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DNA Phosphorothioate (PT), replacing a non-bridging phosphate oxygen atom with a sulfur atom, is one kind of common DNA modification in bacteria. Whole genome scale description of the location and frequency of PT modification is the key to understand its biological function. Herein we developed a novel method, named with iodine-induced cleavage quantitative real-time PCR (IC-qPCR), to evaluate the frequency of PT modification at a given site in bacterial DNA. The efficiency, dynamic range, sensitivity, reproducibility and accuracy of IC-qPCR were well tested and verified employing an E. coli B7A strain as example. The amplification efficiency of IC-qPCR assay ranged from 91% to 99% with a high correlation coefficient ≥0.99. The limit of quantification was determined as low as 10 copies per reaction for the 607710 and 1818096 sites, and 5 copies for the 302695 and 4120753 sites. Based on the developed IC-qPCR method, the modification frequency of four PTs in E. coli B7A was determined with high accuracy, and the results showed that the PT modification was partial and that the modification frequency varied among investigated PT sites. All these results showed that IC-qPCR was suitable for evaluating the PT modification, which would be helpful to further understand the biological function of PT modification.

With the large-scale release of genetically modified (GM) crops, there are ecological concerns on transgene movement from GM crops to non-GM counterparts and wild relatives. In this research, we conducted greenhouse experiments to measure pollen-mediated gene flow (PGF) in the absence and presence of pollinators (Bombus ignitus, Apis mellifera and Pieris rapae) in one GM cotton (resistant to the insect Helicoverpa armigera and the herbicide glyphosate) and two non-GM lines (Shiyuan321 and Hai7124) during 2012 and 2013. Our results revealed that: (1) PGF varied depending on the pollinator species and was highest with B. ignitus (10.83%) and lowest with P. rapae (2.71%); (2) PGF with B. ignitus depended on the distance between GM and non-GM cottons; (3) total PGF to Shiyuan321 (8.61%) was higher than to Hai7124 (4.10%). To confirm gene flow, we tested hybrids carrying transgenes for their resistance to glyphosate and H. armigera and most hybrids showed strong resistance to the herbicide and insect. Our research confirmed that PGF depended on pollinator species, distance between plants and the receptor plant.