Skip to main content
SpringerLink
Log in

Genotyping and development of single-nucleotide polymorphism (SNP) markers associated with blast resistance genes in rice using GoldenGate assay

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is required to use identified sequence variation in the alleles of different genes to determine their functional relevance to the candidate gene(s). In the present study, Illumina GoldenGate assay was used to validate and genotype SNPs in a set of six major rice blast resistance genes, viz. Pi-ta, Piz(t), Pi54, Pi9, Pi5(1) and Pib, distributed over five chromosomes, to understand their functional relevance and study the population structure in rice. All the selected SNPs loci (96) of six blast (Magnaporthe oryzae) resistance genes were genotyped successfully in 92 rice lines with an overall genotype call rate of 92.0 % and minimum GenTrain cutoff score of ≥0.448. The highest genotyped SNPs were found in japonica type (97.1 %) rice lines, followed by indica (92.12 %), indica basmati (91.84 %) and minimum in case of wild species (82.0 %). Among the genotyped loci, the highest score (98.68 %) was observed in case of Piz(t), followed by Pi-ta, Pi5(1), Pib, Pi54 and Pi9. Polymorphism was obtained in 87.5 % SNPs loci producing 7,728 genotype calls. Minor allele frequency ranged from 0.01 to 0.49 and has good differentiating power for distinguishing different rice accessions. Population structure analysis revealed that a set of genotypes from four rice subpopulations had “admix” ancestry (>26 %) with more than one genetic background of indica, japonica and wild types. SNPs markers were validated in a set of 92 rice lines and converted into CAPS markers which can be used in blast resistance breeding programme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Agrama HA, Eizenga GC, Yan W (2007) Association mapping of yield and its components in rice cultivars. Mol Breed 19:341–356

    Article  Google Scholar 

  • Akhunov E, Nicolet C, Dvorak J (2009) Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay. Theor Appl Genet 119:507–517

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Bhattramakki D, Dolan M, Hanafey M, Wineland R, Vaske D, Register JC, Tingey SV, Rafalski A (2002) Insertion–deletion polymorphisms in 3′ regions of maize genes occur frequently and can be used as highly informative genetic markers. Plant Mol Biol 48:539–547

    Article  PubMed  CAS  Google Scholar 

  • Boualaphanh C, Daygon VD, Calingacion MN et al (2011) Use of new generation single nucleotide polymorphism genotyping for rapid development of near isogenic lines in rice. Crop Sci 51:2067–2071

    Article  Google Scholar 

  • Bradbury LMT, Fitzgerald TL, Henry RJ, Jin Q, Waters DLE (2005) The gene for fragrance in rice. Plant Biotechnol J 3:363–370

    Article  PubMed  CAS  Google Scholar 

  • Bryan BT, Wu KS, Farrall L, Jia Y, Hershey H, McAdams S, Tarchini R, Donaldson G, Faulk K, Valent B (2000) A single amino acid difference distinguishes resistant and susceptible allele of the rice blast resistance gene Pi-ta. Plant Cell 12:2033–2045

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Caicedo AL, Williamson SH, Hernandez RD, Boyko A, Fledel-Alon A, York TL, Polato NR, Olsen KM, Nielsen R, McCouch SR, Bustamante CD, Purugganan MD (2007) Genome-wide patterns of nucleotide polymorphism in domesticated rice. PLoS Genet 3:e163

    Article  PubMed Central  Google Scholar 

  • Chen H, Shan Z, Sha A, Wu B, Yaung Z, Chen S, Zhou R, Zhou X (2011) Quantitative trait loci analysis of stem strength and related traits in soybean. Euphytica 179:485–497

    Article  Google Scholar 

  • Close TJ, Bhat PR, Lonardi S, Wu Y, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Waugh R et al (2009) Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics 10:582

    Article  PubMed  PubMed Central  Google Scholar 

  • Das A, Soubam D, Singh PK, Thakur S, Singh NK, Sharma TR (2012) A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae. Funct Integr Genomics 12:215–228

    Article  PubMed  CAS  Google Scholar 

  • Enoch MA, Shen PH, Ducci F, Yuan Q, Liu J, White KV, Albaugh B, Hodgkinson CA, Goldman D (2008) Common genetic origins for EEG, alcoholism and anxiety: the role of CRH-BP. PLoS One 3(10):e3620

    Article  PubMed  PubMed Central  Google Scholar 

  • Fan JB, Oliphant A, Shen R, Kermani B, Garcia F, Gunderson K, Hansen M, Steemers F, Butler SL, Deloukas P et al (2003) Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol 68:69–78

    Article  PubMed  CAS  Google Scholar 

  • Feltus A, Wan J, Schulze SR, Estill JC, Jiang N, Paterson AH (2004) An SNP resource for rice genetics and breeding based on subspecies indica and japonica genome alignments. Genome Res 14:1812–1819

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Garris AJ, McCouch SR, Kresovich S (2003) Population structure and its effect on haplotype diversity and linkage disequilibrium surrounding the xa5 locus of rice (Oryza sativa L.). Genetics 165:759–769

    PubMed  PubMed Central  Google Scholar 

  • Garris AJ, Tai TH, Coburn J, Kresovich S, McCouch SR (2005) Genetic structure and diversity in Oryza sativa L. Genetics 169:1631–1638

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Geng XS, Yang MZ, Huang XQ, Cheng ZQ, Fu J, Sun TLJ (2008) Cloning and analyzing of rice blast resistance gene Pi-ta+ allele from Jinghong erect type of common wild rice (Oryza rufipogon Griff) in Yunnan. Yi Chuan 30(1):109–114

    Article  PubMed  CAS  Google Scholar 

  • Gopalakrishnan S, Sharma RK, Anand Rajkumar K, Joseph M, Singh VP, Singh AK, Bhat KV, Singh NK, Mohapatra T (2008) Integrating marker assisted background analysis with foreground selection for identification of superior bacterial blight resistant recombinants in Basmati rice. Plant Breed 127:131–139

    Article  CAS  Google Scholar 

  • Haining L, Ouyang S, Egan A, Nobuta K, Haas BJ, Zhu W, Gu X, Silva JC, Meyers BC, Buell CR (2008) Characterization of paralogous protein families in rice. BMC Plant Biol 8:18

    Article  Google Scholar 

  • Huang CL, Hwang SY, Chiang YC, Lin TP (2008) Molecular evolution of the Pi-ta gene resistant to rice blast in wild rice (Oryza rufipogon). Genetics 179:1527–1538

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hyten DL, Song Q, Choi IY, Yoon MS, Specht JE, Matukumalli LK, Nelson RL, Shoemaker RC, Young ND, Cregan PB (2008) High-throughput genotyping with the GoldenGate assay in the complex genome of soybean. Theor Appl Genet 116:945–952

    Article  PubMed  CAS  Google Scholar 

  • Hyten DL, Smith JR, Frederick RD, Tucker ML, Song Q, Cregan PB (2009) Bulked segregant analysis using the GoldenGate assay to locate the Rpp3 locus that confers resistance to soybean rust in soybean. Crop Sci 49:265–271

    Article  CAS  Google Scholar 

  • IRGSP (International Rice Genome Sequencing Project.) (2005) The map-based sequence of the rice genome. Nature 436:793–800

    Article  Google Scholar 

  • Jin L, Lu Y, Xiao P, Sun M, Corke H, Bao J (2010) Genetic diversity and population structure of a diverse set of rice germplasm for association mapping. Theor Appl Genet 121(3):475–487

    Article  PubMed  Google Scholar 

  • Konishi S, Izawa T, Lin SY, Ebana K, Fukuta Y et al (2006) An SNP caused loss of seed shattering during rice domestication. Science 312:1392–1396

    Article  PubMed  CAS  Google Scholar 

  • Kumari A, Das A, Devanna BN, Thakur S, Singh PK, Upreti HC, Singh NK, Sharma TR (2013) Mining of rice blast resistance gene Pi54 shows effect of single nucleotide polymorphism on phenotypic expression of alleles. Eur J Plant Pathol 137(1):55–65

    Article  CAS  Google Scholar 

  • Liu K, Muse SV (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21:2128–2129

    Article  PubMed  CAS  Google Scholar 

  • McNally KL, Childs KL, Bohert R, Davidson RM, Zhao K, Ulat BJ, Zeller GG, Clark RM, Hoen DR, Bureau TE et al (2009) Genome-wide SNP variation reveals relationships among landraces and modern varieties of rice. Proc Natl Acad Sci USA 106:12273–12278

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Monna L, Ohta R, Masuda H, Koike A, Minobe Y (2006) Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.). DNA Res 13:43–51

    Article  PubMed  CAS  Google Scholar 

  • Nasu S, Suzuki J, Ohta R, Hasegawa K, Yui R, Kitazawa N, Monna L, Minobe Y (2002) Search in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers. DNA Res 9:163–171

    Article  PubMed  CAS  Google Scholar 

  • Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York

    Google Scholar 

  • Ni J, Colowitb PM, Mackill DJ (2002) Evaluation of genetic diversity in rice subspecies using microsatellite markers. Crop Sci 42:601–607

    Article  CAS  Google Scholar 

  • Pasam RK, Sharma R, Malosetti M, Van Eeuwisk FA, Haseneyer G, Kilian V, Graner A (2012) Genome wide association studies for agronomical traits in a worldwide spring barley collection. BMC Plant Biol 12:16

    Article  PubMed  PubMed Central  Google Scholar 

  • Pavy N, Pelgas B, Beauseigle S, Blais S, Gagnon F, Gosselin I, Lamothe M, Isabel N, Bousquet J (2008) Enhancing genetic mapping of complex genomes through the design of highly-multiplexed SNP arrays: application to the large and unsequenced genomes of white spruce and black spruce. BMC Genomics 9:21

    Article  PubMed  PubMed Central  Google Scholar 

  • Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959

    PubMed  CAS  PubMed Central  Google Scholar 

  • Roorkiwal M, Sawargaonkar SL, Chitikineni A et al (2013) Single nucleotide polymorphism genotyping for breeding and genetics applications in Chickpea and Pigeonpea using beadXpress platform. The Plant Genome 6:2. doi:10.3835/plantgenome2013.05.0017

  • Rostoks N, Ramsay L, MacKenzie K, Cardle L, Bhat PR, Roose ML, Svensson JT, Stein N, Varshney RK, Marshall DF, Graner A, Close TJ, Waugh R (2006) Recent history of artificial out-crossing facilitates whole-genome association mapping in elite inbred crop varieties. Proc Natl Acad Sci USA 103:18656–18661

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Roy JK, Smith KP, Muehlbauer GJ, Chao S, Close TJ, Steffenson BJ (2010) Association mapping of spot blotch resistance in wild barley. Mol Breed 26(2):243–256

    Article  PubMed  PubMed Central  Google Scholar 

  • Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497

    Article  PubMed  CAS  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer 3 on the WWW for general users for biologist programmers. Methods Mol Biol 132:365–386

    PubMed  CAS  Google Scholar 

  • Schranz ME, Windsor AJ, Song BH, Lawton-Rauh A, Mitchell-Olds T (2007) Comparative genetic mapping in Boechera stricta (Brassicaceae). Plant Physiol 144:286–298

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Sharma TR, Madhav MS, Singh BK, Shanker P, Jana TK, Dalal V, Pandit A, Singh A, Gaikwad K, Upreti HC, Singh NK (2005) High resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea. Mol Genet Genomics 274:569–578

    Article  PubMed  CAS  Google Scholar 

  • Sharma TR, Rai AK, Gupta SK, Singh NK (2010) Broad spectrum blast resistance gene Pi-k h designated as Pi54. J Plant Biochem Biotechnol 19:87–89

    Article  CAS  Google Scholar 

  • Sharma TR, Rai AK, Gupta SK, Vijayan J, Devanna BN, Ray S (2012) Rice blast management through host resistance: retrospect and prospects. Agric Sci 1:37–52

    Google Scholar 

  • Shen YJ, Jiang H, Jin JP, Zhang Z-B, Xi B, He Y-Y, Wang G, Wang C, Qian L, Li X et al (2004) Development of genome wide DNA polymorphism database for map-based cloning of rice genes. Plant Physiol 135:1198–1205

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Wickham Garcia E, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A (2005) High-throughput SNP genotyping on universal bead arrays. Mutat Res 573:70–82

    Article  PubMed  CAS  Google Scholar 

  • Singh RK, Singh US, Khush GS (2000) Aromatic rices. In: Singh VP (ed) Basmati rice of India. IBH Publisher, Oxford

    Google Scholar 

  • Syvanen AC (2005) Toward genome-wide SNP genotyping. Nat Genet 37:S5–S10

    Article  PubMed  Google Scholar 

  • Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595

    PubMed  CAS  PubMed Central  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  • Thakur S, Gupta YK, Singh PK, Rathour R, Variar M, Prashanthi SK, Singh AK, Singh UD, Chand D, Singh NK, Sharma TR (2013a) Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae. Funct Integr Genomics 13(3):309–322

    Article  PubMed  CAS  Google Scholar 

  • Thakur S, Singh PK, Rathour R, Variar M, Prashanthi SK, Singh AK, Singh UD, Chand D, Singh NK, Sharma TR (2013b) Positive selection pressure on rice blast resistance allele Piz-t makes it divergent in Indian land races. J Plant Interact 8(1):34–44

    Article  CAS  Google Scholar 

  • Thiel T, Kota R, Grosse I, Stein N, Graner A (2004) SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development. Nucleic Acids Res 32:e5

    Article  PubMed  PubMed Central  Google Scholar 

  • Thompson MJ, Zhao K, Wright M et al (2012) High throughput single nucleotide polymorphism genotyping for breeding applications in rice using beadXpress platform. Mol Breed 29:875–886

    Article  Google Scholar 

  • Waters DE, Henry RJ, Reinke RF, Fitzgerald MA (2005) Gelatinization temperature of rice explained by polymorphisms in starch synthase. Plant Biotechnol J 4:115–122

    Article  Google Scholar 

  • Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7:256–276

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto T, Nagasaki H, Yonemaru J, Ebana K, Nakajima M, Shibaya T, Yano M (2010) Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms. BMC Genomics 11:267

    Article  PubMed  PubMed Central  Google Scholar 

  • Yan J, Shah T, Warburton ML, Buckler ES, McMullen MD et al (2009) Genetic characterization and linkage disequilibrium estimation of a global maize collection using SNP markers. PLoS One 4(12):e8451. doi:10.1371/journal.pone.0008451

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang MZ, Cheng ZQ, Chen SN, Qian J, Xu LL, Huang XQ (2007) A rice blast resistance genetic resource from wild rice in Yunnan, China. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 33:589–595

    PubMed  Google Scholar 

  • Zhao K, Wright M, Kimball KJ, Eizenga G, McClung A, Kovach M, Tyagi W, Ali L, Tung CW, Reynolds A et al (2010) Genomic diversity and introgression in O. sativa reveal the impact of domestication and breeding on the rice genome. PLoS One 5:e10780

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

T. R. Sharma is thankful to the National Agricultural Innovation Project (NAIP) (C4/C1071), ICAR, for financial support. The authors are thankful to the Officer in Charge, National Phytotron Facility, Indian Agricultural Research Institute, New Delhi, for providing basic facilities for growing and maintaining rice lines.

Author information

Authors and Affiliations

  1. Room No 39, LBS Centre, National Research Centre on Plant Biotechnology (NRCPB), IARI, New Delhi, 110012, India

    Shallu Thakur, Pankaj K. Singh, Nagendra K. Singh & Tilak R. Sharma

  2. CSK Himachal Pradesh Agricultural University (CSKHPKV), Palampur, 176062, Himachal Pradesh, India

    Rajeev Rathour

  3. Central Rainfed Upland Rice Research Station (CRRI), Hazaribagh, 825301, Jharkhand, India

    Mukund Variar

  4. University of Agricultural Sciences (UAS), Dharwad, 58001, Karnataka, India

    Sangam K. Prashanthi

  5. Indian Agricultural Research Institute (IARI), New Delhi, 110012, India

    S. Gopalakrishnan, A. K. Singh & U. D. Singh

  6. Himachal Pradesh University (HPU), Shimla, 171005, India

    Shallu Thakur & Duni Chand

Authors
  1. Shallu Thakur
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pankaj K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rajeev Rathour
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mukund Variar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sangam K. Prashanthi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S. Gopalakrishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. U. D. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Duni Chand
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nagendra K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Tilak R. Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tilak R. Sharma.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thakur, S., Singh, P.K., Rathour, R. et al. Genotyping and development of single-nucleotide polymorphism (SNP) markers associated with blast resistance genes in rice using GoldenGate assay. Mol Breeding 34, 1449–1463 (2014). https://doi.org/10.1007/s11032-014-0129-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-014-0129-9

Keywords

Search

Navigation