Galbács, Z.

Galli, Z.

Halász, G.

Heszky, L.

Hoffmann, S.

Kiss, E.

Kozma, P.

Molnár, S.

Szöke, A.

Veres, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 46 (4), 212–213 (2007)Research NoteMarker assisted selection (MAS) for powdery mildew resistance in a grape-vine hybrid familyS. MOLNÁR1), Z. GALBÁCS1), G. HALÁSZ2), S. HOFFMANN3), E. KISS1), P. KOZMA3),  A. VERES1), Z. GALLI1),  A. SZŐKE1) and L. HESZKY1)1)Szent István University, Institute of Genetics and Biotechnol-ogy, Gödöllő, Hungary2) Höhere Bundeslehranstalt und Bundesamt für Wein- und Obst-bau, Klosterneuburg, Austria3)Research Institute of Viticulture and Enology, Pécs, HungaryK e y   w o r d s :  Vitis vinifera L., Muscadinia rotundifo-lia Michx., powdery mildew resistance, Run1 locus, molecular markers, MAS.Introduction: Fungal disease resistance became a car-dinal point of the grape breeding in the 19th century, when pathogens such as powdery mildew (Uncinula or Erysiphe necator Schwein) and downy mildew (Plasmopara vitico-la Berk. et Curtis ex. de Bary Berl. et de Toni) were carried to Europe from North America. European viticulture faced a new challenge and a significant environmental risk factor because of the necessity of repeated fungicide applications during the vegetation period, since traditional cultivars of Vitis vinifera origin do not carry any resistance to the mildew fungi. All sources providing high or partial resist-ance to these pathogens are low-quality wild Vitis species, therefore introgression of the resistance genes from them requires many back-crosses with Vitis vinifera in order to produce high quality cultivars (FISCHER et al. 2004). Mus-cadinia rotundifolia was described as totally resistant to powdery mildew (PM) species due to a single locus called Run1 (for Resistance to Uncinula Necator 1). This domi-nant gene was introduced into the V. vinifera genome using a pseudo-backcross strategy (BOUQUET 1986). Molecular markers closely linked to Run1 locus have been identified (PAUQUET et al. 2001; DONALD et al. 2002), allowing MAS to be used in a breeding program.Our research aimed at the application and validation of molecular markers linked to Run1 PM resistance locus in BC5 individuals based on the results of DONALD et al. (2002) and BARKER et al. (2005) with PCR-RFLP (GLP1-12P1-P3), and SSR (simple sequence repeat) markers (VMC4f3.1, VMC8g9), respectively. The objective of the involvement of the SSR markers was to find a one-step way for identification of the PM resistant lines beside the reliable two-step PCR-RFLP.Material and Methods:  P l a n t  m a t e r i a l :  Indi-viduals of BC5 hybrid family (named 02-2) deriving from the {(M. rotundifolia x V. vinifera) BC4} x Cardinal (Table) were applied (KOZMA 2002).E v a l u a t i o n   o f   p o w d e r y   m i l d e w s y m p t o m s :  The seedlings (4-6 leaf stage) were gravi-tationally infected with the conidia of the pathogen (in ad-dition to the continuous abundance of the fungus) in the greenhouse in Pécs. Based on the symptoms after 3 weeks the plants were grouped as symptomless and PM-suscep-tible.I s o l a t i o n   o f   D N A :  The DNA was isolated with DNeasy® Plant Mini Kit (Qiagen, Biomarker Ltd., Gödöllő, Hungary) as described by the manufacturer.P C R - R F L P   a n d   S S R   a n a l y s e s :  For PCR-RFLP the GLP1-12P1-P3 primers and EcoRI restric-tion analysis were applied (DONALD et al. 2002). In SSR analyses VMC4f3.1 and VMC8g9 (DI GASPERO et al. 2000; pers. comm.) were used. PCRs and SSR allele size deter-Correspondence to: Dr. E. KISS, Institute of Genetics and Biotechnology, Páter Károly str 1. Gödöllő, 2100 Hungary, Fax: +36-28-522069. E-mail: Kiss.Erzsebet@mkk.szie.huT a b l eComparison of the results of phenotyping for powdery mildew symptoms and genotyping with molecular markers (shaded numbers indicate the „resistant allele” sizes)Variety/populationPhenotype Molecular markersSymptom-less/ resistantSuscep-tible/ sensitiveGLP1-12P1-P3Endonucleolytic  cleavage of PCR fragment with EcoRI enzymeVMC4f3.1alleles (bp)VMC8g9alleles (bp)R SRyesSnoR186S184R160S167Cardinal - + - + 164:164 179:179VRH 3082-1-42 BC4+ - + - 184:186 160:16702-2 hybrid family BC5 67 62 66 63164:18661164:18468160:17966167:17963Ratio of recombinants 1/129 = 0.007 13/129 = 0.100 5/129 = 0.038 213 S. MOLNÁR et al.minations were carried out as it was described by HALÁSZ et al. (2005). The VMC8g9 PCR products were separated on a 3.5 % Metaphor agarose gel (Cambrex Bio Science, Rockland, ME, USA) in 1xTBE buffer.Results and Discussion:  As a fi rst step 20 resistant and 20 susceptible BC5 plants of the 02-2 hybrid family were selected according to PM symptoms on leaves and were tested with PCR-RFLP markers. One 870 bp DNA fragment was amplifi ed both in healthy and susceptible plants (Figure, A). Symptomless and susceptible individu-als could only be discriminated by restriction analysis of the PCR product. EcoRI cleaved the DNA amplicon of the symptomless leaves into two pieces (670 and 200 bp), while it did not split the PCR product of the susceptible samples (Figure, B). Based on these results, altogether 142 seedlings from the BC5 family were screened. SSR primers provided a way to monitor outcrosses, too. As a result, lines carrying „alien” alleles could be excluded from further analyses. The Table summarizes the data concerning the two parents (VRH 3082-1-42 (BC4) and Cardinal) and the 129 prog-eny. In the case of all three markers recombinants were obtained (Table): PM resistant individuals, whose GLP1-12P1-P3 (BC4) PCR amplicons remained uncut after EcoRI cleavage or the SSR alleles coupled with the resistance were missing from them. In the case of VMC4f3.1 SSR a 186 bp, while in case of VMC8g9 a 160 bp allele proved to be a PM resistance linked marker (Table). In spite of the fact that the linkage of the applied markers proved to be lower than 100 %, they can be successfully applied in MAS since 90-99 % of the plants selected in this way will carry the Run1 PM resistance gene. With regard to economy the VMC8g9 is the most favourable of the three markers because the discriminative 160-167 bp fragments can be separated on an agarose gel of high resolution (Figure, C) following a simple PCR allowing of the routine analyses of many samples at the same time.Based on physical and molecular mapping of Run1 lo-cus (BARKER et al. 2005) it can be expected that more tightly linked SSR or gene-specifi c markers will be available mak-ing MAS even more effi cient and reliable. MAS benefi ts the breeders, providing a possibility for early screening of PM resistant seedlings and accelerating the selection in the case of pyramiding of resistance genes of different origin (e.g. Run1 and Ren1; KOZMA et al. 2007).The research is supported by the grants from the Hungar-ian Scientifi c Research Fund (OTKA K 62535, M 36630 and M 45633) and GrapeGen06 EU project.BARKER, C. L.; DONALD, T.; PAUQUET, J.; RATNAPARKHE, M. B.; BOUQUET, A.; ADAM-BLONDON, A. F.; THOMAS, M.; DRY, I.; 2005: Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artifi cial chromosome library. Theor. Appl. Genet. 111, 370-377.BOUQUET, A.; 1986: Introduction dans l’espece Vitis vinifera L. d’un caractere de resistance a l’oidium (Uncinula necator Schw. Burr) issu l’espece Muscadinia rotundifolia (Michx) Small. Vignevini 12 (suppl), 141-146.DI GASPERO, G.; PETERLUNGER, E.; TESTOLIN, R.; EDWARDS, K. J.; CIPRIANI, G.; 2000: Conservation of microsatellite loci within the genus Vitis. Theor. Appl. Genet. 101, 301-308.DONALD, T. M.; PELLERONE, F.; ADAM-BLONDON, A. F.; BOUQUET, A.; THO-MAS, M. R.; DRY, I. B.; 2002: Identification of resistance gene ana-logs linked to a powdery mildew resistance locus in grapevines. Theor. Appl. Genet. 104, 610-618.FISCHER, B. M; SALAKHUTDINOV, I.; AKKURT, M.; EIBACH, R.; EDWARDS, K. J.; TÖPFER, R.; ZYPRIAN, E. M.; 2004: Quantitative trait locus analysis of fungal disease resistance factor on a molecular map of grapevine. Theor. Appl. Genet. 108, 501-515.HALÁSZ, G.; VERES, A.; KOZMA, P.; KISS, E.; BALOGH, A.; GALLI, Z.; SZÖKE, A.; HOFFMANN, S.; HESZKY, L.; 2005: Microsatellite fingerprinting of grapevine (Vitis vinifera L.) varieties of the Carpathian Basin. Vitis 44, 173-180.KOZMA, P.; 2002: Goals and methods in grape resistance breeding in Hun-gary. Int. J. Hortic. Sci. 8, 41-46. KOZMA, P.; KISS, E.; HOFFMANN, S.; GALBÁCS, Z.; DULA, .T.; 2007: Using the powdery mildew resistant Muscadinia rotundifolia and Vitis vin-ifera cv. Kishmish vatkana for breeding new cultivars. Acta Hortic. (in press).PAUQUET, J.; BOUQUET, A.; THIS, P.; ADAM-BLONDON, A. F.; 2001: Estab-lishment of local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection. Theor. Appl. Genet. 103, 1201-1210.Received February 19, 2007Figure: Distinction of resistant/symptomless and sensitive/sus-ceptible genotypes by PCR-RFLP (GLP1-12P1-P3) and VMC8g9 SSR primers. (A): R1, R2: resistant and S1, S2 sensitive lines pro-duced PCR fragments of the same size (870 bp); M: DNA molec-ular weight marker (Fermentas GeneRuler™ 100 bp ladder plus). (B): EcoRI restriction pattern of resistant (R1, R2) and sensitive (S1, S2) lines. Amplicons of the sensitive lines remained uncut. M: DNA molecular weight marker (Fermentas GeneRuler™ 100 bp ladder plus). (C): SSR profi le of Cardinal (Cd), BC4 (VRH 3082-1-42), R1, R2 resistant and S1, S2 sensitive genotypes. M: DNA molecular weight marker (Fermentas GeneRuler™ 50 bp DNA ladder).

Marker assisted selection (MAS) for powdery mildew resistance in a grapevine hybrid family

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Marker assisted selection (MAS) for powdery mildew resistance in a grapevine hybrid family

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