, 213:68 | Cite as

Fine mapping of Ren3 reveals two loci mediating hypersensitive response against Erysiphe necator in grapevine

  • Daniel Zendler
  • Pierre Schneider
  • Reinhard Töpfer
  • Eva ZyprianEmail author


Grapevine (Vitis vinifera L.) is economically very important for the production of wine, table grapes and raisins. However, grapevine is threatened by a brought range of pathogens. A destructive disease worldwide is powdery mildew caused by the ascomycete Erysiphe necator. In the grapevine cultivar `Regent’ a resistance locus against E. necator, Ren3, was previously reported. It spans an interval of approximately seven Mb on chromosome 15. We attempted to delimit this interval to facilitate its further molecular analysis. New simple sequence repeat markers targeted to the Ren3 region were designed. They were applied for fine mapping in the cross populations of ‘Regent’ × ‘Lemberger’ and ‘Regent’ × ‘Cabernet Sauvignon’ that segregate for E. necator resistance. Complementarily we scored E. necator infection levels of ‘Regent’ × ‘Lemberger’ progeny at different time points over the course of the vegetation period in 2015 and 2016. Subsequent QTL analysis revealed a maximum LOD value that shifted during the season from marker GF15-10 located at 2.2 Mb to marker GF15-53 located at 3.5 Mb and to marker ScORA7* located at 9.4 Mb on chromosome 15 (positions according to the grapevine reference genome of PN40024). To investigate the Ren3-encoded resistance mechanism we performed detached leaf infection assays for microscopic studies. These revealed that Ren3 carrying individuals react with a hypersensitive response. Results of detached leaf assays on recombinants in the Ren3 locus indicate that not only one, but two distinct genetic regions on chromosome 15 mediate hypersensitive response against E. necator.


Vitis sp. Powdery mildew Resistance Genetic mapping Ren9 ‘Regent’ 



We wish to thank the JKI Institute for Plant Protection in Fruit Crops and Viticulture for the provision of microscopes. Heike Bennek, Margit Schneider and Claudia Welsch contributed expert technical assistance. Ludger Hausmann and Florian Schwander made NGS data from ‘Regent’, ‘Villard Blanc’ and GF.GA-47-42 available. Rudolf Eibach provided some genotypic data on the germplasm collection of the JKI Institute for Grapevine Breeding Geilweilerhof. This project was funded by Deutsche Forschungsgemeinschaft (DFG; Zy11/9-1).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

10681_2017_1857_MOESM1_ESM.pdf (259 kb)
Supplementary Figure 1 Parental and integrated maps of the Ren3 locus on linkage group 15 in the populations ‘Regent’ × ‘Lemberger’ (upper part) and ‘Regent’ × ‘Cabernet Sauvignon’ (lower part). Map distances are given in centiMorgan (cM) units at the left side, marker designations are on the right sides of the bars representing chromosome 15. Lines link the positions of identical markers (PDF 259 kb)
10681_2017_1857_MOESM2_ESM.pdf (299 kb)
Supplementary Figure 2 Barplot of phenotypic datasets for Erysiphe necator resistance of leaves in the ‘Regent’ × ‘Lemberger’ population from previous years 2006, 2007, 2009 and 2010. The median score of the datasets lies between 5 and 9. The number of individuals assigned to each score is given in the table below the graph (PDF 299 kb)
10681_2017_1857_MOESM3_ESM.pdf (204 kb)
Supplementary Figure 3 QTL analysis of resistance to powdery mildew in the cross population of ‘Regent’ × ‘Lemberger’ with the genetic map for ‘Lemberger’ for the four time points of phenotypic evaluation 2015-1, -2 and 2016-1, -2. The LOD blots along the linkage group of IMs are shown by the continuous colored line graphs. The horizontal dotted line indicates the LG specific threshold of 2.5. Kruskal–Wallis significance for each marker is indicated by minus next to the marker name (PDF 204 kb)
10681_2017_1857_MOESM4_ESM.pdf (205 kb)
Supplementary Figure 4 Microscopic pictures of ‘Chambourcin’ inoculated with E. necator (5 dpi). A shows Calcofluor-White staining and B Trypan-Blue staining. Size standard is given in each picture. gs = germinated spore, ap = appressoria, hy = hyphea, HR = hypersensitive response (PDF 204 kb)
10681_2017_1857_MOESM5_ESM.pdf (230 kb)
Suppelemtary Table 1 Identification of recombinants by the presence/absence of Ren3- resistance linked alleles in accessions of the germplam repository at the Institute. DEU numbers refer to Presence or absence of the resistance associated alleles are indicated by (+) and (-). (?) indicates that no allele could be called (PDF 230 kb)
10681_2017_1857_MOESM6_ESM.pdf (278 kb)
Supplementary Table 2 List of resistance gene analogs (RGA) in the reference genome PN40024 (12×) chromosome 15 corresponding to the Ren3 interval (PDF 278 kb)


  1. Adam-Blondon A-F, Roux C, Claux D, Butterlin G, Merdinoglu D, This P (2004) Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics. Theor Appl Genet 109:1017–1027CrossRefPubMedGoogle Scholar
  2. Akkurt M, Welter L, Maul E, Töpfer R, Zyprian E (2007) Development of SCAR markers linked to powdery mildew (Uncinula necator) resistance in grapevine (Vitis vinifera L. and Vitis sp.). Mol Breed 19:103–111. doi: 10.1007/s11032-006-9047-9 CrossRefGoogle Scholar
  3. Barba P, Cadle-Davidson L, Galarneau E, Reisch B (2015) Vitis rupestris B38 confers isolate-specific quantitative resistance to penetration by Erysiphe necator. Plant Pathol 105:1097–1103. doi: 10.1094/PHYTO-09-14-0260-R Google Scholar
  4. Bellin D, Peressotti E, Merdinoglu D et al (2009) Resistance to Plasmopara viticola in grapevine Bianca is controlled by a major dominant gene causing localised necrosis at the infection site. Theor Appl Genet 120:163–176CrossRefPubMedGoogle Scholar
  5. Brewer MT, Milgroom MG (2010) Phylogeography and population structure of the grape powdery mildew fungus, Erysihe necator, from diverse Vitis species. BMC Evol Biol 10:268CrossRefPubMedPubMedCentralGoogle Scholar
  6. Brewer MT, Cadle-Davidson L, Cortesi P, Spanu PD, Milgroom MG (2011) Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi. Fungal Genet Biol 48:704–713CrossRefPubMedGoogle Scholar
  7. Brewer MT, Frenkel O, Milgroom MG (2012) Linkage disequilibrium and spatial aggregation of genotypes in sexually reproducing populations of Erysiphe necator. Phytopathology 102:997–1005CrossRefPubMedGoogle Scholar
  8. Casagrande K, Falginella L, Castellarin SD, Testolin R, Di Gaspero G (2011) Defence responses in Rpv3-dependent resistance to grapevine downy mildew. Planta 234:1097–1109. doi: 10.1007/s00425-011-1461-5 CrossRefPubMedGoogle Scholar
  9. Coleman C, Copetti D, Cipriani G, Hoffmann S, Kozma P, Kovác L, Di Gaspero G (2009) The powdery mildew resistance gene REN1 co-segregates with an NBS-LRR gene cluster in two Central Asian grapevines. BMC Genet 10:89. doi: 10.1186/1471-2156-10-89 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Coll NS, Epple P, Dangl JL (2011) Programmed cell death in the plant immune system. Cell Death Differ 18:1247–1256. doi: 10.1038/cdd.2011.37 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dalbo MA, Ye GNY, Weeden NF, Steinkellner H, Sefc KM, Reisch BI (2000) A gene controlling sex in grapevines placed on a molecular marker-based genetic map. Genome 43:333–340CrossRefPubMedGoogle Scholar
  12. DeYoung BJ, Innes RW (2006) Plant NBS-LRR proteins in pathogen sensing and host defense. Nat Immunol 7:1243–1249. doi: 10.1038/ni1410 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Di Gaspero G, Cipriani G, Marrazzo MT et al (2005) Isolation of (AC)n-microsatellites in Vitis vinifera L. and analysis of genetic background in grapevines under marker assisted selection. Mol Breed 15:11–20CrossRefGoogle Scholar
  14. Eibach R, Zyprian E, Welter L, Töpfer R (2007) The use of molecular markers for pyramiding resistance genes in grapevine breeding. Vitis 46:120–124Google Scholar
  15. Fechter I, Hausmann L, Zyprian E, Daum M, Holtgräwe D, Weisshaar B, Töpfer R (2014) QTL analysis of flowering time and ripening traits suggests an impact of a genomic region on linkage group 1 in Vitis. Theor Appl Genet 127:1857–1872. doi: 10.1007/s00122-014-2310-2 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Feechan A, Anderson C, Torregrosa L, Jermakow A, Mestre P, Wiedemann-Merdinoglu S, Merdinoglu D, Walker A, Cadle-Davidson L, Reisch B, Aubourg S, Bentahar N, Shrestha B, Bouquet A, Adam-Blondon A-F, Thomas MR, Dry IB (2013) Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine. Plant J 76:661–674. doi: 10.1111/tpj.12327 CrossRefPubMedGoogle Scholar
  17. Fischer BM, Salakhutdinov I, Akkurt M, Eibach R, Edwards KJ, Töpfer R, Zyprian EM (2004) Quantitative trait locus analysis of fungal disease resistance factors on a molecular map of grapevine. Theor Appl Genet 108:501–515. doi: 10.1007/s00122-003-1445-3 CrossRefPubMedGoogle Scholar
  18. Frenkel O, Portillo I, Brewer MT, Peros JP, Cadle-Davidson L, Milgroom MG (2012) Development of microsatellite markers from the transcriptome of Erysiphe necator for analysing population structure in North America and Europe. Plant Pathol 61:106–119CrossRefGoogle Scholar
  19. Galet P, Dehan EP (1956) Cépages et Vignobles de France, vol. 1, MontpellierGoogle Scholar
  20. Gao Y, Han Y, Zhao F, Li Y, Cheng Y, Ding Q, Wang Y, Wen Y (2016) Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves. Plant Physiol Biochem 98:12–24. doi: 10.1016/j.plaphy.2015.11.003 CrossRefPubMedGoogle Scholar
  21. Grattapaglia D, Sederoff R (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross mapping strategy and RAPD markers. Genetics 137:1121–1137PubMedPubMedCentralGoogle Scholar
  22. Heintz C (1986) Infection mechanism of grapevine powdery mildew (Oidium tuckeri): comparative studies of the penetration process on artificial membranes and leaf epidermis. Vitis 25:215–225Google Scholar
  23. Hoffmann S, Di Gaspero G, Kovács L, Howard S, Kiss E, Galbács Z, Testolin R, Kozma P (2008) Resistance to Erysiphe necator in the grapevine ‘Kishmish vatkana’ is controlled by a single locus through restriction of hyphal growth. Theor Appl Genet 116:427–438. doi: 10.1007/s00122-007-0680-4 CrossRefPubMedGoogle Scholar
  24. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, Felice N, Paillard S, Juman I, Moroldo M, Scalabrin S, Canaguier A, Le Clainche I, Malacrida G, Durand E, Pesole G, Laucou V, Chatelet P, Merdinoglu D, Delledonne M, Pezzotti M, Lecharny A, Scarpelli C, Artiguenave F, Pe ME, Valle G, Morgante M, Caboche M, Adam-Blondon AF, Weissenbach J, Quetier F, Wincker P, The French-Italian Public Consortium for Grapevine Genome Characterization (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467. doi: 10.1038/nature06148 CrossRefPubMedGoogle Scholar
  25. Jones L, Riaz S, Morales-Cruz A, Amrine KCH, McGuire B, Gubler WD, Walker MA, Cantu D (2014) Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator. BMC Genom 15:1081CrossRefGoogle Scholar
  26. Koch E, Slusarenko A (1990) Arabidopsis is susceptible to infection by a Downy Mildew fungus. Plant Cell 2:437–445CrossRefPubMedPubMedCentralGoogle Scholar
  27. Komárek M, Čadková E, Chrastný V, Bordas F, Bollinger JC (2010) Contamination of vineyard soils with fungicides: a review of environmental and toxicological aspects. Environ Int 36:138–151. doi: 10.1016/j.envint.2009.10.005 CrossRefPubMedGoogle Scholar
  28. Kortekamp A, Wind R, Zyprian E (1998) Investigation of the interaction of Plasmopara viticola with susceptible and resistant grapevine cultivars. J Plant Dis Protect 105:475–488Google Scholar
  29. Martins W, Lucas D, Neves K, Bertioli D (2009) WebSat—a web software for microsatellite marker development. Bioinformation 3:282–283CrossRefPubMedPubMedCentralGoogle Scholar
  30. Merdinoglu D, Butterlin G, Bevilacqua L, Chiquet V, Adam-Blondon AF, Decroocq S (2005) Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol Breed 15:349–366. doi: 10.1007/s11032-004-7651-0 CrossRefGoogle Scholar
  31. Miazzi M, Hajjeh H, Faretra F (2003) Observations on the population biology of the grape powdery mildew fungus Uncinula necator. J Plant Pathol 85:123–129Google Scholar
  32. Miclot A-S, Wiedemann-Merdinoglu S, Duchêne E, Merdinoglu D, Mestre P (2011) A standardised method for the quantitative analysis of resistance to grapevine powdery mildew. Eur J Plant Pathol 133:483–495. doi: 10.1007/s10658-011-9922-z CrossRefGoogle Scholar
  33. Moreira FM, Madini A, Marino R, Zulini L, Stefanini M, Velasco R, Grando MS (2011) Genetic linkage maps of two interspecific grape crosses (Vitis spp.) used to localize quantitative trait loci for downy mildew resistance. Tree Genet Genomes 7:153–167. doi: 10.1007/s11295-010-0322-x CrossRefGoogle Scholar
  34. Oliveira M, Cunha M (2015) Study of the portuguese populations of powdery mildew fungus from diverse grapevine cultivars (Vitis vinifera). J Int Sci Vigne Vin 49:173–182Google Scholar
  35. Pap D, Riaz S, Dry IB, Jermakow A, Tenscher AC, Cantu D, Oláh R, Walker MA (2016) Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii. BMC Plant Biol 16:170. doi: 10.1186/s12870-016-0855-8 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Peros J-P, Troulet C, Guerriero M, Michel-Romiti C, Notteghem J-L (2005) Genetic variation and population structure of the grape powdery mildew fungus, Erysiphe necator, in Southern France. Eur J Plant Pathol 113:407–416CrossRefGoogle Scholar
  37. Qiu W, Feechan A, Dry I (2015) Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease. Hort Res 2:15020. doi: 10.1038/hortres.2015.20 CrossRefGoogle Scholar
  38. Ramming DW, Gabler F, Smilanick J, Cadle-Davidson M, Barba P, Mahanil S, Cadle-Davidson L (2011) A single dominant locus, Ren4, confers rapid non-race-specific resistance to grapevine Powdery mildew. Phytopathology 101:502–508. doi: 10.1094/PHYTO-09-10-0237 CrossRefPubMedGoogle Scholar
  39. Riaz S, Tenscher AC, Ramming DW, Walker MA (2011) Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding. Theor Appl Genet 122:1059–1073. doi: 10.1007/s00122-010-1511-6 CrossRefPubMedGoogle Scholar
  40. RStudio Team (2015). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL
  41. Töpfer R, Hausmann L, Eibach R (2011) Molecular breeding. In: Adam-Blondon AF, Martinez-Zapater JM, Kole C (eds) Genetics, genomics and breeding of grapes. Science Publishers, Enfield, pp 160–185CrossRefGoogle Scholar
  42. van Heerden CJ, Burger P, Vermeulen A, Prins R (2014) Detection of downy and powdery mildew resistance QTL in a ‘Regent’ × ‘RedGlobe’ population. Euphytica 200:281–295. doi: 10.1007/s10681-014-1167-4 CrossRefGoogle Scholar
  43. Van Ooijen JW (2006). JoinMap®4, Software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen, The NetherlandsGoogle Scholar
  44. Van Ooijen JW (2009). MapQTL6®, Software for the mapping of quantitative trait loci in experimental populations of diploid species. Kyazma BV, Wageningen, The NetherlandsGoogle Scholar
  45. Weeden NF (1994) Approaches to mapping in horticultural crops. In: Gresshoff PM (ed) Plant genome analysis. CRC Press, Boca Raton, pp 57–68Google Scholar
  46. Welter LJ, Göktürk-Baydar N, Akkurt M, Maul E, Eibach R, Töpfer R, Zyprian EM (2007) Genetic mapping and localization of quantitative trait loci affecting fungal disease resistance and leaf morphology in grapevine (Vitis vinifera L). Mol Breed 20:359–374. doi: 10.1007/s11032-007-9097-7 CrossRefGoogle Scholar
  47. Zyprian E, Ochßner I, Schwander F, Šimon S, Hausmann L, Bonow-Rex M, Moreno-Sanz P, Grando MS, Wiedemann-Merdinoglu S, Merdinoglu D, Eibach R, Töpfer R (2016) Quantitative trait loci affecting pathogen resistance and ripening of grapevines. Mol Genet Genom 291(1573):1594. doi: 10.1007/s00438-016-1200-5 Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Julius-Kühn Institut, Institut für Rebenzüchtung GeilweilerhofSiebeldingenGermany

Personalised recommendations