Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton

Abstract

Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.

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Abbreviations

Bt:

Bacillus thuringiensis

HRMA:

high-resolution melting curve analysis

References

  1. Bernard PS, Ajioka RS, Kushner JP and Wittwer CT 1998 Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am. J. Pathol. 153 1055–1061

    PubMed  Article  CAS  Google Scholar 

  2. Chagné D, Gasic K, Crowhurst RN, Han Y, Bassett HC, Bowatte DR, Lawrence TJ, Rikkerink EHA, Gardiner SE and Korban SS 2008 Development of a set of SNP markers present in expressed genes of the apple. Genomics 92 353–358

    PubMed  Article  Google Scholar 

  3. Croxford AE, Rogers T, Caligari PDS and 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. 180 594–607

    PubMed  Article  CAS  Google Scholar 

  4. Dabo MS, Mitchell DE and Melcher U 1993 A method for isolation of nuclear DNA from cotton (Gossypium) leaves. Anal. Biochem. 210 34–38

    PubMed  Article  CAS  Google Scholar 

  5. Horn EC, Kumpatla SP, Patterson KA, Gupta M and Thompson SA 2004 Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity. Plant Mol. Biol. Reptr. 22 83a–83i

    Article  Google Scholar 

  6. Lehmensiek A, Sutherland MW and McNamara RB 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. 117 721–728

    PubMed  Article  CAS  Google Scholar 

  7. Loomis WD 1974 Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 31 528–544

    PubMed  Article  CAS  Google Scholar 

  8. Mader E, Lukas B and Novak J 2008 A Strategy to setup codominant microsatellite analysis for high resolution melting curve analysis. BMC Genet. 9 69–77

    PubMed  Article  Google Scholar 

  9. Paterson HA, Brubaker, LC and Wendel FJ 1993 A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol. Biol. Reptr. 11 122–127

    Article  CAS  Google Scholar 

  10. Porebski SL, Baily G, Baum RB 1997 Modification of CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Reptr. 12 8–15

    Article  Google Scholar 

  11. Ririe KM, Rasmussen RP and Witttwer CT 1997 Product differentiation by analysis of melting curves during the polymerase chain reaction. Anal. Biochem. 245 154–160

    PubMed  Article  CAS  Google Scholar 

  12. Vossen HAM R, Aten E, Roos A and Dunnen JTD 2009 High-resolution melting analysis (HRMA)—more than just sequence variant screening. Hum. Mut. 30 860–866

    PubMed  Article  CAS  Google Scholar 

  13. Wittwer CT, Reed GH, Gundry, CN, Vandersteen JG and Pryor RJ 2003 High-resolution genotyping by amplicon melting analysis using LC Green. Clin. Chem. 49 853–60

    PubMed  Article  CAS  Google Scholar 

  14. Wu S, Franks TK, Hunt P, Wirthensohn MG, Gibson JP and Sedgley M 2010 Discrimination of SNP genotypes associated with complex haplotypes by high resolution melting analysis in almond: implications for improved marker efficiencies. Mol. Breed. 25 351–357

    Article  CAS  Google Scholar 

  15. Yashitola J, Thirumurugan T, Sundaram RM, Naseerullah MK, Ramesha MS, Sarma NP and Sonti RV 2002 Assessment of purity of rice hybrids using microsatellite and STS markers. Crop Sci. 42 1369–1373

    Article  CAS  Google Scholar 

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Acknowledgements

The first author acknowledges the financial assistance of Department of Biotechnology (DBT), India, through its post-doctoral program.

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Correspondence to Ramesh V Sonti.

Additional information

MS received 12 March 2012; accepted 10 October 2012

Corresponding editor: Utpal Nath

[Rao PS Kumar PS and Sonti RV 2013 Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton. J. Biosci. 38 1–6] DOI10.1007/s12038-012-9285-4

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Rao, P.S., Kumar, P.S. & Sonti, R.V. Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton. J Biosci 38, 167–172 (2013). https://doi.org/10.1007/s12038-012-9285-4

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Keywords

  • Cotton
  • cry1Ac
  • genomic DNA isolation
  • high-resolution melting curve analysis
  • radicle tip
  • seed purity testing