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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1157-1165.doi: 10.3724/SP.J.1006.2020.92059


CRISPR/Cas9-mediated editing of the thermo-sensitive genic male-sterile gene TMS5 in rice

CHEN Ri-Rong1,ZHOU Yan-Biao2,3,WANG Dai-Jun5,ZHAO Xin-Hui3,TANG Xiao-Dan3,XU Shi-Chong4,TANG Qian-Ying3,FU Xing-Xue3,WANG Kai3,LIU Xuan-Ming1,*(),YANG Yuan-Zhu1,2,3,4,*()   

  1. 1Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation/College of Biology, Hunan University, Changsha 410082, Hunan, China
    2Yuan Longping High-Tech Agriculture Co. Ltd./Key Laboratory of Rice Germplasm Enhancement in Southern China, Ministry of Agriculture and Rural Affairs, Changsha 410001, Hunan, China
    3Yahua Seeds Science Academy of Hunan, Changsha 410119, Hunan, China
    4College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    5College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
  • Received:2019-11-09 Accepted:2020-04-15 Online:2020-08-12 Published:2020-04-26
  • Contact: Xuan-Ming LIU,Yuan-Zhu YANG E-mail:xml05@hnu.edu.cn;yzhuyah@163.com
  • Supported by:
    National Natural Science Foundation of China(31901516);National Major Project for Developing New GM Crops(2016ZX08001-004);Project of Hunan Natural Science Youth Foundation(2019JJ50414)


Thermo-sensitive genic male-sterile (TGMS) gene tms5 is most widely used in the two-line hybrid breeding system in China. To develop novel rice thermo-sensitive male sterile lines, we knocked out the TMS5 genes of six elite japonica and four indica rice varieties by using CRISPR/Cas9 gene editing technology. By analyzing the critical sterility-inducing temperature (CIST) of the newly TGMS lines, it was found that the CIST of japonica TGMS lines ZG75S, CYGS, YG0618S, ZG07S, T0361S, and 7679S were between 28°C and 32°C, the CIST of indica TGMS lines 2537S, 6150S and 6379S were between 24°C and 28°C, and the CIST of indica TGMS line 1109S was lower than 23.5°C. These results indicated that the CIST of tms5 mutant from different genetic background materials was different. The TGMS lines with lower CIST could be obtained by knocking out the TMS5 from different genetic background materials. A hybrid rice combination 1109S/8048 had high quality and high yield. The yield of 1109S/8048 was 13.1% higher than that of Fengliangyou 4. The creation of the TGMS 1109S and the high-yield cross combination 1109S/8048 provides a new way for high-yield breeding.

Key words: rice, CRISPR/Cas9, thermo-sensitive genic male sterile, TMS5, cross combination

Fig. 1

Schematic diagram of the CRISPR/Cas9-TMS5 vector construction A: Schematic diagram of the targeted site in TMS5. The red letters are the target genome sequences. The blue letters are the protospacer adjacent motif (PAM) sequences. B: Schematic diagram of the pCAMBIA1301-Cas9-TMS5-gRNA construct. LB: left border; RB: right border."

Table 1

Primer sequences used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′)

Fig. 2

Identification of the T0 transgenic plants Identification of the positive transgenic plants by PCR (upper) and GUS histochemical staining (lower) in ZG75(A), CYG(B), YG0618(C), ZG07(D), T0361(E), 7679(F), 2537(G), 6150(H), 6379(I), and 1109(J). M: DNA marker; 1-10: transgenic plants."

Fig. 3

Homozygous mutation types of T0 generation The red letters are the target genome sequences; the blue letters are PAM; the green letters are the insert base; the horizontal line indicates the missing base; +: insertion; -: deletion; WT: wild-type."

Supplementary Fig. 1

Identification of the marker-free T1 transgenic plants by PCR Identification of the marker-free transgenic plants by PCR in ZG75(A), CYG(B), YG0618(C), ZG07(D), T0361(E), 7679(F), 2537(G), 6150(H), 6379(I), 1109(J). M: DNA marker; WT: wild-type; 1: positive control; 2-6: the leaf of T1 plants did not show blue by GUS staining."

Fig. 4

Pollen fertility of tms5 mutant under different temperatures A: Pollen fertility of tms5 mutant of japonica rice; B: Pollen fertility of tms5 mutant of indica rice; C: Pollen fertility of 1109S under cold irrigation conditions."

Fig. 5

Identification of phenotype of 1109S/8048 hybrid combination A: Phenotype of 1109S/8048 hybrid combination, Bar = 20 cm; B: Panicle performance of 1109S/8048 hybrid combination, Bar = 5 cm."

Table 2

Agronomic traits of hybrid combination"

Plant height (cm)
Panicle number per plant
1000-grain weight (g)
Length of main panicle (cm)
Grain number per panicle
Seed-setting rate (%)
Grain yield
(kg hm-2)
8048 110.4±3.5 10.7±1.0 16.8±1.6 27.5±1.0 229.3±15.8 74.6±8.2 8857.5±526.5
Fengliangyou 4
133.1±4.5 11.5±1.2 27.5±2.1 25.3±1.1 193.5±14.6 75.7±5.6 9835.5±387.0
1109S/8048 120.6±4.0** 13.5±2.0** 22.6±1.5** 30.7±1.5** 258.4±23.6** 75.9±4.5 11124.0±279.0**
[1] 黄忠明, 周延彪, 唐晓丹, 赵新辉, 周在为, 符星学, 王凯, 史江伟, 李艳峰, 符辰建, 杨远柱. 基于CRISPR/Cas9技术的水稻温敏不育基因tms5突变体的构建. 作物学报, 2018,44:844-851.
doi: 10.3724/SP.J.1006.2018.00844
Huang Z M, Zhou Y B, Tang X D, Zhao X H, Zhou Z W, Fu X X, Wang K, Shi J W, Li Y F, Fu C J, Yang Y Z. Construction of tms5 mutants in rice based on CRISPR/Cas9 technology. Acta Agron Sin, 2018,44:844-851 (in Chinese with English abstract).
doi: 10.3724/SP.J.1006.2018.00844
[2] 周延彪, 赵新辉, 唐晓丹, 周在为, 庄楚雄, 杨远柱. 基于CRISPR/Cas9技术的水稻反光敏不育基因csa突变体的获得. 杂交水稻, 2018,33(6):68-74.
Zhou Y B, Zhao X H, Tang X D, Zhou Z W, Zhuang C X, Yang Y Z. Acquisition of mutants of the reverse photoperiod-sensitive genic male sterility gene csa in rice based on CRISPR/Cas9 technology. Hybrid Rice, 2018,33(6):68-74 (in Chinese with English abstract).
[3] Zhou H, Zhou M, Yang Y Z, Li J, Zhu L Y, Jiang D G, Dong J F, Liu Q J, Gu L F, Zhou L Y, Feng M J, Qin P, Hu X C, Song C L, Shi J F, Song X W, Ni E D, Wu X J, Deng Q Y, Liu Z L, Chen M S, Liu Y G, Cao X F, Zhuang C X. RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice. Nat Commun, 2014,5:1-9.
[4] 周海, 周明, 杨远柱, 曹晓风, 庄楚雄. RNase ZS1加工UbL40 mRNA控制水稻温敏雄性核不育. 遗传, 2014,36:1274.
Zhou H, Zhou M, Yang Y Z, Cao X F, Zhuang C X. RNase ZS1 processes UbL40 mRNA and controls thermosensitive genic male sterility in rice. Hereditas (Beijing), 2014,36:1274 (in Chinese with English abstract).
[5] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotech, 2015,32:76-84.
[6] Baltes N J, Voytas D F. Enabling plant synthetic biology through genome engineering. Trends Biotechnol, 2015,33:120-131.
doi: 10.1016/j.tibtech.2014.11.008 pmid: 25496918
[7] Li M, Li X, Zhou Z, Wu P, Fang M, Pan X, Lin Q, Luo W, Wu G, Li H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3 and IPA1 in rice using a CRISPR/Cas9 system. Front Plant Sci, 2016,7:377.
pmid: 27066031
[8] Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, Du W, Du J, Francis F, Zhao Y, Xia L. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Front Plant Sci, 2017,8:298.
doi: 10.3389/fpls.2017.00298 pmid: 28326091
[9] Zhou Y B, Liu C, Tang D Y, Lu Y, Wang D, Yang Y Z, Gui J S, Zhao X Y, Li L G, Tang X D, Yu F, Li J L, Liu L L, Zhu Y H, Lin J Z, Liu X M. The receptor-like cytoplasmic kinase STRK1 phosphorylates and activates CatC, thereby regulating H2O2 homeostasis and improving salt tolerance in rice. Plant Cell, 2018,30:1100-1118.
doi: 10.1105/tpc.17.01000 pmid: 29581216
[10] Wang H, Chu Z, Ma X, Li R, Liu Y. A high through-put protocol of plant genomic DNA preparation for PCR. Acta Agron Sin, 2013,39:1200-1205.
[11] 姜树坤, 张喜娟, 王嘉宇, 张凤鸣. 水稻幼穗-颖花发育的研究进展. 植物遗传资源学报, 2012,13:1018-1022.
Jiang S K, Zhang X J, Wang J Y, Zhang F M. Research advancement on young panicle and spikelet development in rice (Oryza sativa L.). J Plant Genet Resour, 2012,13:1018-1022 (in Chinese with English abstract).
[12] Zhou Y B, Liu H, Zhou X C, Yan Y Z, Du C Q, Li Y X, Liu D R, Zhang C S, Deng X L, Tang D Y, Zhao X Y, Zhu Y H, Lin J Z, Liu X M. Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase PcGDH improves nitrogen assimilation and growth quality in rice. Mol Breed, 2014,34:335-349.
[13] 李希陶, 刘耀光. 基因组编辑技术在水稻功能基因组和遗传改良中的应用. 生命科学, 2016,28:1243-1249.
Li X T, Liu Y G. Genome editing technology for functional genomics and genetic improvement in rice. Chin Bull Life Sci, 2016,28:1243-1249 (in Chinese with English abstract).
[14] Zhou H, He M, Li J, Chen L, Huang Z F, Zheng S Y, Zhu L Y, Ni E D, Jiang D G, Zhao B R, Zhuang C X. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep, 2016,6:1-12.
pmid: 28442746
[15] Svitashev S, Schwartz C, Lenderts B, Young J K, Ciqan A M. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun, 2016,7:13274.
doi: 10.1038/ncomms13274 pmid: 27848933
[16] Wang Y P, Cheng X, Shan Q W, Zhang Y, Liu J X, Gao C X, Qiu J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol, 2014,32:947-951.
doi: 10.1038/nbt.2969 pmid: 25038773
[17] Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol, 2015,15:16.
doi: 10.1186/s12896-015-0131-2 pmid: 25879861
[18] Zirkle C. Some forgotten records of hybridization and sex in plants 1716-1739. J Hered, 1932,23:433-447.
[19] Stiles W. A short history of the plant sciences. Nature, 1942,150:672-673.
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