利用CRISPR/Cas9技术创制糯小麦新种质OA

Jimai 44 is a new green strong-gluten wheat variety developed by the Crop Institute of Shan-dong Academy of Agricultural Sciences in recent years,which exhibits excellent traits such as strong gluten,high yield,wide adaptability,disease resistance and stress resistance.Jizimai 5 is a new purple wheat variety independently developed by the Crop Institute of Shandong Academy of Agricultural Sciences.In this study,a multi-target editing system based on the CRISPR/Cas9 system and polycistronic tRNA self-cleavage system were used to perform targeted editing of the waxy gene in Jimai 44 and Jizimai 5.Two specific sgRNA target sequences were selected in the coding regions of the three homologous genes waxy-7A,waxy-4A and waxy-7D,respectively.The gene editing vector pLGYE03-waxy was constructed and then introduced into wheat via the Agrobacterium-mediated method.Hi-TOM sequencing analysis showed that five homozygous mutant lines at the target sites were obtained for each variety at the T0 generation.The results of staining assays revealed that the edited lines of both varieties displayed waxy endosperm,thus forming new waxy wheat germplasms.This study not only enriched the germplasm resources of waxy wheat,but also provided a new idea for the targeted improve-ment of wheat varieties,as well as a new approach for waxy wheat breeding.