Whole-genome Sequence Analysis to Confirm the Absence of Transgene in a Rice Line Made by Gene Targeting

Abstract

The recent development of homologous recombination-mediated gene targeting (GT) techniques has made it easy to modify nucleotide sequences in plant genomes. Backcross breeding following GT provides transgene-free plant lines with high probability. However, owing to the possibility of unintentional transgene introduction during genetic transformation, analytical methods may be necessary to reliably and comprehensively detect transgenes remaining on a plant genome. Herein, we conducted an analysis based on whole-genome sequencing. We analyzed the GT rice DNA using a next-generation sequencer, and we performed data analysis to find the transgene integration on the genome. All results supported the absence of transgenes in GT rice. Our genome integrity evaluation method could also be applicable to plant lines produced using other genome-editing technologies such as CRISPR/Cas9.