Advertisement

Theoretical and Applied Genetics

, Volume 107, Issue 8, pp 1384–1390 | Cite as

A fluorogenic 5′ nuclease (TaqMan) assay to assess dosage of a marker tightly linked to red skin color in autotetraploid potato

Article

Abstract

We have recently identified an allele of dihydroflavonol 4-reductase (dfr) that cosegregates with the ability of potato (Solanum tuberosum L) to produce red pelargonidin-based anthocyanin pigments. A rapid assay to assess dosage of this allele in cultivated potato, an autotetraploid, would be useful for breeding programs that develop red-skinned cultivars. To identify regions of dfr that are conserved between alleles, as well as regions that are variable, a portion of the gene was sequenced from several cultivated and wild potato clones. In one region the sequence of the 'red' dfr allele differed at two nucleotide positions from the three other sequence classes observed. A fluorogenic oligonucleotide probe labeled with 6-FAM was designed to anneal specifically to the red allele in this region, while a second probe labeled with VIC was designed to anneal to the 'not-red' dfr alleles. PCR primers that annealed to conserved sequences flanking the variable region were also developed. When subjected to a fluorogenic 5′ nuclease (TaqMan) allelic discrimination assay all diploid clones tested clustered into three distinct groups based on the relative amounts of FAM and VIC released. These three groups represented clones homozygous for the red allele, heterozygous for the red allele, and homozygous for the not-red allele(s). When tetraploid clones were tested they separated into five distinct clusters, three of which were shared with diploid clones. The five clusters were interpreted to represent clones quadruplex, triplex, duplex, simplex and nulliplex for the red dfr allele. This interpretation was supported by monitoring the segregation of red allele dosage in several tetraploid crosses. To the best of our knowledge this is the first report of a fluorogenic 5′ nuclease assay being used for allelic discrimination in an autopolyploid.

Keywords

Potato Cultivar Potato Clone Genotypic Classis Allelic Discrimination Assay Double Reduction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We thank S. Kresovich for suggesting the 5′ nuclease assay, H. De Jong for providing diploid clones and for reviewing the manuscript, K. Paddock for assistance in maintaining potato clones, and the US Potato Genebank (NRSP-6) for providing accessions of cultivated Solanum spp. This work was supported in part by Federal Hatch Funds and a grant from the Empire State Potato Growers, Incorporated.

References

  1. Bracho MA, Moya A, Barrio E (1998) Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity. J Gen Virol 79:2921–2928PubMedGoogle Scholar
  2. De Jong H (1987) Inheritance of pigmented tuber flesh in cultivated diploid potatoes. Am Potato J 64:337–343Google Scholar
  3. De Jong H, Burns VJ (1993) Inheritance of tuber shape in cultivated diploid potatoes. Am Potato J 70:267–283Google Scholar
  4. De Jong WS, De Jong DM, De Jong H, Kalazich J, Bodis M (2003) An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.). Theor Appl Genet (in press)Google Scholar
  5. Dodds KS, Long DH (1955) The inheritance of colour in diploid potatoes. I. Types of anthocyanidins and their genetic loci. J Genet 53:136–149Google Scholar
  6. Dodds KS, Long DH (1956) The inheritance of colour in diploid potatoes. II. Three-factor linkage group. J Genet 54:27–41Google Scholar
  7. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349PubMedGoogle Scholar
  8. Flipse E, Keetels CJAM, Jacobsen E, Visser RGF (1996) The dosage effect of the wild-type GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch. Theor Appl Genet 92:121–127CrossRefGoogle Scholar
  9. Hollick JB, Patterson GI, Asmundsson IM, Chandler VL (2000) Paramutation alters regulatory control of the maize pl1 locus. Genetics 154:1827–1838PubMedGoogle Scholar
  10. Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410PubMedGoogle Scholar
  11. Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000) 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 2000:655–661CrossRefGoogle Scholar
  12. Livak KJ (1999) Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Gen Anal: Biomol Eng 14:143–149Google Scholar
  13. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362PubMedGoogle Scholar
  14. Meksem K, Ruben E, Hyten DL, Schmidt ME, Lightfoot DA (2001) High-throughput genotyping for a polymorphism linked to soybean cyst nematode resistance gene Rhg4 by using Taqman probes. Mol Breed 7:63–71CrossRefGoogle Scholar
  15. Mendoza HA, Mihovilovich EJ, Saguma F (1996) Identification of triplex (YYYy) potato virus Y (PVY) immune progenitors derived from Solanum tuberosum ssp. andigena. Am Potato J 73:13–19Google Scholar
  16. Patterson GI, Thorpe CJ, Chandler VL (1993) Paramutation, an allelic interaction, is associated with a stable and heritable reduction of transcription of the maize b regulatory gene. Genetics 135:881–894PubMedGoogle Scholar
  17. Rickert AM, Premstaller A, Gebhardt C, Oefner PJ (2002) Genotyping of SNPs in a polyploid genome by pyrosequencing (TM). Biotechniques 32:592–603PubMedGoogle Scholar
  18. Salvi S, Tuberosa R, Phillips RL (2001) Development of PCR-based assays for allelic discrimination in maize by using the 5′ nuclease procedure. Mol Breed 8:169–176CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Department of Plant BreedingCornell UniversityIthacaUSA

Personalised recommendations