Skip to main content
Log in

Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement

  • Review
  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Single nucleotide polymorphism (SNP) data can be obtained using one of the numerous uniplex or multiplex SNP genotyping platforms that combine a variety of chemistries, detection methods, and reaction formats. Kompetitive Allele Specific PCR (KASP) is one of the uniplex SNP genotyping platforms, and has evolved to be a global benchmark technology. However, there are no publications relating either to the technology itself or to its application in crop improvement programs. In this review, we provide an overview of the different aspects of the KASP genotyping platform, discuss its application in crop improvement, and compare it with the chip-based Illumina GoldenGate platform. The International Maize and Wheat Improvement Center routinely uses KASP, generating in excess of a million data points annually for crop improvement purposes. We found that (1) 81 % of the SNPs used in a custom-designed GoldenGate assay were transferable to KASP; (2) using KASP, negative controls (no template) consistently clustered together and rarely produced signals exceeding the threshold values for allele calling, in contrast to the situation observed using GoldenGate assays; (3) KASP’s average genotyping error in positive control DNA samples was 0.7–1.6 %, which is lower than that observed using GoldenGate (2.0–2.4 %); (4) KASP genotyping costs for marker-assisted recurrent selection were 7.9–46.1 % cheaper than those of the BeadXpress and GoldenGate platforms; and (5) KASP offers cost-effective and scalable flexibility in applications that require small to moderate numbers of markers, such as quality control analysis, quantitative trait loci (QTL) mapping in bi-parental populations, marker-assisted recurrent selection, marker-assisted backcrossing, and QTL fine mapping.

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

Access this article

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
Fig. 4

Similar content being viewed by others

References

  • Babu R, Rojas NP, Gao S, Yan J, Pixley K (2013) Validation of the effects of molecular marker polymorphisms in LcyE and CrtRB1 on provitamin A concentrations for 26 tropical maize populations. Theor Appl Genet 126:389–399

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Beissinger TM, Hirsch CN, Sekhon RS, Foerster JM, Johnson JM, Muttoni G, Vaillancourt B, Buell CR, Kaeppler SM, de Leon N (2013) Marker density and read-depth for genotyping populations using genotyping-by-sequencing. Genetics 193:1073–1081

    Article  CAS  PubMed  Google Scholar 

  • Bernardo R, Charcosset A (2006) Usefulness of gene information in marker-assisted recurrent selection: a simulation appraisal. Crop Sci 46:614–621

    Article  Google Scholar 

  • Bernardo R, Yu JM (2007) Prospects for genomewide selection for quantitative traits in maize. Crop Sci 47:1082–1090

    Article  Google Scholar 

  • Buckler ES, Holland JB, Bradbury PJ, Acharya CB, Brown PJ, Browne C, Ersoz E, Flint-Garcia S, Garcia A, Glaubitz JC, Goodman MM, Harjes C, Guill K, Kroon DE, Larsson S, Lepak NK, Li H, Mitchell SE, Pressoir G, Peiffer JA, Rosas MO, Rocheford TR, Romay MC, Romero S, Salvo S, Sanchez VH, da Silva HS, Sun Q, Tian F, Upadyayula N, Ware D, Yates H, Yu J, Zhang Z, Kresovich S, McMullen MD (2009) The genetic architecture of maize flowering time. Science 325:714–718

    Article  CAS  PubMed  Google Scholar 

  • Chen X, Sullivan PF (2003) Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput. Pharmacogenomics J 3:77–96

    Article  PubMed  Google Scholar 

  • Darvasi A (1998) Experimental strategies for the genetic dissection of complex traits in animal models. Nat Genet 18:19–24

    Article  CAS  PubMed  Google Scholar 

  • Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6:e19379

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fan JB, Oliphant A, Shen R, Kermani BG, Garcia F, Gunderson KL, Hansen M, Steemers F, Butler SL, Deloukas P, Galver L, Hunt S, McBride C, Bibikova M, Rubano T, Chen J, Wickham E, Doucet D, Chang W, Campbell D, Zhang B, Kruglyak S, Bentley D, Haas J, Rigault P, Zhou L, Stuelpnagel J, Chee MS (2003) Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol 68:69–78

    Article  CAS  PubMed  Google Scholar 

  • Gut IG (2001) Automation in genotyping of single nucleotide polymorphisms. Hum Mutat 17:475–492

    Article  CAS  PubMed  Google Scholar 

  • Hamblin MT, Warburton ML, Buckler ES (2007) Empirical comparison of simple sequence repeats and single nucleotide polymorphisms in assessment of maize diversity and relatedness. PLoS ONE 2:e1367

    Article  PubMed Central  PubMed  Google Scholar 

  • Harjes CE, Rocheford TR, Bai L, Brutnell TP, Kandianis CB, Sowinski SG, Stapleton AE, Vallabhaneni R, Williams M, Wurtzel ET, Yan J, Buckler ES (2008) Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science 319:330–333

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hartl DL, Clark AG (1989) Principles of population genetics, 2nd edn. Sinauer Associates, Inc., Sunderland

    Google Scholar 

  • Heckenberger M, Bohn M, Ziegle JS, Joe LK, Hauser JD, Hutton M, Melchinger AE (2002) Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties. I. Genetic and technical sources of variation in SSR data. Mol Breed 10:181–191

    Article  CAS  Google Scholar 

  • Henshall J, Hawken R, Dominik S, Barendse W (2012) Estimating the effect of SNP genotype on quantitative traits from pooled DNA samples. Genet Sel Evol 44:1–13

    Article  Google Scholar 

  • Hyten D, Cannon S, Song Q, Weeks N, Fickus E, Shoemaker R, Specht J, Farmer A, May G, Cregan P (2010) High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence. BMC Genomics 11:38

    Article  PubMed Central  PubMed  Google Scholar 

  • Kumpatla SP, Buyyarapu R, Abdurakhmonov IY, Mammadov JA (2012) Genomics-assisted plant breeding in the 21st century: technological advances and progress. In: Abdurakhmonov I (ed) Plant breeding. InTech publishers, Available from http://www.intechopen.com/books/plant-breeding/genomics-assisted-plant-breeding-in-the-21st-centurytechnological-advances-and-progress

  • Le Hellard S, Ballereau SJ, Visscher PM, Torrance HS, Pinson J, Morris SW, Thomson ML, Semple CAM, Muir WJ, Blackwood DHR, Porteous DJ, Evans KL (2002) SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis. Nucleic Acids Res 30:e74

    Article  PubMed Central  PubMed  Google Scholar 

  • Li H, Luo J, Hemphill JK, Wang J, Gould JH (2001) A rapid and high yielding DNA miniprep for cotton (Gossypium spp.). Plant Mol Biol Report 19:183

    Article  CAS  Google Scholar 

  • Low YL, Wedrén S, Liu J (2006) High-throughput genomic technology in research and clinical management of breast cancer. Evolving landscape of genetic epidemiological studies. Breast Cancer Res 8:209

    Article  PubMed Central  PubMed  Google Scholar 

  • Lu Y, Yan J, Guimaraes C, Taba S, Hao Z, Gao S, Chen S, Li J, Zhang S, Vivek B, Magorokosho C, Mugo S, Makumbi D, Parentoni S, Shah T, Rong T, Crouch J, Xu Y (2009) Molecular characterization of global maize breeding germplasm based on genome-wide single nucleotide polymorphisms. Theor Appl Genetics 120:93–115

    Article  CAS  Google Scholar 

  • Meuwissen THE, Hayes BJ, Goddard ME (2001) Prediction of total genetic value using genome-wide dense marker maps. Genetics 157:1819–1829

    CAS  PubMed  Google Scholar 

  • Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88:9828–9832

    Article  CAS  PubMed  Google Scholar 

  • Neelam K, Brown-Guedira G, Huang L (2013) Development and validation of a breeder-friendly KASPar marker for wheat leaf rust resistance locus Lr21. Mol Breed 31:233–237

    Article  CAS  Google Scholar 

  • Paterson AH, Brubaker CL, Wendel JF (1993) A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Report 11:122–127

    Article  CAS  Google Scholar 

  • Phillips C, Lareu M, Sanchez J, Brion M, Sobrino B, Morling N, Schneider P, Syndercombe Court D, Carracedo A (2004) Selecting single nucleotide polymorphisms for forensic applications. Int Congr Ser 1261:18–20

    Article  CAS  Google Scholar 

  • Poland JA, Brown PJ, Sorrells ME, Jannink JL (2012) Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS ONE 7:e32253

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pumphrey MO, Bernardo R, Anderson JA (2007) Validating the Fhb1 QTL for Fusarium head blight resistance in near-isogenic wheat lines developed from breeding populations. Crop Sci 47:200–206

    Article  CAS  Google Scholar 

  • Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100

    Article  CAS  PubMed  Google Scholar 

  • Ragoussis J (2006) Genotyping technologies for all. Drug Discov Today Technol 3:115–122

    Article  Google Scholar 

  • Reif JC, Hamrit S, Heckenberger M, Schipprack W, Peter MH, Bohn M, Melchinger AE (2005) Genetic structure and diversity of European flint maize populations determined with SSR analyses of individuals and bulks. Theor Appl Genet 111:906–913

    Article  CAS  PubMed  Google Scholar 

  • Schlotterer C (2004) The evolution of molecular markers—just a matter of fashion? Nat Rev Genet 5:63–69

    Article  PubMed  Google Scholar 

  • Semagn K, Bjornstad A, Ndjiondjop MN (2006a) Principles, requirements and prospects of genetic mapping in plants. Afr J Biotechnol 5:2569–2587

    CAS  Google Scholar 

  • Semagn K, Bjornstad A, Ndjiondjop MN (2006b) Progress and prospects of marker assisted backcrossing as a tool in crop breeding programs. Afr J Biotechnol 5:2588–2603

    CAS  Google Scholar 

  • Semagn K, Bjornstad A, Xu Y (2010) The genetic dissection of quantitative traits in crops. EJB 13:article 14. doi:10.2225/vol13-issue5-fulltext-21

  • Semagn K, Beyene Y, Makumbi D, Mugo S, Prasanna BM, Magorokosho C, Atlin G (2012a) Quality control genotyping for assessment of genetic identity and purity in diverse tropical maize inbred lines. Theor Appl Genet 125:1487–1501

    Article  PubMed  Google Scholar 

  • Semagn K, Magorokosho C, Vivek BS, Makumbi D, Beyene Y, Mugo S, Prasanna BM, Warburton ML (2012b) Molecular characterization of diverse CIMMYT maize inbred lines from eastern and southern Africa using single nucleotide polymorphic markers. BMC Genomics 13:113. doi:10.1186/1471-2164-13-113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Semagn K, Beyene Y, Warburton M, Tarekegne A, Mugo S, Meisel B, Sehabiague P and Prasanna BM (2013) Meta-analyses of QTL for grain yield and anthesis silking interval in 18 maize populations evaluated under water-stressed and well-watered environments. BMC Genomics 14:313. doi:10.1186/1471-2164-14-313

  • Shelton CA (2006) Quantitative PCR approach to SNP detection and linkage mapping in Caenorhabditis elegans. Biotechniques 41:583–588

    Article  CAS  PubMed  Google Scholar 

  • Sobrino B, Brión M, Carracedo A (2005) SNPs in forensic genetics: a review on SNP typing methodologies. Forensic Sci Int 154:181–194

    Article  CAS  PubMed  Google Scholar 

  • Syvanen A-C (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2:930–942

    Article  CAS  PubMed  Google Scholar 

  • Van Inghelandt D, Melchinger A, Lebreton C, Stich B (2010) Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers. Theor Appl Genet 120:1289–1299

    Article  PubMed Central  PubMed  Google Scholar 

  • Vignal A, Milan D, SanCristobal M, Eggen A (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 34:275–305

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang XL, Lopez-Valenzuela JA, Gibbon BC, Gakiere B, Galili G, Larkins BA (2007) Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm. J Exp Bot 58:2653–2660

    Article  CAS  PubMed  Google Scholar 

  • Warburton ML, Setimela P, Franco J, Cordova H, Pixley K, Banziger M, Dreisigacker S, Bedoya C, MacRobert J (2010) Toward a cost-effective fingerprinting methodology to distinguish maize open-pollinated varieties. Crop Sci 50:467–477

    Article  Google Scholar 

  • Wen W, Araus JL, Trushar S, Cairns J, Mahuku G, Banziger M, Torres JL, Sanchez C, Yan J (2011) Molecular characterization of a diverse maize inbred line collection and its potential utilization for stress tolerance improvement. Crop Sci 51:2569–2581

    Google Scholar 

  • Yan J, Kandianis CB, Harjes CE, Bai L, Kim EH, Yang X, Skinner DJ, Fu Z, Mitchell S, Li Q, Fernandez MGS, Zaharieva M, Babu R, Fu Y, Palacios N, Li J, DellaPenna D, Brutnell T, Buckler ES, Warburton ML, Rocheford T (2010a) Rare genetic variation at Zea mays crtRB1 increases β-carotene in maize grain. Nat Genet 42:322–327

    Article  CAS  PubMed  Google Scholar 

  • Yan J, Yang X, Shah T, Sanchez-Villeda H, Li J, Warburton M, Zhou Y, Crouch JH, Xu Y (2010b) High-throughput SNP genotyping with the GoldenGate assay in maize. Mol Breed 25:441–451

    Article  CAS  Google Scholar 

  • Zhang J, Stewart JM (2000) Economical and rapid method for extracting cotton genomic DNA. J Cotton Sci 4:193–201

    CAS  Google Scholar 

  • Zheng PZ, Allen WB, Roesler K, Williams ME, Zhang SR, Li JM, Glassman K, Ranch J, Nubel D, Solawetz W, Bhattramakki D, Llaca V, Deschamps S, Zhong GY, Tarczynski MC, Shen B (2008) A phenylalanine in DGAT is a key determinant of oil content and composition in maize. Nat Genet 40:367–372

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This manuscript was prepared based on a wide range of SNP data generated for the “Drought-Tolerant Maize for Africa” projects, which is funded by the Bill & Melinda Gates Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kassa Semagn.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 15 kb)

Supplementary material 2 (DOCX 260 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Semagn, K., Babu, R., Hearne, S. et al. Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breeding 33, 1–14 (2014). https://doi.org/10.1007/s11032-013-9917-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-013-9917-x

Keywords

Navigation