Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

Yi, Cheng; Zhang, Jun; Chan, Ka Man; Liu, Xiao Kun; Hong, Yan

doi:10.1016/j.ab.2007.11.022

Cited by 37 publications

(30 citation statements)

References 8 publications

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“…We followed the real-time qPCR (qRT-PCR) methodology 38 and calculations 39 to estimate the copy number of the PHYA1 RNAi vector integrated into the transformed cotton genomes. We cloned the GhUBC1 fragment into the plasmid vector pCR4 TOPO-TA following the manufacturer's protocol and instructions (Invitrogen), which contains nptII gene as a selectable marker.…”

Section: Methodsmentioning

confidence: 99%

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

et al. 2014

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Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to B70% for the PHYA1 transcript, and compensatory overexpression of up to B20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

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Section: Methodsmentioning

confidence: 99%

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

et al. 2014

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“…To date, qPCR technology has been applied to analyze copy number of transgenic plants, including soybean and peanut (Schmidt and Parrott, 2001), tomato (Mason et al, 2002), maize (Ingham et al, 2001;Shou et al, 2004;Song et al, 2002), rapeseed (Weng et al, 2004), wheat (Doshi et al, 2007;Li et al, 2004), rice (Jiang et al, 2009;Yang et al, 2005), tobacco (Subr et al, 2006), cotton (Yi et al, 2008), citrus (Omar et al, 2008), grape (Costa et al, 2009) and cassava (Beltran et al, 2009). To speed up the molecular analysis of transgenic plants, we describe here the development and application of a qPCR method that utilizes an endogenous calibrator and the threshold crossing point (Ct) calculated by the Applied Biosystem 7500 system for determination of transgene copy numbers in L. fendleri.…”

Section: Introductionmentioning

confidence: 99%

Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in <i>Lesquerella fendleri</i>

Chen¹

2010

American Journal of Agricultural and Biological Sciences

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Problem statement:In transgenic plants, the number of transgene copies could greatly influence the level of expression and genetic stability of the target gene, thus it is important to develop an efficient method for accurate estimation of transgene copies. The quantitative Polymerase Chain Reaction (qPCR) technique is becoming more efficient nowadays to determine copy numbers of transgenes in transgenic plants, being used here, for the first time in quantifying copy numbers of transgenes in Lesquerella fendleri. Approach: The system utilized a known one copy gene, LfKCS4/5, from L. fendleri as an endogenous calibrator and the threshold Crossing point (Ct) measured by Applied Biosystem 7500 system to calculate the copy numbers of transgenes in primary transgenic lines (T0 generation). Results: The qPCR condition was optimized and each primer set had a PCR efficiency of 0.99 or 1.01. Our data demonstrated unambiguous 2-fold discrimination of the copy number of ß-glucuronidase gene (gusA) and hygromycine phosphotransferase II (hptII) genes in 12 T0 lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7%) showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of the Transferred (T)-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed based on main models of T-DNA integration in the plant genome. Conclusion: The qPCR described in this study is an efficient method and it is particularly useful in identification and selection of transgenic plants with desirable copy numbers at early stage.

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“…Transgenic plants with one or two transgene copies are generally preferred for stable and high-level expression of the transgene (Yi et al, 2008). The Southern blotting method is the most commonly used for copy number determination but is time-consuming and labor-intensive.…”

Section: Discussionmentioning

confidence: 99%

“…Southern blotting hybridization is the most common method for copy number determination, but it is time-consuming and labor-intensive. Some investigators have attempted to simplify the method for copy number determination using real-time polymerase chain reaction (PCR) (Yang et al, 2005;Yi et al, 2008). This is the most accurate method to determine transgene copy number.…”

Section: Introductionmentioning

confidence: 99%

Rapid determination of transgene copy number in tobacco by competitive PCR using a pair of SSR primers

Wang²,

Xi³

et al. 2010

Genet. Mol. Res.

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ABSTRACT. We developed a straightforward, rapid, and inexpensive method to determine transgene copy number in tobacco. The plasmid (pSSRCopy) used for tobacco transformation contains a simple sequence repeat (SSR) locus, PT1199, which was partially deleted in the middle, a homogenous SSR locus in tobacco K326. A 168-bp segment of the cloned PT1199 was shortened to 95 bp by deleting a 73-bp internal fragment. Using a pair of SSR primers, competitive PCR was amplified from genomic DNA from transgenic tobacco harboring pSSRCopy, and the two expected bands were found. The 168-bp band (SSR-168) corresponds to endogenous PT1199 and the 95-bp band (SSR-95) comes from the integrated pSSRCopy. A single copy of a transgene can be easily distinguished from multiple copies by comparing band densities.

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Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

Cited by 37 publications

References 8 publications

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in <i>Lesquerella fendleri</i>

Rapid determination of transgene copy number in tobacco by competitive PCR using a pair of SSR primers

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