Development of multiplex PCR system for identification of glyphosate-tolerant sugar beet
Journal Title: Plant varieties studying and protection - Year 2016, Vol 0, Issue 4
Abstract
Purpose. To create a multiplex system for identification glyphosate-tolerant sugar beet by using PCR. Methods. Molecular genetic analysis. Results. The article presents the results of studies to determine the parameters of the polymerase chain reaction (PCR) in order to develop a multiplex system for identification of the structural elements of the design of transgenic gene cp 4 epsps, which provides tolerance to glyphosate. For amplicon target DNA sequences, the following values of temperature conditions of PCR were determined: step 1 (initial denaturation) 95 °C – 3 min; step 2 (specific reaction products accumulation): denaturation 95 °C – 45 s; hybridization of primers 55 °C – 50 s; elongation 72 °C – 1 min; number of cycles – 40; step 3 (final elongation) 72 °C – 6 min. A series of PCR were carried out for the purpose of selecting the optimal amount of DNA matrix for efficient estimate of transgenic sugar beet plants for the presence of specific sequences. Conclusions. To identify transgenic glyphosate-tolerant sugar beet, it is advisable to determine 35S promoter and gene cp 4 epsps in individual genotypes. It was found that during the selection of temperature parameters of multiplex reaction a 5 °C rise in primer hybridization temperature did not affect the identification of gene als that allowed to include specific primers for determination of this sequence as an internal control. Based on the results of test multiplex reactions, concentrations of dNTPs and Mg2+ ions were determined that allowed to exclude the possibility of non-specific fragments and false-negative results. The optimum amount of matrix DNA (100–150 ng) for an efficient estimate of transgenic sugar beet plants for the presence of specific sequences was determined. Obtained results allowed to develop a multiplex test system for identification of transgenic glyphosate-tolerant sugar beet which can be used for simultaneous determination of the 35S promoter, cp 4 epsps gene and als gene as an internal reaction control.
Authors and Affiliations
Larysa Prysiazhniuk, Yuliia Shytikova, Olexandr Volchkov
Keywords
Related Articles
- EP ID EP257658
- DOI 10.21498/2518-1017.4(33).2016.88686
- Views 111
- Downloads 0
Seed quality of winter wheat varieties after black fallow depending on organo-mineral fertilizer application in the Left-Bank Forest-Steppe zone of Ukraine
Purpose. To determine the effect of the basic organo-mineral fertilizer on the formation and stability of grain quality of winter wheat varieties in multiple crop rotation after black fallow as a predecessor. Methods. F...
Adaptive capacity of different potato varieties under the conditions of the Right-Bank Polissia of Ukraine
Purpose. To study the adaptive ability and to determine the main criteria for adaptability assessment of potato varieties selected at the Institute for Potato Research of the National Academy of Agricultural Sciences un...
Cis-, intra-, subgenesis, genome editing as modern technologies for modifying the crop genomes (review)
Purpose. Reviewing the literature on modern technologies of genetic modification of crop genomes. Results. The current state of genetically modified plants creation is analyzed. The information on cis-, intra- and subgen...
Resistance of raspberry promising varieties (Rubus idaeus L.) to main diseases of stem expression type
The article features summary of studying the resistance of rasberry promising varieties to main diseases (Didymella applanata Sacc., Gloeosporium venetum Speg. і Septoria rubi Sacc.) of stem expression type under the con...
Features of accumulation of inorganic elements in seeds of white mustard (Sinapis alba L.) and black mustard (Brassica nigra L.)
Purpose. To investigate special aspects of accumulation of inorganic elements including heavy metals in seeds of white and black mustard to be grown for obtaining drugs. Methods. Field experiments, microwave digestion,...