An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Li, F.; Gan, S.

doi:10.1515/sg-2011-0003

Abstract

Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with simple sequence repeats (SSRs) using an automated sequence analyser. However, the method has not been explored in terms of performance optimisation and cost control. In this paper, we optimised the protocol for fluorescent-dUTP based SSR genotyping in a case study with Eucalyptus. A combination of low dNTP concentration (25 μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescent intensity of SSR amplicons, thereby facilitating accurate and multiplexed scoring of SSR alleles. The usefulness of the optimised protocol was demonstrated in its application to genetic mapping of SSR loci onto E. urophylla and E. tereticornis linkage maps constructed previously. The protocol optimised here would provide a reliable and economical assay for sequencer-based SSR genotyping in a wide range of biological applications.

References

Agrama, H. A., T. L. George and A. F. Salah (2002): Construction of genome map for Eucalyptus camaldulensis Dehn. Silvae Genet. 51: 201–206.
Search in Google Scholar Back to article
Brondani, R. P. V., C. Brondani, R. Tarchini and D. Grattapaglia (1998): Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla. Theor. Appl. Genet. 97: 816–827.
Search in Google Scholar Back to article
Brondani, R. P. V., E. R. Williams, C. Brondani and D. Grattapaglia (2006): A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. BMC Plant Biol. 6: 20.10.1186/1471-2229-6-20
Search in Google Scholar Back to article
Busch, J. D., R. Benford, T. Pearson, E. Palmer, R. P. Balda and P. Keim (2009): Development of polymorphic tetranucleotide microsatellites for pinyon jays (Gymnorhinus yanocephalus). Conserv. Genet. 10: 689–691.
Search in Google Scholar Back to article
Doyle, J. J. and D. J. Doyle (1990): Isolation of plant DNA from fresh tissue. Focus 12: 13–15.
Search in Google Scholar Back to article
FAO (2000): Global forest resources assessment 2000 – main report. FAO, Rome.
Search in Google Scholar Back to article
Gan, S., J. Shi, M. Li, K. Wu, J. Wu and J. Bai (2003): Moderate-density molecular maps of Eucalyptus urophylla S. T. Blake and E. tereticornis Smith genomes based on RAPD markers. Genetica 118: 59–67.10.1023/A:1022966018079
Search in Google Scholar Back to article
Gilmore, S. and R. Peakall (2003): Isolation of microsatellite markers in Cannnabis sativa L. (marijuana). Mol. Ecol. Notes 3: 105–107.
Search in Google Scholar Back to article
Gion, J.-M., P. Rech, J. Grima-Pettenati, J. Verhaegen and C. Plomion (2000): Mapping candidate genes in Eucalyptus with emphasis on lignification genes. Mol. Breed. 6: 441–449.10.1023/A:1026552515218
Search in Google Scholar Back to article
Grattapaglia, D. and M. Kirst (2008): Eucalyptus applied genomics: from gene sequences to breeding tools. New Phytol. 179: 911–929.10.1111/j.1469-8137.2008.02503.x
Search in Google Scholar Back to article
Jones, R. C., D. A. Steane, B. M. Potts and R. E. Vaillancourt (2002): Microsatellite and morphological analysis of Eucalyptus globulus populations. Can. J. For. Res. 32: 59–66.
Search in Google Scholar Back to article
Klevytska, A. M., L. B. Price, J. M. Schupp, P. L. Worsham, J. Wong and P. Keim (2001): Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome. J. Clin. Microbiol. 39: 3179–3185.
Search in Google Scholar Back to article
Lincoln, S., M. Daly and E. Lander (1992): Constructing genetic maps with MAPMAKER/EXP 3.0. Whitehead Institute technical report, 3rd edition. Whitehead Institute, Cambridge, MA.
Search in Google Scholar Back to article
MacAvoy, E. S., L. M. McGibbon, J. P. Sainsbury, H. Lawrence, C. A. Wilson, C. H. Daugherty and G. K. Chambers (2007): Genetic variation in island populations of tuatara (Sphenodon spp) inferred from microsatellite markers. Conserv. Genet. 8: 305–318.
Search in Google Scholar Back to article
Magnusson, P. K. E., E. Wilander and U. Gyllensten (1996): Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products. BioTechniques 21: 844–847.10.2144/96215st01
Search in Google Scholar Back to article
Nagaraju, J., M. Kathirvel, R. R. Kumar, E. A. Siddiq and S. E. Hasnain (2002): Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescence-based ISSR-PCR and SSR markers. Proc. Natl. Acad. Sci. USA 99: 5836–5841.10.1073/pnas.042099099
Search in Google Scholar Back to article
Nguyen, H. T. and X. Wu (2005): Molecular marker systems for genetic mapping. In: The handbook of plant genome mapping: genetic and physical mapping, eds. K. Meksem and G. Kahl. WILEY-VCH Verlag GmbH & Co. KgaA, Weinheim, Germany. 23–52.10.1002/3527603514.ch2
Search in Google Scholar Back to article
Ottewell, K. M., S. C. Donnellan, G. F. Moran and D. C. Paton (2005): Multiplexed microsatellite markers for the genetic analysis of Eucalyptus leucoxylon (Myrtaceae) and their utility for ecological and breeding studies in other Eucalyptus species. J. Hered. 96: 445–451.10.1093/jhered/esi057
Search in Google Scholar Back to article
Pashley, C. H., J. R. Ellis, D. E. McCauley and J. M. Burke (2006): EST databases as a source for molecular markers: lessons from Helianthus. J. Hered. 97: 381–388.10.1093/jhered/esl013
Search in Google Scholar Back to article
Powell, W., G. C. Machray and J. Provan (1996): Polymorphisms revealed by simple sequence repeats. Trend. Plant Sci. 1: 215–222.10.1016/S1360-1385(96)86898-0
Search in Google Scholar Back to article
Schlötterer, C. (2004): The evolution of molecular markers-just a matter of fashion? Nat. Rev. Genet. 5: 63–69.
Search in Google Scholar Back to article
Schuelke, M. (2000): An economic method for the fluorescent labeling of PCR fragments. Nat. Biotech. 18: 233–234.
Search in Google Scholar Back to article
Tauz, D. (1989): Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 16: 6463–6471.10.1093/nar/17.16.64633183412780284
Search in Google Scholar Back to article
Thamarus, K., K. Groom, J. Murrell, M. Byrne and G. F. Moran (2002): A genetic linkage map for Eucalyptus globulus with candidate loci for wood, fibre and floral traits. Theor. Appl. Genet. 104: 379–387.
Search in Google Scholar Back to article
Williams, R. D. and J. A. Dewoody (2003): Fluorescent dUTP helps characterize 10 novel tetranucleotide microsatellites from an enriched salamander (Ambystoma texanum) genomic library. Mol. Ecol. Notes 4: 17–19.
Search in Google Scholar Back to article
Woolbright, S. A., S. P. DiFazio, T. Yin, G. D. Martinsen, X. Zhang, G. J. Allan, T. G. Whitham and P. Keim (2008): A dense linkage map of hybrid cottonwood (Populus fremontii × P. angustifolia) contributes to long-term ecological research and comparison mapping in a model forest tree. Heredity 100: 59–70.10.1038/sj.hdy.680106317895905
Search in Google Scholar Back to article
Yasodha, R., R. Sumathi, P. Chezhian, S. Kavitha and M. Ghosh (2008): Eucalyptus microsatellites mined in silico: survey and evaluation. J. Genet. 87: 21–25.10.1007/s12041-008-0003-918560170
Search in Google Scholar Back to article

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Abstract

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