Establishment of a rapid assay for sequencing of carried DNA and edited sites in gene-editing tomato plants

Gene editing can modify gene function by inserting or deleting nucleotides in a specific region in a gene, which can alter the desired trait in the plant. In the selection process of gene-editing plants, many populations are created and need to be checked for the edited genes, but traditional gDNA extraction methods are laborious. Here, we used Biocube to extract gDNA and designed a specific primers to identify Cas9 in tomato [marker genes]. Biocubes are directly pressed into plant tissues to absorb and store DNA, and allow for quick and easy gDNA extraction. In this study, using template DNA obtained from tomato ( Solanum lycopersicum L.) leaves by the traditional gDNA and the Biocube extraction methods, we confirmed carried DNA and editing in gene-editing plants, and compared the results of the two methods. Template DNA was obtained from cvs. Micro-Tom and M82 by the two extraction methods. Cas9, a gene-specific primer, and rbcl , an internal control, were used in PCR to confirm the presence of carried DNA. To verify the carried DNA, four samples were selected and whole-genome sequencing was performed. High-resolution melting analysis was used to check whether the target gene sequence was edited. Editing was compared using the targeted deep-sequencing results. The results showed no differences between the two gDNA extraction methods. In conclusion, we established a rapid assay for carried DNA and edited sites in gene-editing tomato plants.