Use of real-time PCR for determining copy number and zygosity in transgenic plants

Bubner, Ben; Baldwin, Ian Thomas

doi:10.1007/s00299-004-0859-y

Cited by 208 publications

(172 citation statements)

References 40 publications

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“…We adapted the comparative method with double-dye oligonucleotides (TaqMan probes, Applied Biosystems, Foster City, CA, USA) of Bubner and Baldwin. 8 We found no evidence for hemizygosity in our sample.…”

mentioning

confidence: 49%

Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis

Kéri

Kiss

Kelemen³

2009

Mol Psychiatry

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mentioning

confidence: 49%

Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis

Kéri

Kiss

Kelemen³

2009

Mol Psychiatry

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“…The wild type (DJ or HJ) was used as the recipient for Agrobacterium-mediated transformation as described previously to generate the transgenic rice (Hiei et al, 1994). The T-DNA copy numbers of relative transgenic rice plants were confirmed by RT-qPCR described previously (Bubner and Baldwin, 2004;Yang et al, 2005), and the results are presented in the Supplemental Table 3. Homozygous T3 or T4 plants were taken for the following field test.…”

Section: Generation Of Transgene Constructs and Plant Transformationmentioning

confidence: 99%

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

et al. 2018

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Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

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“…Southern hybridization is laborious, takes a lot of time and requires a variety of equipment. When quantitative PCR (PCR-RT) is used to solve the task, the results may be inadequate if the necessary conditions are not met during the test and subsequent statistical processing of the data [15,18,19]. However, with appropriate execution, this method is highly technological and allows for a short time to accumulate a large array of information.…”

Section: Introductionmentioning

confidence: 99%

“…The determination of genotypes КК and Kk by PCR-RT is reduced to the problem of discrimination of two copies of a gene in the genome from one [8,15,16]. It can be solved by quantitative PCR [8,17], as well as by hybridization of nucleic acids [15,18,19].…”

Section: Introductionmentioning

confidence: 99%

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MOLECULAR GENOTYPING OF CHICKEN (Gallus gallus L.) FEATHERING GENES IN CONNECTION WITH SEPARATION BY SEX

Alekseev¹,

Borodin²,

Nikulin³

et al. 2017

S-h. biol.

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A b s t r a c tTraditional breeding is time and material consuming. Modern laboratory techniques significantly speed up and reduce the costs for breeding animals with the desired properties. A test based on the quantitative real-time PCR (qPCR) technology was developed to distinguish between homozygous and heterozygous state of the genes from alleles K and k which are responsible for the rate of wing feather growth in day-old chicks. The use of quantitative real-time PCR for the analysis of genotypes is aimed at the discrimination between one and two copies of the target gene in a genome. To obtain reliable results, certain rules must be followed when conducting the assay: the efficiency of the PCR should be close to the maximum; it is possible to obtain a significant number of false results without the appropriate statistical analysis. A new assay algorithm was proposed to overcome the limitations of qPCR: all samples are subjected to two successive independent analyses in parallel with the reference samples of both genotypes; if the two runs produce divergent results then the assay is repeated and the previous results are discarded. This approach allows to reduce assay error probability down to zero. The new system consists of three (instead of four) primers for amplification of two genes and two probes, allowing efficient analysis of various allele K genotypes. Quantitative realtime PCR data analysis was performed by ΔΔCt method using the statistics software package SPSS for ROC analysis. Using the method developed, the percentage of KK, Kk and kk genotypes was determined in 145 cocks of original lines B5, B6, B7 and B9 of domestic meat chicken of cross Smena 8. It was shown that 19 cocks of line B5 and 15 cocks of line B6 had kk genotype. From the 46 cocks of line B7, none had kk genotype, 17 cocks (37 %) had Kk genotype, and 29 cocks (63 %) had KK genotype. From the 65 cocks of line B9, none had kk genotype, 17 cocks (26 %) had Kk genotype, and 48 cocks (74 %) had KK genotype. Analyzed fragments were sequenced to exclude the effects of possible nucleotide sequence variability on the assay. The sequences did not contain any nucleotide substitutions in the sites of the primers and probes annealing. The data obtained will accelerate selection of new domestic meat chicken breeds with possibility of sexing based on feather length in day-old chicken. Further breeding work involves the assessment of the offspring using traditional and molecular genetic methods.

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Use of real-time PCR for determining copy number and zygosity in transgenic plants

Cited by 208 publications

References 40 publications

Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis

Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

MOLECULAR GENOTYPING OF CHICKEN (Gallus gallus L.) FEATHERING GENES IN CONNECTION WITH SEPARATION BY SEX

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