varieties (Pauquet et al. 2001), it is possible that the plants inherited, in addition to resistance alleles of locus Run1 (that is linked to Rpv1), some factor that cause the deviations of segregation on the region where the allele of resistance Rpv1 is located.In this same cross, Bouquet (1986) observed a high distorted segregation ratio due to chromosome disequilibrium and plant mortality.Likewise, Pauquet et al. (2001), studying populations of the fourth and fifth backcross to V. vinifera from this same cross (M.rotundifolia x M. seedling), also found two populations with an excess of susceptible genotypes that caused a distorted segregation ratio.The authors hypothesized that this was the result of the influence of genetic background from which the gene was taken.A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1 (chromosome 12), possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the locus Run1 (Backer et al. 2005), which is linked to Rpv1.
Segregation distortions were also found in other interspecific crosses when one of the homozygotes was favored, which may suggest the involvement of gametophytic and zygotic factors.In grapes, segregation distortions were found in the crosses V. rupestres x V. arizonica/girdiana (Riaz et al. 2006), and V. vinifera x V. arizonica/candicans (Riaz et al. 2008).In the chromosomal region where the allele Run1/Rpv1 is located, the zygotic selection acted in favor of susceptible homozygous or heterozygous genotypes in the two studied populations, which can prevent the development of a larger number of plants homozygous with resistance alleles.Thus, the deficit of homozygote genotypes found in the present work for the resistance alleles suggests the occurrence of zygotic selection.

Phenotyping
One-fourth of the genotyped plants were tested phenotypically for resistance to downy mildew, whereby all nine obtained allelic combinations for the two populations are represented (Table 1).The analysis of the average number of sporangia per leaf disc resulted in the formation of three general groups: the first group showed the highest incidences of downy mildew infection and were associated with absence of resistance alleles; the second group showed moderate infection and was associated with resistance alleles in one locus, regardless if the alleles were homozygous or heterozygous According to the methodology employed by Lorieux et al. (1995).*** P<0.001 FD Sánchez-Mora et al.
Table 1 includes also the phenotypic mean for downy mildew resistance, in which the number of sporangiophores was converted to 9 to 1 Scale, according to OIV 452.Again, it was possible to verify that the resistance alleles of the locus Rpv3 have higher contribution to resistance to downy mildew when compared to Rpv1.
Resistance to P viticola in grape is greatly increased through pyramiding resistance alleles (Figure 3).Plants with absence of resistance alleles showed the highest level of infection (μ = 2.1; s=1.7).Intermediate resistance was shown by genotypes with resistance alleles in one of the loci (Rpv1, μ =4.0; s=2.1 and Rpv3, μ =4.9; s=2.2), and the highest level of resistance was obtained with the presence of resistance alleles in both loci (μ =7.2; s= 2.0).However, the presence of a specific resistance allele was not enough to consistently be associated with a specific phenoype (Figure 3).The frequency distribution indicated that, in addition to the resistance alleles, the genetic background of the plant could be responsible for the variation of phenotypes associated with plants with the same alleles.In this study, plants with the same genotypic class showed varying response to the pathogen in each inoculation (Figure 2).addition, the effect of single locus or combination of resistance genes was associated with degrees of resistance.Thus, when the plants carried resistance alleles from only one locus, regardless whether it homozygous or heterozygous, the disease control was less than or equal to 45.1%.However, when the plants carried a combination of resistance alleles of two loci the infections control by P. viticola was between 76.8 to 88.9%.
The inoculation technique on leaf discs is widely used in genetic resistance studies against P. viticola.In grape, this strategy is effective for distinguishing phenotypic differentiation between resistant and susceptible plants.With the use of first bioassays in grape leaves, it was possible to select genotypes with reasonable accuracy.This approach could possibly be used as a diagnostic marker for selecting which individual plants with disease resistance are most suitable to be planted in the field (Calonnec et al. 2013).The combination of alleles at loci Rpv1 and Rpv3 showed better disease control when compared to the intermediate resistance of individual loci.These results suggest that each locus has a partial and additive effect on grape resistance to downy mildew and the dominance of the resistance allele within a locus.
Phenotype variations in plants carrying the same resistance alleles might be partially due to the methodology and the different genetic backgrounds of each resistance alleles.It is also possible that some minor QTLs or other coding sequences, which also participate in plant pathogen response, segregated in the studied populations.Therefore, different genotypes with the same major resistance alleles can show different degrees of disease resistance, as based on observed average confidence levels for disease resistance in each genotype in this study.
Previously, Eibach et al. (2007) demonstrated that the combination of alleles from both genes Rpv1 and Rpv3 in the F 1 progeny of the cross VRH3082-1-42 x Regent, resulted in an additive effect in disease resistance.Likewise, Schwander et al. (2012) observed an additive effect on resistance to downy mildew by combining the Rpv3 and Rpv10 resistance loci, indicating that they are applicable for pyramiding of resistance pathway MAS.Introgression of resistance alleles Rpv3 and Rpv12 from V. amurensis to V. vinifera also showed the positive effects of pyramiding resistance genes; plants heterozygous for both alleles restricted pathogen growth much more effectively than plants with only one or neither of the resistance alleles (Venuti et al. 2013).
The analyzed plants in the current study containing resistance alleles in homozygous state at the two loci showed the best results in terms of disease control.However, these genotypes did not react significantly different to P. viticola from other genotypic classes that have pyramided resistance alleles at the two loci, suggesting other type of gene action than additive effect between distinct alleles.
The results of the phenotypic evaluations showed that the loci Rpv1 and Rpv3 contribute significantly to disease control against downy mildew.The creation of plants homozygous with resistance alleles Rpv1 and Rpv3 is greatly useful to breeders and may contribute to creation of new elite cultivars.These selected plants can be crossed with other varieties of economic interest, ensuring that the F 1 plants will carry at least one resistant allele in each locus and ensuring some level of disease resistance.In addition, they can speed up the breeding programs due to the reduction  9).The intensity of sporangiophore formation was rated (9: zero to three, 7: four to 10, 5: ten to thirty, 3: thirty to fifty, 1: more than fifty), according the methodology described by Schwander et al. (2012).Y axis is label for the presence (+) or absence (-) of the resistance correlating alleles Rpv1 and Rpv3.Mean of the downy mildew resistance level in each group with standard deviation (s) in brackets is given in the X axis label.
FD Sánchez-Mora et al. of time in getting resistant varieties.Moreover, the selected resistant plants could be used to introduce resistant alleles in currently used tolerant cultivars, such as BRS Vitoria, which is tolerant to downy mildew (Maia et al. 2014), since it is of the interest of a local viticulture industry, especially those ones focused on the fruit export.

Figure 1 .
Figure 1.Genotypic frequency distribution (in percentage) of two loci conferring resistance to downy mildew in 622 plants of UFSC-2013-1 and UFSC-2013-2 populations.Numbers above the bars indicate quantity of plants.

Figure 2 .
Figure 2. Average values of the number of sporangiophores per leaf of nine genotypic combinations of the loci Rpv1 and Rpv3.Leaf discs of plants with genotype rpv1/rpv1, rpv3/rpv3 could have 50 or more sporangiophores.Bars represent a 95% confidence interval.

Figure 3 .
Figure 3. Frequency distribution (%) for the degree of infection of P. viticola in 142 plants of UFSC-2013-1 and UFSC-2013-2 populations.The average values number of sporangiophores per leaf of genotypic combinations of the loci Rpv1 and Rpv3 were classified according to the OIV descriptor 452 from susceptible (1) to resistant (9).The intensity of sporangiophore formation was rated (9: zero to three, 7: four to 10, 5: ten to thirty, 3: thirty to fifty, 1: more than fifty), according the methodology described bySchwander et al. (2012).Y axis is label for the presence (+) or absence (-) of the resistance correlating alleles Rpv1 and Rpv3.Mean of the downy mildew resistance level in each group with standard deviation (s) in brackets is given in the X axis label.

Table 1 .
Number of plants and phenotypic mean evaluated for each genotypic class * = The Capital letter identifies the resistance allele; ** = Based on Scale OIV 452 FD Sánchez-Mora et al.

Table 2 .
Result of segregation the two populations and chi-square test revealed by SSR markers