Molecular Breeding

, 25:67 | Cite as

Application of high-resolution DNA melting for genotyping and variant scanning of diploid and autotetraploid potato

  • David De KoeyerEmail author
  • Katheryn Douglass
  • Agnes Murphy
  • Sean Whitney
  • Lana Nolan
  • Yong Song
  • Walter De Jong


The ideal marker system for tetraploid potato would be dosage-sensitive and have the ability to distinguish heterozygous genotypes with multiple haplotypes within the genomic region targeted by the marker. The objective of this study was to evaluate the utility of high-resolution DNA melting (HRM) for genotyping and polymorphism detection in diploid and tetraploid potato. Amplicon scanning, unlabelled probe, and short amplicon assays were developed for four candidate genes affecting tuber skin and flesh colour, and starch, and a marker linked to nematode resistance. Genotyping a set of 95 potato clones revealed several examples of clones with three distinct haplotypes. Combined probe and amplicon analysis identified between 29 and 44 unique genotypes for the same assays. Assays developed for four of the five target genes are suitable for marker-assisted selection in potato breeding programs. This study illustrates the use of HRM in potato genetics. Further advances in the technology and associated data analysis should make HRM a useful tool for basic and applied studies of potato.


Potato High-resolution DNA melting Haplotype dosage Autotetraploid genetics Marker-assisted selection Variant scanning 



We thank Valerie Burns and Stephen Allaby for maintaining the plants and for providing trait descriptions. This work was supported, in part, with funds from the New Brunswick Department of Agriculture and Aquaculture Embracing Innovation in Agriculture program.

Supplementary material

11032_2009_9309_MOESM1_ESM.doc (794 kb)
(DOC 795 kb)


  1. Barone A (2004) Molecular marker-assisted selection for potato breeding. Am J Potato Res 81:111–117CrossRefGoogle Scholar
  2. Bormann CA, Rickert AM, Ruiz RA, Paal J, Lubeck J, Strahwald J, Buhr K, Gebhardt C (2004) Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers. Mol Plant Microbe Interact 17:1126–1138CrossRefPubMedGoogle Scholar
  3. Brodie B (2003) The loss of expression of the H1 gene in Bt transgenic potatoes. Am J Potato Res 80:135–139CrossRefGoogle Scholar
  4. Brown C, Kim T, Ganga Z, Haynes K, De Jong D, Jahn M, Paran I, De Jong W (2006) Segregation of total carotenoid in high level potato germplasm and its relationship to beta-carotene hydroxylase polymorphism. Am J Potato Res 83:365–372CrossRefGoogle Scholar
  5. Chagné D, Gasic K, Crowhurst RN, Han Y, Bassett HC, Bowatte DR, Lawrence TJ, Rikkerink EHA, Gardiner SE, Korban SS (2008) Development of a set of SNP markers present in expressed genes of the apple. Genomics 92:353–358CrossRefPubMedGoogle Scholar
  6. Colton LM, Groza HI, Wielgus SM, Jiang J (2006) Marker-assisted selection for the broad-spectrum potato late blight resistance conferred by gene RB derived from a wild potato species. Crop Sci 46:589–594CrossRefGoogle Scholar
  7. Croxford AE, Rogers T, Caligari PDS, Wilkinson MJ (2008) High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar. New Phytol 180:594–607CrossRefPubMedGoogle Scholar
  8. De Jong H (1991) Inheritance of anthocyanin pigmentation in the cultivated potato: a critical review. Am J Potato Res 68:585–593CrossRefGoogle Scholar
  9. De Jong WS, De Jong DM, Bodis M (2003a) A fluorogenic 5′ nuclease (TaqMan) assay to assess dosage of a marker tightly linked to red skin color in autotetraploid potato. Theor Appl Genet 107:1384–1390CrossRefPubMedGoogle Scholar
  10. De Jong WS, De Jong DM, De Jong H, Kalazich J, Bodis M (2003b) An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.). Theor Appl Genet 107:1375–1383CrossRefPubMedGoogle Scholar
  11. Erali M, Voelkerding KV, Wittwer CT (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58CrossRefPubMedGoogle Scholar
  12. Flinn B, Rothwell C, Griffiths R, Lägue M, DeKoeyer D, Sardana R, Audy P, Goyer C, Li X-Q, Wang-Pruski G, Regan S (2005) Potato expressed sequence tag generation and analysis using standard and unique cDNA Libraries. Plant Mol Biol 59:407–433CrossRefPubMedGoogle Scholar
  13. Gallais A (2003) Quantitative genetics and breeding methods in autopolyploid plants. Institut National de la Recherche Agronomique, ParisGoogle Scholar
  14. Gebhardt C, Bellin D, Henselewski H, Lehmann W, Schwarzfischer J, Valkonen JPT (2006) Marker-assisted combination of major genes for pathogen resistance in potato. Theor Appl Genet 112:1458–1464CrossRefPubMedGoogle Scholar
  15. Hämäläinen JH, Watanabe KN, Valkonen JPT, Arihara A, Plaisted RL, Pehu E, Miller L, Slack S (1997) Mapping and marker-assisted selection for a gene for extreme resistance to potato virus Y. Theor Appl Genet 94:192–197CrossRefGoogle Scholar
  16. Helgeson JP, Pohlman JD, Austin S, Haberlach GT, Wielgus SM, Ronis D, Zambolim L, Tooley P, McGrath JM, James RV, Stevenson WR (1998) Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight. Theor Appl Genet 96:738–742CrossRefGoogle Scholar
  17. Huang H, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877CrossRefPubMedGoogle Scholar
  18. Hutten R, Soppe W, Hermsen J, Jacobsen E (1995) Evaluation of dihaploid populations from potato varieties and breeding lines. Potato Res 38:77–86CrossRefGoogle Scholar
  19. Jung CS, Griffiths HM, De Jong DM, Cheng S, Bodis M, De Jong WS (2005) The potato P locus codes for flavonoid 3′, 5′-hydroxylase. Theor Appl Genet 110:269–275CrossRefPubMedGoogle Scholar
  20. Kasai KY, Morikawa Y, Sorri VA, Valkonen JPT, Gebhardt C, Watanabe KN (2000) Development of SCAR markers to the PVY resistance gene Ryadg based on a common feature of plant disease resistance genes. Genome 43:1–8CrossRefPubMedGoogle Scholar
  21. Lehmensiek A, Sutherland M, McNamara R (2008) The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley. Theor Appl Genet 117:721–728CrossRefPubMedGoogle Scholar
  22. Mackay J, Wright C, Bonfiglioli R (2008) A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4:8CrossRefPubMedGoogle Scholar
  23. Montgomery J, Wittwer CT, Palais R, Zhou L (2007) Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc 2:59–66CrossRefPubMedGoogle Scholar
  24. Morgante M, Salamini F (2003) From plant genomics to breeding practice. Curr Opin Biotechnol 14:214–219CrossRefPubMedGoogle Scholar
  25. Neigenfind J, Gyetvai G, Basekow R, Diehl S, Achenbach U, Gebhardt C, Selbig J, Kersten B (2008) Haplotype inference from unphased SNP data in heterozygous polyploids based on SAT. BMC Genomics 9:356CrossRefPubMedGoogle Scholar
  26. Oberacher H, Parson W, Hölzl G, Oefner PJ, Huber CG (2004) Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry. J Am Soc Mass Spectrom 15:1897–1906CrossRefPubMedGoogle Scholar
  27. Pineda O, Bonierbale MW, Plaisted RL, Brodie BB, Tanksley SD (1993) Identification of RFLP markers linked to the HI gene conferring resistance to the potato cyst nematode Globodera rostochiensis. Genome 36:152–156CrossRefPubMedGoogle Scholar
  28. Poulson MD, Wittwer CT (2007) Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis. Biotechniques 43:87–91CrossRefPubMedGoogle Scholar
  29. Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597–608CrossRefPubMedGoogle Scholar
  30. Rickert AM, Premstaller A, Gebhardt C, Oefner PJ (2002) Genotyping of Snps in a polyploidy genome by pyrosequencing. Biotechniques 32:592–598PubMedGoogle Scholar
  31. Rickert AM, Kim JH, Meyer S, Nagel A, Ballvora A, Oefner PJ, Gebhardt C (2003) First generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome. Plant Biotechnol J 1:399–410CrossRefPubMedGoogle Scholar
  32. Rickert AM, Ballvora A, Matzner U, Klemm M, Gebhardt C (2005) Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays. Biotechnol Appl Biochem 42:93–96CrossRefPubMedGoogle Scholar
  33. SantaLucia J Jr (2007) Physical principles and visual-OMP software for optimal PCR design. Methods Mol Biol 402:3–34CrossRefPubMedGoogle Scholar
  34. Schlueter SD, Dong Q, Brendel V (2003) GeneSeqer@PlantGDB: gene structure prediction in plant genomes. Nucl Acids Res 31:3597–3600CrossRefPubMedGoogle Scholar
  35. Seipp MT, Durtschi JD, Liew MA, Williams J, Damjanovich K, Pont-Kingdon G, Lyon E, Voelkerding KV, Wittwer CT (2007) Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting. J Mol Diagn 9:284–289CrossRefPubMedGoogle Scholar
  36. Seipp MT, Pattison D, Durtschi JD, Jama M, Voelkerding KV, Wittwer CT (2008) Quadruplex genotyping of F5, F2, and MTHFR variants in a single closed tube by high-resolution amplicon melting. Clin Chem 54:108–115CrossRefPubMedGoogle Scholar
  37. Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM, Geigenberger P (2002) Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14:2191–2213CrossRefPubMedGoogle Scholar
  38. Unneberg P, Stromberg M, Sterky F (2005) SNP discovery using advanced algorithms and neural networks. Bioinformatics 21:2528–2530CrossRefPubMedGoogle Scholar
  39. Usuka J, Zhu W, Brendel V (2000) Optimal spliced alignment of homologous cDNA to a genomic DNA template. Bioinformatics 16:203–211CrossRefPubMedGoogle Scholar
  40. Van Ooijen JW (2006) JoinMap® 4. Software for the calculation of genetic linkage maps in experimental populations. Kyazma B.V., WageningenGoogle Scholar
  41. Wu S-B, Wirthensohn M, Hunt P, Gibson J, Sedgley M (2008) High resolution melting analysis of almond SNPs derived from ESTs. Theor Appl Genet 118:1–14CrossRefPubMedGoogle Scholar
  42. Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT (2004) Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem 50:1328–1335CrossRefPubMedGoogle Scholar
  43. Zhou L, Wang L, Palais R, Pryor R, Wittwer CT (2005) High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51:1770–1777CrossRefPubMedGoogle Scholar
  44. Zhou L, Errigo RJ, Lu H, Poritz MA, Seipp MT, Wittwer CT (2008) Snapback primer genotyping with saturating DNA dye and melting analysis. Clin Chem 54:1648–1656CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • David De Koeyer
    • 1
    Email author
  • Katheryn Douglass
    • 1
  • Agnes Murphy
    • 1
  • Sean Whitney
    • 1
  • Lana Nolan
    • 1
    • 2
  • Yong Song
    • 1
    • 3
  • Walter De Jong
    • 4
  1. 1.Potato Research CentreAgriculture and Agri-Food CanadaFrederictonCanada
  2. 2.Faculty of Forestry and Environmental ManagementUniversity of New BrunswickFrederictonCanada
  3. 3.Hunan Provincial Key Laboratory of Crop Germplasm Innovation and UtilizationHunan Agricultural UniversityChangshaPeoples Republic of China
  4. 4.Department of Plant Breeding and GeneticsCornell UniversityIthacaUSA

Personalised recommendations