With the wide application of genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model s...With the wide application of genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model system, we developed a non-invasive method to accurately identify genome-edited mutants by using DNA from insect exuviae. We compared the quantity and quality of genomic DNA from exuviae in five instar hoppers and found that the 1st instar exuviae had the highest DNA yield and content, while the 3rd instar exuviae had the best quality. Consensus genotypes were identified from genomic DNA of hoppers at different developmental stages in the same individuals. Moreover, we demonstrated that the amplification products from DNA extracted from locust exuviae are the consensus sequences with those from the hemolymph and foreleg pre-tarsus. Therefore, non-invasive samples provide the same genotyping results as minimally invasive and invasive samples of the same individuals. Furthermore, this identification method that uses genomic DNA from exuviae can be used for early screening of positive genome-edited individuals in each generation for adult crossing. In our study, the non-invasive identification method was not only simpler and provided results earlier than existing methods, but also had a better reproducibility and accuracy. This non-invasive identification approach using genomic DNA from exuviae can be adapted to meet the growing demand for genetic analysis and will find wide application in insect genome editing research.展开更多
基金This research was supported by the National Natural Science Foundation of China(Grant No.31702064)Natural Science Foundation of Hebei Province(Grant No.C2018201135)+5 种基金Youth Foundation of Hebei Educational Committee(Grant No.QN2019117)The State Key Laboratory of Integrated Management of Pest Insects and Rodents(Grant No.ChineseIPM1709)Natural Science Interdisciplinary Research Program of Hebei University(Grant No.DXK201910)Advanced Talents Incubation Program of Hebei University(Grant No.050001-521000981003)“Giant Plan”of Hebei Province(Grant No.070006-702800116049,070006-702800117003)Post-graduate’s Innovation Fund Project of Hebei Province(Grant No.CXZZSS2019004).
文摘With the wide application of genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model system, we developed a non-invasive method to accurately identify genome-edited mutants by using DNA from insect exuviae. We compared the quantity and quality of genomic DNA from exuviae in five instar hoppers and found that the 1st instar exuviae had the highest DNA yield and content, while the 3rd instar exuviae had the best quality. Consensus genotypes were identified from genomic DNA of hoppers at different developmental stages in the same individuals. Moreover, we demonstrated that the amplification products from DNA extracted from locust exuviae are the consensus sequences with those from the hemolymph and foreleg pre-tarsus. Therefore, non-invasive samples provide the same genotyping results as minimally invasive and invasive samples of the same individuals. Furthermore, this identification method that uses genomic DNA from exuviae can be used for early screening of positive genome-edited individuals in each generation for adult crossing. In our study, the non-invasive identification method was not only simpler and provided results earlier than existing methods, but also had a better reproducibility and accuracy. This non-invasive identification approach using genomic DNA from exuviae can be adapted to meet the growing demand for genetic analysis and will find wide application in insect genome editing research.
