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

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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)

Abstract:

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′)
TMS5-target-F TGGCCAGCGGCAAGTCATCGCCGG
TMS5-target-R AAACCCGGCGATGACTTGCCGCTG
M13F GTAAAACGACGGCCAG
GUS-F CGTCCGTCCTGTAGAAACCC
GUS-R GTGCGGATTCACCACTTGC
TMS5-CX-F TCCAACGCATAGCAGTAGTCG
TMS5-CX-R TGCCATCGTATCTCCGGTAAA
Cas9-F ACCGAGGGAATGAGAAAGCC
Cas9-R CCTTCTGGGTGGTCTGGTTC
HPT-F GAAGTGCTTGACATTGGGAGT
HPT-R AGATGTTGGCGACCTCGTATT

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"

品种
Variety
株高
Plant height (cm)
有效穗
Panicle number per plant
千粒重
1000-grain weight (g)
穗长
Length of main panicle (cm)
穗粒数
Grain number per panicle
结实率
Seed-setting rate (%)
产量
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
丰两优4号
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**
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