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.
雜交水稻育種是提高水稻產(chǎn)量的重要戰(zhàn)略，培育雄性不育系是雜交成功的關(guān)鍵。CRISPR/CAS9系統(tǒng)已廣泛應(yīng)用于目標(biāo)基因的編輯，而其在作物遺傳改良中的應(yīng)用卻鮮有報道。本文，我們利用CRISPR/CAS9系統(tǒng)，誘導(dǎo)了中國應(yīng)用最廣泛的溫敏雄性不育（TGMS）基因TMS5的特異性突變，并開發(fā)了新的“轉(zhuǎn)基因清潔”TGMS株系。我們利用CRISPR/CAS9系統(tǒng)設(shè)計了TMS5編碼區(qū)的10個靶位點進行靶向誘變，并評估了靶上和靶外效應(yīng)的潛在發(fā)生率。最后，我們建立了最有效的構(gòu)建物，TMS5ab構(gòu)建物，用于培育有潛在應(yīng)用價值的“無轉(zhuǎn)基因標(biāo)簽”的TGMS株系。根據(jù)不同目標(biāo)序列的特點，討論了影響編輯效率的因素。值得注意的是，利用tms5ab結(jié)構(gòu)，我們在兩個水稻亞種中開發(fā)了11個新的無轉(zhuǎn)基因標(biāo)簽的TGMS株系，并在一年內(nèi)應(yīng)用于潛在的雜交育種。該系統(tǒng)的應(yīng)用不僅大大加快了不育系的選育，而且有利于雜種優(yōu)勢的開發(fā)。