Molecular Breeding

, Volume 26, Issue 4, pp 595–617 | Cite as

Polymorphism analysis of genomic regions associated with broad-spectrum effective blast resistance genes for marker development in rice

  • G. Tacconi
  • V. Baldassarre
  • C. Lanzanova
  • O. Faivre-Rampant
  • S. Cavigiolo
  • S. Urso
  • E. Lupotto
  • G. Valè


Cultivated European rice germplasm is generally characterized by moderate to high sensitivity to blast, and blast resistance is therefore one of the most important traits to improve in rice breeding. We collected a panel of 25 rice genotypes containing 13 broad range rice resistance genes that are commonly used in breeding programs around the world: Pi1, Pi2, Pi5, Pi7, Pi9, Pi33, Pib, Pik, Pik-p, Pita, Pita 2 , Piz and Piz-t. The efficiency of the selected Pi genes towards Italian blast pathotypes was tested via artificial inoculation and under natural field infection conditions. To characterize haplotypes present in the chromosomal regions of the blast resistance genes, a polymorphism search was conducted in the sequence regions adjacent to the blast resistance by examining DNA from the Pi gene donors with a panel of 5–7 potential receivers (cultivated European rice genotypes). Seven InDel and 8 presence/absence polymorphisms were directly detected by gel analysis after DNA amplification, while sequencing of 12.870 bp through 32 loci in different genotypes revealed 85 SNP (one SNP every 151 bp). Seven SSRs were additionally tested revealing 5 polymorphic markers between donors and receivers. Polymorphisms were used to develop 35 PCR-based molecular markers suitable for introgressing of Pi genes into a set of the European rice germplasm. For this last purpose, allelic molecular marker variation was evaluated within a representative collection of about 95 rice genotypes. Polymorphic combinations allowing introgression of the broad spectrum resistance genes into a susceptible genetic background have been identified, thus confirming the potential of the identified markers for molecular-assisted breeding.


Rice (Oryza sativaBlast (Magnaporthe oryzae) resistance Haplotype analysis Marker-assisted selection (MAS) Gene pyramiding 



The authors wish to thank the following persons for the gift of rice genotypes and blast isolates: Dr. Harold Bockelman (USDA, Agricultural Research Service, National Small Grains Research Facility, National Small Grains Collection, USA) for C101LAC, Bala, Carreon, Te Qing, Saber, Katy, Kaybonnet, Zenith, Jefferson; Dr. Didier Tharreau (UMR BGPI, CIRAD, France) for C101LAC, IR64, IR1529, K1, K60 and for the three Italian blast strains; Dr. Jong-Seong Jeon (Graduate School of Biotechnology & Plant Metabolism Research Center Kyung Hee University Korea) for RIL260, IRBL5-M, IRBL7-M; Dr. Kazutoshi Okuno (National Institute of Agrobiological Sciences NIAS, Japan) for Kanto 51, Kusabue, BL1, K1, Fukunishiki, Toride 1 and Dr. Guo-Liang Wang (Department of Plant Pathology, The Ohio State University, USA) for 75-1-127 and C101A-51. This study acknowledges financial support of the project VALORYZA (Italian Ministero per le Politiche Agricole e Agroalimentari, DM 301/7303/06), CEREALAB and SITEIA (Italian Region Emilia Romagna) and of the EU co-funded project EURIGEN (049 AGRI GEN RES).


  1. Ballini E, Berruyer R, Morel JB, Lebrun MH, Notteghem JL, Tharreau D (2007) Modern elite rice varieties of the ‘Green Revolution’ have retained a large introgression from wild rice around the Pi33 rice blast resistance locus. New Phytol 175:340–350CrossRefPubMedGoogle Scholar
  2. Ballini E, Morel JB, Droc G, Price A, Courtois B, Notteghem JL, Tharreau D (2008) A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Mol Plant Microbe Interact 21:859–868CrossRefPubMedGoogle Scholar
  3. Berruyer R, Adreit H, Milazzo J, Gaillard S, Berger A, Dioh W, Lebrun MH, Tharreau D (2003) Identification and fine mapping of Pi33, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene ACE1. Theor Appl Genet 107:1139–1147CrossRefPubMedGoogle Scholar
  4. Bonman JM, Khush GS, Nelson RJ (1992) Breeding rice for resistance to pest. Ann Rev Phytopathol 30:507–528CrossRefGoogle Scholar
  5. Bryan GT, Wu KS, Farrall L, Jia Y, Hershey HP, McAdams SA, Faulk KN, Donaldson GK, Tarchini R, Valent B (2000) A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pita. Plant Cell 12:2033–2046CrossRefPubMedGoogle Scholar
  6. Campbell MA, Chen D, Ronald PC (2004) Development of co-dominant amplified polymorphic sequence markers in rice that flank the Magnaporthe grisea resistance gene Pi7(t) in recombinant inbred line 29. Phytopathol 94:302–307CrossRefGoogle Scholar
  7. Chen D, Zeigler RS, Nelson RJ (1995) Population structure of Pyricularia grisea at two screening sites in the Philippines. Phytopathol 85:1011–1020CrossRefGoogle Scholar
  8. Couch BC, Kohn LM (2002) A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycology 94:683–963CrossRefGoogle Scholar
  9. Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833CrossRefPubMedGoogle Scholar
  10. Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H, Read ND, Lee YH, Carbone I, Brown D, Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E, Rehmeyer C, Li W, Harding M, Kim S, Lebrun MH, Bohnert H, Coughlan S, Butler J, Calvo S, Ma LJ, Nicol R, Purcell S, Nusbaum C, Galagan JE, Birren BW (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980–986CrossRefPubMedGoogle Scholar
  11. Fjellstrom R, Conaway-Bormans CA, McClung AM, Marchetti MA, Shank AR, Park WD (2004) Development of DNA markers suitable for marker assisted selection of three Pi genes conferring resistance to multiple Pyricularia grisea pathotypes. Crop Sci 44:1790–1798CrossRefGoogle Scholar
  12. Fjellstrom R, McClung AM, Shank AR (2006) SSR markers closely linked to the Pi-z locus are useful for selection of blast resistance in a broad array of rice germplasm. Mol Breed 17:149–157CrossRefGoogle Scholar
  13. Francia E, Tacconi G, Crosatti C, Barabaschi D, Bulgarelli D, Dellaglio E, Valè G (2005) Marker assisted selection in crop plants. Plant Cell Tissue Org Cult 82:317–342CrossRefGoogle Scholar
  14. Fukuta Y, Telebanco-Yanoria MJ, Imbe T, Tsunematsu H, Kato H, Ebron LA, Hayashi N, Ando I, Khush GS (2003) Development of new international standard differential variety series to integrate blast resistance of rice (Oryza sativa L). JIRCAS Res Highlights 2003:16–17Google Scholar
  15. Hayashi K, Hashimoto N, Daigen M, Ashikawa I (2004) Development of PCR-based SNP markers for rice blast resistance genes at the Pi-z locus. Theor Appl Genet 108:1212–1220CrossRefPubMedGoogle Scholar
  16. Hayashi K, Yoshida H, Ashikawa I (2006) Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes. Theor Appl Genet 113:251–260CrossRefPubMedGoogle Scholar
  17. Hittalmani Parco, Mew TV, Zeigler RS (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theor Appl Genet 100:1121–1128CrossRefGoogle Scholar
  18. Jeon JS, Chen D, Yi GH, Wang GL, Ronald PC (2003) Genetic and physical mapping of Pi5(t), a locus associated with broad-spectrum resistance to rice blast. Mol Genet Genomics 269:280–289PubMedGoogle Scholar
  19. Jia Y, Wang Z, Fjellstrom RG, Moldenhauer KAK, Azam MA, Correl J, Lee FN, Xia Y, Rutger JN (2004) Rice Pita gene confers resistance to the major pathotypes of the rice blast fungus in the United States. Phytopathol 94:296–301CrossRefGoogle Scholar
  20. Kiyosawa S (1972) Genetics of blast resistance. In: Rice breeding IRRI. Manila, Phillipines pp 203–225Google Scholar
  21. Lee SK, Song MY, Seo YS, Kim HK, Ko S, Cao PJ, Suh JP, Yi G, Roh JH, Lee S, An G, Hahn TR, Wang GL, Ronald P, Jeon JS (2009) Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes. Genetics 181:1627–1638CrossRefPubMedGoogle Scholar
  22. Li L, Wang L, Jing J, Li Z, Lin F, Huang L, Pan Q (2007) The Pikm gene, conferring stable resistance to isolates of Magnaporthe oryzae, was finely mapped in a crossover-cold region on rice chromosome 11. Mol Breed 20:179–188CrossRefGoogle Scholar
  23. Liu G, Lu G, Zeng L, Wang GL (2002) Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Mol Genet Genomics 267:472–480CrossRefPubMedGoogle Scholar
  24. Liu XQ, Wang L, Chen S, Lin F, Pan QH (2005) Genetic and physical mapping of Pi36(t), a novel rice blast resistance gene located on rice chromosome 8. Mol Genet Genomics 274:394–401CrossRefPubMedGoogle Scholar
  25. Mackill D, Bonman JM (1992) Inheritance of blast resistance in near-isogenic line of rice. Phytopathol 82:746–749CrossRefGoogle Scholar
  26. Michelmore RW (2003) The impact zone: genomics and breeding for durable disease resistance. Curr Opin Plant Biol 6:397–404CrossRefPubMedGoogle Scholar
  27. Picco AM, Rodino D, Rodolfi M, Sala F (2001) Biologia di Pyricularia grisea (Cooke) Saccardo. http://www agricoltura regione lombardia it/admin/rla_Documenti/1-1235/biologia_di_p grisea_ok pdf
  28. Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang GL (2006) The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice. Genetics 172:1901–1914CrossRefPubMedGoogle Scholar
  29. Roumen E, Levy M, Notteghem JL (1997) Characterisation of the European pathogen population of Magnaporthe grisea by DNA fingerprinting and pathotype analysis. Eur J Plant Pathol 103:363–371CrossRefGoogle Scholar
  30. Saghai-Maroof MA, Soliman KM, Jogensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. PNAS 81:8014–8018CrossRefPubMedGoogle Scholar
  31. Sallaud C, Lorieux M, Roumen E, Tharreau D, Berruyer R, Svestasrani P, Garsmeur O, Ghesquiere A, Notteghem JL (2003) Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy. Theor Appl Genet 106:794–803PubMedGoogle Scholar
  32. Scardaci SC, Webster RK, Greer JE, Hill JF, Williams JF, Mutters RG, Brandon DM, McKenzie KS, Oster JJ (1997) Rice blast: a new disease in California. http://www plantsciences ucdavis edu/uccerice/AFS/agfs0297 htm
  33. Sere Y, Onasanya A, Afolabi A, Mignouna HD, Akator K (2007) Genetic diversity of the blast fungus, Magnaporthe grisea (Hebert) Barr, in Burkina Faso. African J Biotechnol 6:2568–2577Google Scholar
  34. Singh S, Sidhu JS, Huang N, Vikal Y, Li Z, Brar DS, Dhaliwal HS, Khush GS (2001) Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106. Theor Appl Genet 102:1011–1015CrossRefGoogle Scholar
  35. Sivaraj R, Gnanamanickam SS, Levy M (1996) Pyricularia grisea: a molecular approach for management of rice blast, vol 132. Rice Genetics III International Rice Research Institute, Manila, pp 958–962 Khush GS (ed)Google Scholar
  36. Staden R, Beal KF, Bonfield JK (1999) The staden package, 1998. Meth Mol Biol 132:115–130Google Scholar
  37. Temnykh S, Park WD, Ayres N, Cartinhour S, Hauck N, Lipovich L, Cho YC, Ishii T, McCouch SR (2000) Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.). Theor Appl Genet 100:697–712CrossRefGoogle Scholar
  38. Wang GL, Mackill DJ, Bonman JM, McCouch SR, Champoux MC, Nelson RJ (1994) RFLP mapping of genes conferring complete and partial resistance to blast in a durably resistant rice cultivar. Genetics 136:1421–1434PubMedGoogle Scholar
  39. Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. Plant J 19:55–64CrossRefPubMedGoogle Scholar
  40. Werner K, Friedt W, Ordon F (2005) Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2). Mol Breed 16:45–55CrossRefGoogle Scholar
  41. Yu ZH, Makill DJ, Bonman JM, Tanksley SD (1991) Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor Appl Genet 81:471–476CrossRefGoogle Scholar
  42. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang GL (2006) The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Mol Plant Microbe Interact 19:1216–1228CrossRefPubMedGoogle Scholar
  43. Zhou B, Dolan M, Sakai H, Wang GL (2007) The genomic dynamics and evolutionary mechanism of the Pi2/9 locus in rice. Mol Plant Microbe Interact 20:63–71CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • G. Tacconi
    • 1
  • V. Baldassarre
    • 1
  • C. Lanzanova
    • 2
  • O. Faivre-Rampant
    • 3
    • 4
  • S. Cavigiolo
    • 2
  • S. Urso
    • 1
  • E. Lupotto
    • 2
  • G. Valè
    • 1
  1. 1.CRA-GPG Genomic Research CenterPiacenzaItaly
  2. 2.CRA-RIS Rice Research UnitVercelliItaly
  3. 3.Parco Tecnologico PadanoLodiItaly
  4. 4.CIRAD, UMR BGPIMontpellier Cedex5France

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