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Real-time PCR for the detection of precise transgene copy number in durum wheat

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Abstract

Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.

This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

Abbreviations

FISH:

fluorescence in situ hybridization

GM:

genetically modified

HMW-GS:

high molecular weight glutenin subunits

MGB:

minor groove binder

PCR:

polymerase chain reaction

SDS:

Sequence Detection System

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Correspondence to Agata Gadaleta.

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Key words

  • Durum wheat
  • Transgenic plants
  • Biolistic method
  • Glutenin subunits
  • Real-time PCR
  • Southern analysis
  • Transgene copy number
  • Quantitative PCR assay
  • Transgene stable expression
  • HMW-GS genes