水稻光温敏核不育系的诱变创制

doi:10.16819/j.1001-7216.2026.250613

摘要/Abstract

摘要：

【目的】水稻两系杂交育种技术高度依赖携带 tms5 位点的温敏雄性核不育系。该位点源自自然突变，在不同遗传背景中具有不同的不育起点温度，而且在后代中可能存在遗传漂变，所以不育起点温度偏高和遗传漂变是制约水稻两系杂交技术发展的关键瓶颈。【方法】本研究以粳稻中花 11 种子为材料，建立了光温敏核不育系的创制方法。水稻种子经过化学诱变剂甲基磺酸乙酯(EMS)诱变处理(M₁)自交收种后，在高温环境下的 M₂群体中筛选出不育单株。将这些不育单株割蔸后继续生长，此时环境温度逐渐降低，部分新抽穗的植株育性得到恢复。【结果】经多年实验，约10%的不育株在低温条件下能够自交结实。收集这些育性恢复株系的种子，进一步进行高温不育和低温可育的验证。这些经过验证的光温敏不育系，采用构建遗传群体结合 BSA-seq (混池分离分析)基因定位方法，已获得18个新的中花11背景的光温敏雄性不育位点，其中ostms15，ostms16，ostms18，ostms19已经发表。在这些位点中，ostms15 不育系的育性转换温度显著低于同样遗传背景下在生产上广泛应用的 tms5不育系，目前已有20多家科研单位将该不育系用于水稻杂交育种。【结论】本研究提出的水稻光温敏不育系的创制技术，可以获得一系列新型光温敏不育系，有望彻底突破水稻两系杂交中光温敏不育系缺乏的瓶颈，助力水稻杂交育种的持续发展。

关键词: 水稻, EMS诱变, 光/温敏核雄性不育, 两系杂交法

Abstract:

【Objective】The two-line hybrid rice breeding technology relies on thermo-sensitive genic male sterile (TGMS) lines carrying the tms5 locus. This locus originates from a natural mutation and exhibits varying sterility onset temperatures across different genetic backgrounds. Additionally, genetic drift may occur in subsequent generations. Thus, elevated sterility onset temperatures and genetic drift are key bottlenecks restricting the development of two-line hybrid rice technology.【Method】Using the japonica rice variety Zhonghua 11(ZH11) as material, we established a method for creating P/TGMS lines. Rice seeds were treated with ethylmethanesulfonate (EMS), and sterile individuals were screened from the M₂ population under high-temperature conditions. After ratooning, these fertility-restored plants were obtained under low-temperature conditions.【Result】Through years of experimentation, approximately 10% of the sterile plants were able to self-fertilize under low temperatures. Seeds from these fertility-restored lines were collected and further validated for sterility under high temperatures and fertility under low temperatures. These validated P/TGMS lines were subjected to genetic population construction combined with BSA-seq (Bulked Segregant Analysis sequencing) for gene mapping, resulting in the identification of 18 new P/TGMS lines in the ZH11 background. Among these, ostms15, ostms16, ostms18, and ostms19 have been published. Notably, the fertility transition temperature of ostms15 is significantly lower than that of the widely used tms5 locus in the same genetic background. Currently, more than 20 research institutions have utilized this sterile line for hybrid rice breeding.【Conclusion】The P/TGMS line creation technology proposed in this study can generate a series of novel P/TGMS lines, potentially overcoming the bottleneck of P/TGMS line scarcity in two-line hybrid rice breeding and supporting the sustainable development of hybrid rice breeding.

Key words: rice, EMS mutagenesis, photoperiod/thermo-sensitive genic male sterility, two-line hybrid breeding

朱骏, 杨延铭, 杨仲南. 水稻光温敏核不育系的诱变创制[J]. 中国水稻科学, 2026, 40(4): 460-468.

ZHU Jun, YANG Yanming, YANG Zhongnan. Mutagenesis and Creation of Photoperiod/Thermo-sensitive Genic Male Sterile Lines in Rice[J]. Chinese Journal OF Rice Science, 2026, 40(4): 460-468.

图/表 4

图1 光温敏水稻EMS诱变流程 A: 水稻EMS诱变流程；B: 筛选流程中不同育性水稻植株；C: 不同批次筛选的数据汇总。

Fig. 1. EMS mutagenesis process for P/TGMS lines in rice A, Schematic diagram of the EMS mutagenesis process in rice; B, Illustration of rice plants with different fertility during the screening process; C, Summary data of screening across different batches. HT, High temperature; LT, Low temperature.

图2 光温敏水稻不育系的育性性状验证 A：不同时间种植的光温敏不育系；B：高温条件下三个不同批次的光温敏不育系的结实率统计；C：低温条件下三个不同批次的光温敏不育系的结实率统计; ostms15，ostms18，ostms19：本研究筛选到的三个光温敏不育系；tms5：中花11背景下的tms5不育系；AT：环境温度。

Fig. 2. Fertility validation of rice P/TGMS lines A, P/TGMS lines planted at different times; B, Seed-setting rate of three different batches of P/TGMS lines under high-temperature conditions; C, Seed-setting rate of three different batches of P/TGMS lines under low-temperature conditions; ostms15, ostms18 and ostms19, Three PGMS lines screened in this study; tms5, tms5 sterile line in the ZH11 genetic background; AT, Ambient temperature.

图3 光温敏不育系候选基因BSA-seq定位流程 A: BSA-seq原始数据的VCF格式文件结构及其关键信息标注；B: 测序分析的遗传学基础；C: 筛选后的 SNP 位点分布，其中紫色圆圈标记了候选基因的连锁区域，紫色箭头标注了进一步分析后确认的候选基因具体位点；D: 回补实验流程及回补植株的性状表现。

Fig. 3. Mapping process of the candidate gene for P/TGMS lines through BSA-seq A, Structure of VCF format files from raw data of BSA-seq and annotation of key information; B, Genetic basis of sequencing analysis; C, Distribution map of filtered SNP sites, where purple circles mark the candidate gene linkage regions, and purple arrows indicate the specific locus of candidate gene confirmed through further analysis; D, Complementation process and phenotypic performance of complemented plants. HT, High temperature.

图4 利用本技术克隆到的光温敏新位点利用本文介绍的EMS诱变技术在粳稻中花11中克隆到的光温敏位点在水稻12条染色体上的分布，蓝色标记为已报道的光温敏位点，红色为本实验室克隆的光温敏新位点。

Fig. 4. New P/TGMS loci identified using this technology The localization information of identified P/TGMS loci using the EMS mutagenesis technique described in this study across the 12 chromosomes of rice. Blue markers indicate previously reported P/TGMS loci, while red markers represent new P/TGMS loci cloned in this study.

参考文献 36

[1]	Cheng S H, Zhuang J Y, Fan Y Y, Du J H, Cao L Y. Progress in research and development on hybrid rice: A super-domesticate in China[J]. Annals of Botany, 2007, 100: 959-966.
[2]	袁隆平. 杂交水稻的育种战略设想[J]. 杂交水稻, 1987(1): 1-3.
	Yuan L P. Breeding strategy for hybrid rice[J]. Hybrid Rice, 1987(1): 1-3. (in Chinese with English abstract)
[3]	Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds[J]. Nature, 2019, 565(7737): 91-95.
[4]	Chen L, Liu Y G. Male sterility and fertility restoration in crops[J]. Annual Review of Plant Biology, 2014, 65(1): 579-606.
[5]	程式华, 孙宗修, 斯华敏, 卓丽圣. 水稻两用核不育系育性转换光温反应型的分类研究[J]. 中国农业科学, 1996(4): 12-17.
	Cheng S H, Sun Z X, Si H M, Zhuo L S. Classification of light-temperature-responsive types for fertility conversion in dual-purpose nuclear sterile lines of rice[J]. Scientia Agricultura Sinica, 1996(4): 12-17. (in Chinese with English abstract)
[6]	石明松. 晚粳自然两用系选育及应用初报[J]. 湖北农业科学, 1981(7): 1-3.
	Shi M S. A perliminary report on selection and application of natural dual-purpose lines of late japonica[J]. Hubei Agricultrual Sciences, 1981(7): 1-3. (in Chinese with English abstract)
[7]	袁隆平. 两系法杂交水稻研究的进展[J]. 中国农业科学, 1990, 23(3): 1-6.
	Yuan L P. Research progress on two-line hybrid rice[J]. Scientia Agricultura Sinica, 1990(3): 1-6. (in Chinese with English abstract)
[8]	杨仕华, 程本义, 沈伟峰, 夏俊辉. 中国两系杂交水稻选育与应用进展[J]. 杂交水稻, 2009, 24(1): 5-9.
	Yang S H, Cheng B Y, Shen W F, Xia J H. Progress of application and breeding on two-line hybrid rice in China[J]. Hybrid Rice, 2009, 24(1): 5-9. (in Chinese with English abstract)
[9]	林海, 李红英, 鄂志国, 庞乾林. 2020年我国审定的水稻品种基本特性分析[J]. 中国稻米, 2021, 27(6): 6-11.
	Lin H, Li H Y, Pang Q L. Analysis of basic characteristics of rice varieties to be validated in China in 2020[J]. China Rice, 2021, 27(6): 6-11. (in Chinese with English abstract)
[10]	唐世军, 吴霞, 文斌, 陶诗顺. 两系和三系杂交水稻产量性状差异性比较[J]. 杂交水稻, 2022, 37(4): 139-142.
	Tang S J, Wu X, Wen B, Tao S S. Comparison of difference in yield characters between two-line and three-line hybrid rice[J]. Hybrid Rice, 2022, 37(4): 139-142. (in Chinese with English abstract)
[11]	中国水稻研究所. 2019年中国水稻产业发展报告[M]. 北京: 中国农业科学技术出版社, 2019: 169-170.
	China Rice Research Institute. China Rice Industry Development Report 2019[M]. Beijing: China Agricultural Science and Technology Press, 2019: 169-170.
[12]	Zhou H, Zhou M, Yang Y, Li J, Zhu L, Jiang D, Dong J, Liu Q, Gu L, Zhou L, Feng M, Qin P, Hu X, Song C, Shi J, Song X, Ni E, Wu X, Deng Q, Liu Z, Chen M, Liu Y G, Cao X, Zhuang C. RNase Z^S1 processes Ub_L40 mRNAs and controls thermosensitive genic male sterility in rice[J]. Nature Communications, 2014, 5: 4884.
[13]	Yan B, Liu C, Sun J, Mao Y, Zhou C, Li J, Liu W, Li S, Yan W, Fu C, Qin P, Fu X, Zhao X, Song X, Nie J, Gao F, Yang Y, Chen Y, Cao X. Impaired 2', 3'-cyclic phosphate tRNA repair causes thermo-sensitive genic male sterility in rice[J]. Cell Research, 2024, 34(11): 763-775.
[14]	Peng G, Liu M, Zhu L, Luo W, Wang Q, Wang M, Chen H, Luo Z, Xiao Y, Zhang Y, Hong H, Liu Z, Zhou L, Guo G, Wang Y, Zhuang C, Zhou H. The E3 ubiquitin ligase CSIT1 regulates critical sterility-inducing temperature by ribosome-associated quality control to safeguard two-line hybrid breeding in rice[J]. Molecular Plant, 2023, 16: 1695-709.
[15]	Peng G, Liu M, Luo Z, Deng S, Wang Q, Wang M, Chen H, Xiao Y, Zhang Y, Hong H, Zhu L, Liu Z, Zhou L, Wang Y, Zhuang C, Zhou H. An E3 ubiquitin ligase CSIT2 controls critical sterility-inducing temperature of thermo-sensitive genic male sterile rice[J]. New Phytologist, 2024, 241: 2059-2074.
[16]	Zhou C, Liu C, Yan B, Sun J, Li S, Li J, Wang J, Huang X, Yan W, Yang S, Fu C, Qin P, Fu X, Zhao X, Wu Y, Song X, Wang Y, Qian W, Yang Y, Cao X. tRNA selectivity during ribosome-associated quality control regulates the critical sterility-inducing temperature in two-line hybrid rice[J]. Proceedings of the National Academy of Sciences, 2025, 122(6): e2417526122.
[17]	Ding J, Lu Q, Ouyang Y, Mao H, Zhang P, Yao J, Xu C, Li X, Xiao J, Zhang Q. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice[J]. Proceedings of the National Academy of Sciences, 2012, 109: 2654-2659.
[18]	Zhou H, Liu Q, Li J, Jiang D, Zhou L, Wu P, Lu S, Li F, Zhu L, Liu Z, Chen L, Liu Y G, Zhuang C. Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA[J]. Cell Research, 2012, 22: 649-660.
[19]	Fan Y, Yang J, Mathioni S M, Yu J, Shen J, Yang X, Wang L, Zhang Q, Cai Z, Xu C, Li X, Xiao J, Meyers B C, Zhang Q. PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice[J]. Proceedings of the National Academy of Sciences, 2016, 113: 15144-15149.
[20]	Qi Y, Liu Q, Zhang L, Mao B, Yan D, Jin Q, He Z. Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice[J]. Theoretical and Applied Genetics, 2014, 127: 1173-1182.
[21]	Wu L, Jing X, Zhang B, Chen S, Xu R, Duan P, Zou D, Huang S, Zhou T, An C, Luo Y, Li Y. A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility[J]. Nature Communications, 2022, 13: 2055.
[22]	斯华敏, 付亚萍, 刘文真, 孙宗修, 胡国成. 水稻光温敏雄性核不育系的系谱分析[J]. 作物学报, 2012, 38(3): 394-407.
	Si H M, Fu Y P, Liu W Z, Sun Z X, Hu G C. Pedigree analysis of photoperiod-thermo sensitive genic male sterile rice[J]. Acta Agronomica Sinica, 2012, 38(3): 394-407. (in Chinese with English abstract)
[23]	张华丽, 陈晓阳, 黄建中, 鄂志国, 龚俊义, 舒庆尧. 中国两系杂交水稻光温敏核不育基因的鉴定与演化分析[J]. 中国农业科学, 2015, 48(1): 1-9.
	Zhang H L, Chen X Y, Huang J Z, Gong J Y, Shu Q Y. Identification and transition analysis of photo-/ thermo-sensitive genic male sterile genes in two-line hybrid rice in China[J]. Scientia Agricultura Sinica, 2015, 48(1): 1-9. (in Chinese with English abstract)
[24]	陈立云, 雷东阳, 唐文邦, 肖应辉. 两系法杂交水稻研究和应用中若干问题的思与行[J]. 中国水稻科学, 2010, 24(6): 641-646.
	Chen L Y, Lei D Y, Tang W B, Xiao Y H. Thoughts and practice on some problems about research and application of two-line hybrid rice[J]. Chinese Journal of Rice Science, 2010, 24(6): 641-646. (in Chinese with English abstract)
[25]	Shi Q S, Lou Y, Shen S Y, Wang S H, Zhou L, Wang J J, Liu X L, Xiong S X, Han Y, Zhou H S, Huang X H, Wang S, Zhu J, Yang Z N. A cellular mechanism underlying the restoration of thermo/photoperiod- sensitive genic male sterility[J]. Molecular Plant, 2021, 14(12): 2104-2114.
[26]	Zhang C, Xu T, Ren M Y, Zhu J, Shi Q S, Zhang Y F, Qi Y W, Huang M J, Song L, Xu P, Yang Z N. Slow development restores the fertility of photoperiod-sensitive male-sterile plant lines. Plant Physiology. 2020, 184(2): 923-32.
[27]	Zhu J, Lou Y, Shi Q S, Zhang S, Zhou W T, Yang J, Zhang C, Yao X Z, Xu T, Liu J L, Zhou L, Hou J Q, Wang J Q, Wang S, Huang X H, Yang Z N. Slowing development restores the fertility of thermo-sensitive male-sterile plant lines[J]. Nature Plants, 2020, 6(4): 360-367.
[28]	Wang Y C, Liu X L, Zhang Z, Zhou L, Zhang Y F, Zhu B S, Yang Y M, Zhong X, Su Z X, Ma P Y, Huang X H, Yang ZN, Zhu J. The residual activity of fatty acyl-CoA reductase underlies thermo-sensitive genic male sterility in rice[J]. Plant, Cell & Environment, 2025, 48(2): 1273-1285.
[29]	Zhou L, Mao Y C, Yang Y M, Wang J J, Zhong X, Han Y, Zhang Y F, Shi Q S, Huang X H, Meyers B C, Zhu J, Yang Z N. Temperature and light reverse the fertility of rice P/TGMS line ostms19 via ROS homeostasis[J]. Plant Biotechnology Journal, 2024, 22: 2020-2032.
[30]	Zhang Y F, Li Y L, Zhong X, Wang J J, Zhou L, Han Y, Li D D, Wang N, Huang X H, Zhu J, Yang Z N. Mutation of glucose-methanol-choline oxidoreductase leads to thermosensitive genic male sterility in rice and Arabidopsis[J]. Plant Biotechnology Journal, 2022, 20: 2023-2035.
[31]	Han Y, Jiang S Z, Zhong X, Chen X, Ma C K, Yang Y M, Mao Y C, Zhou S D, Zhou L, Zhang Y F, Huang X H, Zhang H, Li LG, Zhu J, Yang Z N. Low temperature compensates for defective tapetum initiation torestore the fertility of the novel TGMS line ostms15[J]. Plant Biotechnology Journal, 2023, 21: 1659-1670.
[32]	Zhang Z, Guo Y Y, Wang Y C, Zhou L, Fan J, Mao Y C, Yang Y M, Zhang Y F, Huang X H, Zhu J, Zhang C, Yang Z N. A point mutation in a meiotic crossover formation gene HEI10/TFS2 leads to thermos-sensitive genic female sterility in rice[J]. The Plant Journal, 2024, 118: 506-518.
[33]	汪邑晨, 朱本顺, 周磊, 朱骏, 杨仲南. 光/温敏核不育系的不育机理及两系杂交稻的发展与展望[J]. 中国水稻科学, 2024, 38(5): 463-474.
	Wang Y C, Zhu B S, Zhou L, Zhu J, Yang Z N. Sterility mechanism of photoperiod/thermo-sensitive genic male sterile lines and development and prospects of two-line hybrid rice[J]. Chinese Journal of Rice Science, 2024, 38(5): 463-474. (in Chinese with English abstract)
[34]	Till B J, Reynolds S H, Greene E A, Codomo C A, Enns L C, Johnson J E, Burtner C, Odden A R, Young K, Taylor N E, Henikoff J G, Comai L, Henikoff S. Large-scale discovery of induced point mutations with high-throughput TILLING[J]. Genome Research, 2003, 13(3): 524-30.
[35]	叶俊, 吴建国, 杜婧, 郑希, 张志, 石春海. 水稻“9311”突变体筛选和突变体构建[J]. 作物学报, 2006, 32(10): 1525-1529.
	Ye J, Wu J G, Du J, Zheng X, Zhang Z, Shi C H. The screening of mutants and construction of mutant population for cultivar “9311” in rice (Oryza sativa L)[J]. Acta Agronomica Sinica, 2006, 32(10): 1525-1529. (in Chinese with English abstract)
[36]	王芳权, 范方军, 夏士健, 宗寿余, 郑天清, 王军, 李文奇, 许扬, 陈智慧, 蒋彦婕, 陶亚军, 仲维功, 杨杰. 水稻光温敏核不育基因tms5与pms3的互作效应[J]. 作物学报, 2020, 46(3): 317-329.
	Wang F Q, Fan F J, Xia S J, Zong S Y, Zheng T Q, Wang J, Li W Q, Xu Y, Chen Z H, Jiang Y J, Tao Y J, Zhong W G, Yang J. Interactive effects of the photoperiod-/thermo- sensitive genic male sterile genes tms5 and pms3 in rice[J]. Acta Agronomica Sinica, 2020, 46(3): 317-329. (in Chinese with English abstract)