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Scientia Horticulturae 126 (2010) 448–453

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

arker-assisted selection for two dominant powdery mildew resistance genesntrogressed into a hybrid grape population

. Katula-Debrecenia,∗, A.K. Lencsésa, A. Szokea, A. Veresa, S. Hoffmannb, P. Kozmab, L.G. Kovácsc,. Heszkya, E. Kissa

Institute of Genetics and Biotechnology, Szent István University, Gödöllo, Páter K. u. 1. 2100, HungaryResearch Institute of Viticulture and Enology, University of Pécs, Pécs, Pázmány P. u. 4. 7634, HungaryDepartment of Biology, Missouri State University, Springfield, Missouri, 901 South National Avenue, MO 65897, USA

r t i c l e i n f o

rticle history:eceived 3 February 2010eceived in revised form 23 July 2010ccepted 25 August 2010

eywords:rape

a b s t r a c t

Molecular marker-assisted selection (MAS) was carried out to track the inheritance of Run1 and Ren1,two dominant powdery mildew resistance genes that originate from different genetic resources. TheRun1 locus was introgressed from a Muscadinia rotundifolia × Vitis vinifera BC4 hybrid plant derived froma recurrent pseudo-backcross breeding scheme, whereas the Ren1 locus was introgressed from the pow-dery mildew-resistant V. vinifera L. variety Kishmish vatkana. Introduction of the Ren1 resistance gene ofV. vinifera origin into the breeding program makes possible the long-term defence of the dominant Run1

un1en1owdery mildew resistanceASene pyramiding

gene. Using a BC5 (BC4 × Kishmish vatkana) hybrid progeny consisting of 441 plants and applying severalSSR and BAC-derived CB markers, we demonstrated that the powdery mildew-resistant phenotype co-segregated with the presence of at least one resistance locus-linked marker in the genome. Our data alsocorroborated earlier findings that the M. rotundifolia-derived Rpv1 and Run1 loci are closely linked. Tofurther streamline the selection process, we developed a multiplex PCR- and agarose gel electrophoresis-based method for the simultaneous detection of both Run1 and Ren1. The results illustrate that MAS offersa rapid and accurate method to select hybrid genotypes that combine multiple loci of interest in grape.

. Introduction

Breeding new grape (Vitis vinifera L.) varieties is time-onsuming and resource-intensive, since grapes have a longeneration cycle, and because the maintenance of hybrid proge-ies requires extensive area of land and rigorous cultivation. These of DNA-based markers linked to genes of interest consider-bly reduces breeding costs. Molecular marker-assisted selectionMAS) facilitates the precise identification of seedlings that havenherited the desired gene shortly after germination, even beforehe expression of the trait is observable in the progeny. In thisay, unwanted progeny can be eliminated and the size of theybrid population can be reduced early during the breeding pro-ess. In recent years, considerable progress has been made inenerating tools for MAS in grapes. Large number of DNA sequence-

ased markers have been developed which, in turn, made theonstruction of genetic linkage maps possible (Doligez et al., 2006;i Gaspero et al., 2007). Many of the simple sequence repeats-ased (SSR) markers are publicly available in Internet-accessible

∗ Corresponding author. Tel.: +36 28 522 069; fax: +36 28 522 069.E-mail address: Debreceni.Diana@mkk.szie.hu (D. Katula-Debreceni).

304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2010.08.012

© 2010 Elsevier B.V. All rights reserved.

databases. The availability of linkage maps and molecular markersmakes the mapping of agronomically favorable genes increasinglystraightforward. The recent publication of the V. vinifera genomesequence (Jaillon et al., 2007) accelerates the development ofnew SSR markers and allows them to be anchored to physicalmaps.

Simple sequence repeats (SSR)-based markers are particularlyuseful in MAS, because they are co-dominant and, thus, allow theunambiguous identification of both the desired allele and its homo-logue. SSR markers enable breeders to simultaneously select forseveral genes in a progeny. This is particularly useful when multiplegenes that encode the same phenotype are to be introgressed into asingle genome. Combining multiple genes to confer the same phe-notype is termed gene pyramiding. This approach is essential whenbreeders combine several qualitative resistance (R) loci against adisease into a hybrid plant. Different R genes are thought to detectthe pathogen by different mechanisms, therefore, resistance con-ferred by a combination of various R genes is more difficult to

overcome by the pathogen than resistance due to a single R gene(McDonald and Linde, 2002). Maximizing the durability of resis-tance is particularly important when fighting off rapidly evolvingpathogens such as the grape powdery mildew (PM, Erysiphe necatorSchw. Burr).

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The discovery of Run1 gene in the American muscadinerape (Muscadinia rotundifolia Michx. Small) initiating effector-riggered immune responses against PM opened new possibilitiesn grapevine breeding (Bouquet, 1986; Dry et al., 2010). The Run1ocus was mapped to linkage group 12 and it encompasses atring of resistance genes, three of which were found closelyinked to a marker co-segregating with resistance (Pauquet etl., 2001; Donald et al., 2002). Additional markers have beeneveloped on the basis of bacterial artificial chromosome (BAC)lone-derived DNA sequences, and the locus was mapped to theenomic region between SSR marker VMC4f3.1 and CB markerB292.294 (Barker et al., 2005). The mechanism by which Run1onfers resistance is thought to be hypersensitive reaction (HR)-ediated death of the leaf epidermal cell attacked by the pathogen.

nterestingly, a downy mildew (Plasmopara viticola Berk. et Cur-is) resistance locus in M. rotundifolia (Rpv1), was also tracedack to a region near the Run1 locus by Merdinoglu et al.2003).

Although the European grape cultivars are susceptible to downyildew (DM) and powdery mildew (PM) infections, some Centralsian table grape varieties such as Dzhandzhal kara (Korbuly, 1999)nd Kishmish vatkana (Kozma et al., 2006) show a marked resis-ance against powdery mildew.

Another dominant gene for PM resistance, named Ren1, wasecently identified in the Central Asian V. vinifera variety Kish-ish vatkana (Kozma et al., 2006). Using an F1 progeny from a

ross between Kishmish vatkana and a PM-susceptible V. viniferaariety, Hoffmann et al. (2008) performed bulked segregant anal-sis and localized Ren1 to linkage group 13. Taking advantage ofecombinant haplotypes in which crossing-over occurred in theen1 region, they mapped the locus 0.9 cM away from SSR mark-rs UDV020a and VMC9H4.2. The defense mechanism by whichen1 confers resistance appears to be different from that of Run1.

n Kishmish vatkana and its Ren1-containing progeny, E. necators able to parasitize epidermal cells and to form microcolonies

ithout triggering an immediate cell death, although a delayedell death may be part of the plant’s defense response. Hyphalrowth and conidiation are markedly slower in Kishmish vatkanahan in susceptible grapes (Hoffmann et al., 2008). PM resistancen a V. vinifera plant is remarkable not only as a resource forrape breeding, but also as an evolutionary phenomenon, as theild progenitors of V. vinifera are thought to have evolved in

he absence of selection pressure from E. necator in Eurasia. Theiscovery of Ren1 opens up possibilities of finding new diseaseesistance genes within V. vinifera and their rapid incorporationnto the genomes of traditional European cultivars (Kozma et al.,009).

The incorporation of the Ren1 gene into a breeding pro-ram to provide resistance to PM makes possible the long-termupport of Run1 gene and decreases the danger of breakdownf resistance in the event that a new pathogen race appears.ombining and preserving in the backcross progenies the two

ndependently inherited resistance genes does not present a dif-culty compared to a polygenic system because selection of theominant resistance genes can be achieved using molecular mark-rs.

The purpose of our study was to use marker-assisted selectionMAS) to identify the Run1+/Ren1+ genotypes in the hybrid familyroduced by crossing (M. rotundifolia MICH. SMALL × V. vinifera L.)C4 carrying the dominant PM (Run1) and DM (Rpv1) resistanceenes with Kishmish vatkana containing the dominant PM (Ren1)

esistance gene; to prove the pyramiding of the two PM resistanceenes with marker-assisted selection. It was also an objective toevelop a multiplex PCR method for the improvement of MAS effi-iency in order to be able to detect the different resistance genes insingle step.

rticulturae 126 (2010) 448–453 449

2. Materials and methods

2.1. Plant material

To generate grape genotypes that combine the Ren1 and Run1genes, a cross was made between Kishmish vatkana and VRH 3082-1-42, where the former was the male parent and the latter thefemale parent. VRH 3082-1-42 is a progeny plant from the fourthrecurrent pseudo-backcross (BC4) of M. rotundifolia to V. vinifera(Bouquet, 1986). Thus, the progeny resulting from the VRH 3082-1-42 × Kishmish vatkana cross is considered a M. rotundifolia–V.vinifera BC5 hybrid family. Seeds resulting from this cross wereplanted in Jiffy peat pots in the spring. Members of this progenyare identified as Pecs-06-1/1-899, where the word Pecs-06-1 rep-resents the progeny and the numbers 1-899 represent the plant IDwithin the population. Seedlings at the one-leaf-stage were thentransplanted into larger pots and grown in the greenhouse under acanopy of mature PM-infected V. vinifera plants. The phenotype ofthe seedlings was determined as resistant or susceptible at the endof the growing season.

2.2. Genomic DNA extraction and PCR

Genomic DNA was isolated from young leaves with DNeasyPlant Mini Kit (Qiagen, 2006). PCR was performed in a reaction vol-ume of 10 �L in BioRad iCycler thermocycler. The components ofthe reaction mixture were 20 ng of template DNA, 0.6 U of WTB-Taqpolymerase (WestTeam Biotech, Pécs), 0.1 mM dNTP mix, 0.75 �Mof each forward and reverse primer, and 1.25 mM MgCl2 in 1× PCRbuffer.

2.3. Simple sequence repeat and CB marker analysis

VMC8g9 and VMC4f3.1 was used to follow the inheritance of theRun1 gene as described by Barker et al. (2005), and VMC1g3.2 wasused for Rpv1 according to Wiedemann-Merdinoglu et al. (2006).Screening for the Ren1 gene was undertaken using three linked SSRmarkers, UDV020a, VMC9h4.2 and VMCNg4e10.1 (Hoffmann et al.,2008).

CB primers, CB69.70 and CB137.138, have been developed byBarker et al. (2005) using a bacterial artificial chromosome (BAC)library (Dry personal communication).

For the amplification with the SSR markers, we performedtouchdown PCR, which consisted of an initiation cycle at 94 ◦C fortwo 2 min; 10 cycles of denaturation at 94 ◦C for 30 s, primer anneal-ing at 65 ◦C for 30 s, and extension at 72 ◦C for 1 min, where theannealing temperature was decreased by 1 ◦C at each cycle. Thiswas followed by 24 cycles of denaturation at 94 ◦C for 30 s, anneal-ing at 56 ◦C for 30 s, and extension at 72 ◦C for 1 min. The reactionwas completed with a post- amplification extension cycle at 72 ◦Cfor 5 min. Reaction conditions with CB primers were as follows: ini-tiation at 94 ◦C for 2 min; 40 cycles of denaturation at 94 ◦C for 10 s,primer annealing at 57 ◦C for 30 s; and DNA synthesis at 72 ◦C for1 min; post-amplification at 72 ◦C for 5 min.

2.4. Gel electrophoresis

To determine the exact size of PCR amplicons, they were frac-tionated in an 8% polyacrylamide gel (ReproGelTM, GE Healthcare,AP Hungary Kft) in a vertical system (ALF-Express II., AmershamBiosciences, AP Hungary Kft, Budapest). Fragments were detected

by the Cy-5 fluorescent label attached to the forward primer. Theprecise size of the amplified SSR regions was determined relative toexternal and internal standards of known nucleotide length, usingthe ALFwin Fragment Analyser 1.0 software. Products of the CBprimers were detected in 1.2% agarose gel. Products of the multi-

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lex PCR were separated both in ALFExpress and in 4% MetaPhor®

Cambrex Bioproducts, Biocenter Kft, Szeged) agarose gel.

. Results and discussion

In this paper, we report our results of combining the Ren1 andun1 loci into a single grape genome which represents our ini-ial step in breeding resistant grapevines using a gene pyramidingcheme. We created a BC5 hybrid population from a cross betweenishmish vatkana and VRH3082-1-42, a M. rotundifolia × V. viniferaC4 hybrid (Bouquet, 1986) and applied three different Ren1-linkedSR markers and three different Run1-linked SSR and CB markerso follow the inheritance of the resistance genes. We examined 441ndividuals of the BC5 progeny. We present the allele sizes of the

arkers and the possible haplotypes in Fig. 1, where the allelesinked to resistance genes are shown in bold underline.

The Kishmish vatkana × VRH 3082-1-42 cross-resulted in 1,185rogeny plants. Following natural infection under greenhouse con-itions, 286 of these plants were determined to be PM-susceptiblend 899 PM-resistant. The ratio of susceptible to symptomless indi-iduals in the hybrid family was 3:1. Run1+, Ren1+ and Run1+/Ren1+enotypes of identical symptomless phenotypes were identifiedith molecular markers, whose discrimination using traditionalethods would require a long time and complicated procedures.

or MAS analysis, we randomly selected 411 plants from the 899M-resistant and 30 from the PM-susceptible progeny. We usedhese plants to test whether PM-resistance co-segregated with

en1 and/or Run1 specific markers. To find markers that can besed for routine genotyping in MAS, we evaluated SSR markers

inked to Ren1 and Run1. For Ren1-linked markers, we assayedMC9h4.2, VMCNg4e10.1, and UDV020a, which were determinedy Hoffmann et al. (2008) to be located at a genetic distance of

Fig. 1. Position of markers and the allele sizes i

rticulturae 126 (2010) 448–453

approximately 0.9 cM from the Ren1 locus. For all three of thesemarkers, amplicon size differences allowed unambiguous distinc-tion of Ren1 and its homologous allele. Allele sizes for VMC9h4.2,VMCNg4e10.1, and UDV020a for the progeny under study areshown in Fig. 1. The three Ren1-linked alleles were always inher-ited together, confirming their tight linkage (Hoffmann et al.,2008) (Table 1). All plants that carrying the Ren1-linked mark-ers were resistant to PM, and none of the 30 PM-susceptibleplants inherited any of the marker alleles indicating PM resis-tance.

For Run1-linked markers, we applied VMC8g9 and VMC4f3.1 asdescribed by Barker et al. (2005). VMC8g9 was found to consistentlyco-segregate with PM resistance phenotype in VRH3082-1-42 × V.vinifera cv. Cabernet Sauvignon (Barker et al., 2005). The allelesizes of VMC8g9 were 160 (Run1-linked), 167, and 174 bp, andwere readily distinguishable from one-another. At the beginningwe started to use VMC4f3.1, but because of the 2-bp differencebetween the Run1-linked allele and its homologue (186 bp vs.184 bp, respectively) we discontinued the use of this marker (datanot shown). Since we only had one informative Run1-linked SSRmarker, we thought it to be important to confirm the VMC8g9-generated results with an independent method. Thus, we tested ifthis SSR marker co-segregated with Run1-specific dominant mark-ers CB69.70 and CB137.138, which had been designed on the basisof the BAC library clones by Barker et al. (2005). All plants thatharboured the Run1-linked VMC8g9 allele proved to be positivefor CB69.70 and CB137.138. Fig. 2 shows the 277-bp PCR product

amplified with CB137.138 and its association with PM-resistantindividuals. Data of Table 1 prove the tight correlation betweenVMC8g9 and CB markers with the same set of plant samples shownin Fig. 2. In those plants which do not contain the Run1 gene (forexample 16, 26, 36, 76 etc.) and after all are PM symptomless the

n grape genotypes involved in this study.

D. Katula-Debreceni et al. / Scientia Horticulturae 126 (2010) 448–453 451

Table 1Results of 30 randomly selected PM symptomless samples with all the 6 markers used for analyzing the presence of the Run1 or the Ren1 resistance genes. Run1+/Ren1+genotypes are shaded.

Symptomless genotypes Ren1 Run1

06-1/ VMC9h4.2 VMCNg4e10.1 UDV020a CB69.70 CB137.138 VMC8g9.1

6 − − − + + +16 + + + − − −26 + + + − − −36 + + + − − −46 + + + + + +56 − − − + + +76 + + + − − −

106 + + + + + +116 + + + − − −126 − − − + + +136 + + + + + +146 + + + + + +156 + + + + + +176 − − − + + +206 + + + − − −216 + + + + + +226 + + + + + +236 + + + − − −246 − − − + + +256 − − − + + +276 − − − + + +306 + + + − − −316 + + + − − −326 − − − + + +336 + + + − − −346 − − − + + +

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en1 resistance gene from Kishmish vatkana is present. Plants thatre positive for both Run1 and Ren1 (46, 106, 136, 146, 156, 216, 226,06 and 416) are valuable material for grape breeding since theyave two dominant PM resistance genes from different sources andhey are on different chromosomes (Run1 is on LG12 and Ren1 is onG13). All plants that carrying the Run1-linked markers were resis-ant to PM, and none of the 30 PM-susceptible plants was positiveor any of these three Run1-linked alleles.

Merdinoglu et al. (2003) showed that the Rpv1 locus, which con-ers resistance to downy mildew, was tightly linked to the Run1ocus in M. rotundifolia × V. vinifera BC hybrid plants. They were

2ble to link the Rpv1 locus to SSR marker VMC1g3.2. The Rpv1-pecific allele size of BC4 of VMC1g3.2 is 122 bp. As the VMC1g3.2rimers also prime the synthesis of a 122-bp amplicon in Kish-ish vatkana, only the individuals homozygous for the 122 bp allele

ig. 2. PCR products generated with CB137.138 primers. M: DNA molecular weight marungary), 601–605: PM-susceptible plants (Run1−), BC4: PM-resistant parent (Run1+), ki

hat produced no PM symptoms. Presence of the 277 bp fragment indicates PM-resistant gel.

+ − − −+ − − −+ + + ++ + + +

could be identified as Rpv1+ genotypes. The analysis of heterozy-gous individuals will require the involvement of another marker;actually, this analysis has already been started with two new mark-ers located in the vicinity of the VMC1g3.2 marker locus (datanot shown). Nonetheless, we determined that individuals that arehomozygous for this allele (122:122, Rpv1+) are also Run1+, whichcorroborates the findings by Merdinoglu et al. (2003) and Dry et al.(2010).

None of the plants that supported powdery mildew growthon their leaves harboured either of the resistance genes. Amongthe 411 plants that were resistant to powdery mildew 35% con-

tained both the Run1 and Ren1 resistance genes, while 28% wereRun1-positive and 37% Ren1-positive. Marker-assisted selection isindispensable for selecting Run1+/Ren1+ genotypes due to the samephenotypic effect of both resistance genes.

ker (GeneRuler 100 bp Ladder Plus, Fermentas, Lithuania, BIOCENTER Kft, Szeged,s: Kishmish vatkana: Run1−, card: cardinal PM-susceptible (Run1−), 6–416: plantsenotypes (signed with asterisk). PCR products were electrophoresed in 1.2% agarose

452 D. Katula-Debreceni et al. / Scientia Horticulturae 126 (2010) 448–453

Fig. 3. ALF-pattern of multiplex PCR using VMC1g3.2, VMC8g9 and VMC9h4.2 primers. (a) ES: external standard (95, 275, 300, 500 bp), BC4 (Run1+/Ren1−), KV: Kishmishvatkana (Run1−/Ren1+), BC5 progeny: 601: Run1−/Ren1−, 106: Run1+/Ren1+, 176: Run1+/Ren1−, 76: Run1−/Ren1+. The Run1+/Ren1+ genotype is labelled with *. (b) Virtualgel photo of the multiplex PCR.

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ig. 4. (a) Multiplex PCR products (using VMC8g9 and VMC9h4.2 primers) separatedzinta Kft, Budapest, Hungary); KV: Kishmish vatkana: Run1−/Ren1+; BC4: Run1+/Renb) Barcode of multiplex PCR results. The genotype containing both powdery milde

To further streamline the selection process, we developed a mul-iplex PCR-based method and agarose gel electrophoresis of theesulting amplicons. Although our goal was to prove the presencef the two different PM-resistant genes in the progeny, we used anpv1-linked marker (VMC1g3.2) to follow the DM resistant gene.ur results showed that the Rpv1- and Run1-linked markers fre-uently cosegregate. The data illustrated that MAS offers a rapidnd accurate method to select hybrid genomes that combine mul-iple loci in grape.

Having verified markers that reliably co-segregated with theen1 and Run1 loci, we set out to identify a marker combina-ion that would enable us to simultaneously detect the presencef both resistance loci in a multiplex PCR reaction. Such mark-rs need to generate PCR products that differ in size, but annealo genomic DNA at similar temperature. We determined thathe following marker combinations were suitable for multi-lex PCR: VMC9h4.2/VMC8g9, VMCNg4e10.1/VMC8g9, VMC1g3.2/MC9h4.2, VMC1g3.2/VMCNg4e10.1 and VMC1g3.2/VMC8g9/MC9h4.2 (Fig. 3).

A great advantage of the multiplex PCR method is that it enabless to select the valuable genotypes in a single step, saving time,ffort, and resources. This method is ideal to quickly and effi-iently test the presence of resistance genes in the 899 individualsf the VRH 3082-1-42 BC4 × Kishmish vatkana BC5 cross, as wells to study other hybrid families further on. It allows us to select

ene-pyramided genotypes and to perform routine progeny checkshrough multiplex PCR for the joint presence of both the Run1 anden1 genes.

We previously reported that homologues of the VMC8g9 markerould be distinguished in a different M. rotundifolia × V. vinifera

MetaPhor® agarose gel. M: DNA molecular weight marker (BioLine HyperLadder V.,5 progeny: 601: Run1−/Ren1− 90: Run1+/Ren1+, 6: Run1+/Ren1−, 36: Run1−/Ren1+.

stance genes is shaded.

BC5 hybrid progeny when electrophoresed in high-concentrationagarose gels (Molnár et al., 2007). This method has the advantage ofnot requiring the polyacrylamide electrophoresis, the ALF Expressapparatus or the fluorescent labeling of primers.

Thus, we generated multiplex PCR products with VMC1g3.2,VMC8g9 and VMC9h4.2 primers in individuals Pecs-06-1/601, Pecs-06-1/090, Pecs-06-1/006, and Pecs-06-1/036, which representedRun1−/Ren1−, Run1+/Ren1+, Run1+/Ren1−, and Run1−/Ren1+genotypes, respectively. We then fractionated the PCR productsin 1.2% agarose and 4% high-resolution MetaPhor® agarose gels.While the 1.2% routine agarose is suitable for separating the resis-tant genotypes from the sensitive ones only with VMC1g3.2, wecould reliably detect the various allele sizes with VMC8g9 andVMC9h4.2 in 4% MetaPhor® agarose gel (Fig. 4a). The exact allelesizes were already known as they had been previously determinedwith the ALF Express apparatus (Fig. 1). The individual inheritingthe allele linked to the Run1 PM resistance gene (size: 160 bp) ofthe BC4 parent shows Run1+ genotype, the individual inheriting theallele linked to the Ren1 PM resistance gene (size: 286 bp) of Kish-mish vatkana shows Ren1+ genotype, while the individual carryingboth resistance genes shows Run1+/Ren1+ genotype. This methodis suitable for separating the resistant individuals also throughagarose-based electrophoresis. Fig. 4b demonstrates these resultsin a barcode format.

4. Conclusion

The results of the present study demonstrate that SSR markersdeveloped for the mapping of Run1 and Ren1 disease resistance

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in grapevine and assessment of their usefulness for marker assisted selection.Theor. Appl. Genet. 103, 1201–1210.

D. Katula-Debreceni et al. / Scien

oci in grape can be applied for MAS. With these markers we wereble to prove the presence of the pyramided PM resistance genes inhe BC5 hybrid family. Plants carrying pyramided resistance genesRun1, Ren1) for the same phenotype can be identified only withNA level analysis. This is the first time when SSR markers linked

o Ren1 have been used for MAS.

cknowledgement

Research was supported by the Hungarian National Scienceund (OTKA K 62535 and PD 72424). Authors thank Dr. Ian DryCSIRO Plant Industry, Australia) for kindly providing the CB primerequences.

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ouquet, A., 1986. Introduction dans l’espece Vitis vinifera L. d’un caractere deresistance a l’oidium (Uncinula necator Schw. Burr) issu l’espece Muscadiniarotundifolia (Michx.) Small. Vignevini 12 (Suppl.), 141–146.

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