Zheng Wang
Guangming He
Ligeng Ma
Xing Wang Deng
aPeking University Institute of Advanced Agricultural Sciences, Weifang, Shandong, 261325, China
bState Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Advanced Agriculture Sciences and School of Life Sciences, Peking University, Beijing, 100871, China
cCollege of Life Sciences, Capital Normal University, Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement, Beijing, 100048, China
More InformationCorresponding author: E-mail address: ligeng.ma@cnu.edu.cn (Ligeng Ma);E-mail address: deng@pku.edu.cn (Xing Wang Deng)
Publish Date:2020-05-25
Abstract
Abstract
Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that theTaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.Keywords: Wheat,
CRISPR/Cas9,
Male sterility
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