Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System.

Hai Zhou,Shaoyan Zheng,Liya Zhu,Dagang Jiang,Jing Li,Chuxiong Zhuang,Bingran Zhao,Erdong Ni,Ming He,Zhifeng Huang,Liang Chen

doi:10.1038/srep37395

Abstract

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

Highlights

Time savings, better grain quality and higher yields, greater effectiveness and economical use of simpler procedures for breeding and hybrid seed production
The CRISPR/Cas[9] editing system mediates targeted genome editing through the complex of Cas[9] endonuclease and guide RNA, which provides a simple and efficient technique compared with other genome engineering technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)[39,40]
RNA interference (RNAi) and antisense approaches have become more commonly used in reverse genetics systems[40]

Summary

Introduction

Time savings, better grain quality and higher yields, greater effectiveness and economical use of simpler procedures for breeding and hybrid seed production. In a previous work[15], we demonstrated that the TGMS gene tms[5] encodes the endonuclease RNase ZS1 in AnS-1. We established a simple and efficient rice TGMS cultivating system using CRISPR/Cas[9] editing technology to knock out TMS5, which is of great value in new commercial TGMS line applications. Based on this system, we developed 11 commercial “transgene clean” TGMS rice lines within only one year. This work accelerates TGMS line breeding and lays the foundation for large-scale applications in two-line hybrid rice breeding

Methods

Results

Conclusion