Comprehensive molecular characterization and biosafety assessment of TaDOF7.6 transgenic and gene-edited wheat

Abstract

Wheat is one of the major staple food crops worldwide, and stable wheat yield is crucial for ensuring global food security. Transgenic and gene-editing technologies serve as important approaches for wheat genetic improvement, yet further application of related materials requires systematic evaluation of their molecular characteristics, genetic stability and environmental biosafety. In this study, TaDOF7.6 -RNAi transgenic wheat and TaDOF7.6 -CRISPR/Cas9 gene-edited wheat previously constructed in our laboratory were used as experimental materials to evaluate their yield-related traits, molecular profiles and partial biosafety indicators. Both RNAi-mediated silencing and CRISPR/Cas9-mediated knockout of TaDOF7.6 significantly improved wheat grain traits and yield-related agronomic traits, further confirming that TaDOF7.6 acts as a negative regulator in wheat yield regulation. For TaDOF7.6 -RNAi transgenic wheat, the integration site of T-DNA on wheat chromosome 3B was identified via thermal asymmetric interlaced PCR (TAIL-PCR). Vector-derived fragments including Hyg, GUS and dsRNA were consistently detectable across T3 to T5 generations. Moreover, these RNAi lines maintained stable histochemical GUS activity and sustained silencing efficiency of TaDOF7.6 . No significant differences were observed in seed germination, seedling emergence and pollen-related traits between RNAi-25, RNAi-31 lines and wild-type wheat. Within the sampling scope and conventional PCR detection conditions adopted in this research, no target exogenous gene fragments were detected in 24 surrounding plant species. Additionally, there were no obvious disparities in field insect community and weed distribution between transgenic lines and wild-type controls. For TaDOF7.6 -CRISPR/Cas9 gene-edited wheat, the insertion locus of exogenous vector on wheat chromosome 4D was successfully mapped. Transgene-free homozygous mutants were acquired through Hi-TOM high-throughput sequencing, Sanger sequencing and progeny screening. No unintended sequence variations were detected at all predicted off-target sites compared with wild-type plants. Meanwhile, the gene-edited lines exhibited consistent performance with wild-type wheat in seed germination, seedling emergence and pollen-associated phenotypes. This study provides fundamental data for molecular characterization and comprehensive biosafety assessment of TaDOF7.6 -RNAi transgenic wheat and TaDOF7.6 -CRISPR/Cas9 gene-edited wheat, and verifies the application potential of TaDOF7.6 as a promising target gene for wheat yield improvement. The transgene-free gene-edited mutants obtained in this work also supply elite new germplasm resources for subsequent wheat molecular breeding programs.