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| Establishment of Taqman Quantitative PCR System to Estimate Copy Numbers of Exogenous Transgene in Genome Edited Tomato |
| REN Shuang, ZHU Hong-liang |
| College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China |
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Abstract Recently, CRISPR/Cas9 genome editing technology has been broadly utilized to crop breeding. Genome-edited but transgene-free plants can be segregated away from T0 transgenic plants by genetic separation, which will eliminate the risk of transgenic safety. Copy numbers of transgene which has been integrated into the transformed plant genome is a critical factor affecting the genetic separation of the offspring. Copy numbers are currently obtained by Southern blot analysis, but this method is complicated and requires relatively large amounts of plant materials. As to avoid these shortcomings, Real-time Fluorescent Quantitative Polymerase Chain Reaction provides a new solution. The endogenous ascorbate peroxidase (APX) is selected as reference gene. The exogenous hygromycin phosphortransferase (HPT) is selected as target gene. With the Taqman RT-PCR conditions, one copy was significantly estimated in the 12 genome edited tomatos of slyPDS (Phytoene desaturase). Initially, a sensitive and efficient detection system is developed for estimating copy number in genome edited tomato, which laid a foundation for the rapid and reliable screening of improved crops.
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Received: 13 April 2017
Published: 25 October 2017
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