Creating a Fragrant Environment-insensitive Recessive Genic Male Sterile Germplasm Lacking Coleoptile Purple Lines by CRISPR/Cas9 Gene Editing

doi:10.16819/j.1001-7216.2025.240501

Abstract

Abstract:

【Objective】Hybrid rice has developed into the third generation in which environment-insensitive recessive genic male sterility (EI-RGMS) is the essential trait. In many rice breeding programs, fragrance improvement is an important target. Additionally, the purple line in coleoptile is a clearly identifiable anthocyanin trait at the very early germination stage, which facilitates simple, rapid, and accurate identification of hybrid purity. The purpose of this study is to develop a fragrant EI-RGMS material lacking the coleoptile purple line based on maintainer II-32B, providing a new core germplasm for the third-generation hybrid rice.【Methods】Using CRISPR/Cas9, we knocked out the pollen development gene OsbHLH141, fragrance gene OsBadh2, and coleoptile purple line gene OsMYB76 in the elite rice parent II-32B.【Result】Mutant lines with all three genes knocked out showed loss of anthocyanin traits, a distinct aroma, and male sterility.【Conclusion】This study successfully created a fragrant EI-RGMS germplasm without purple line in coleoptile in the II-32B background.

Key words: environment-insensitive, recessive genic male sterility, fragrance, purple line in coleoptile, CRISPR/Cas9

摘要：

【目的】水稻杂种优势利用技术已发展到第三代，环境非敏感型隐性核雄性不育是第三代杂交稻的必需性状，而香味是许多水稻育种计划的重要目标。另外，水稻芽鞘紫线是极早期表现的一种明显稳定的花青素性状，有利于杂交种纯度的简单、快速和准确鉴定。本研究目的是将II-32B制备成香型、无芽鞘紫线的环境非敏感型隐性核雄性不育材料，为研发方便纯度鉴定的香型第三代杂交稻创造核心种质。【方法】利用CRISPR/Cas9技术，同时对优良水稻亲本II-32B的花粉发育相关基因OsbHLH141、香味基因OsBadh2以及芽鞘紫线基因OsMYB76进行敲除。【结果】获得了三个基因同时发生突变的材料，突变体表现为芽鞘紫线等花青素性状缺失，稻米具有香味且雄性不育。【结论】本研究成功获得了以II-32B为背景的香型、无芽鞘紫线的环境非敏感型隐性核雄性不育种质。

关键词: 环境非敏感, 隐性核雄性不育, 香味, 芽鞘紫线, CRISPR/Cas9

YANG Jiaxin, GUAN Yusheng, DU Run, LI Xianyong, CAI Zuokun, WANG Chutao, YANG Qiyang, HE Yongxin, ZHU Zichao, ZHANG Yi. Creating a Fragrant Environment-insensitive Recessive Genic Male Sterile Germplasm Lacking Coleoptile Purple Lines by CRISPR/Cas9 Gene Editing[J]. Chinese Journal OF Rice Science, 2025, 39(5): 643-649.

杨佳欣, 管玉圣, 杜润, 李贤勇, 蔡座坤, 王楚桃, 阳启样, 何永歆, 朱子超, 张毅. 利用CRISPR/Cas9技术创制无芽鞘紫线的香型环境非敏感隐性核雄性不育种质[J]. 中国水稻科学, 2025, 39(5): 643-649.

Figures/Tables 7

References 20

[1]	郭韬, 余泓, 邱杰, 李家洋, 韩斌, 林鸿宣. 中国水稻遗传学研究进展与分子设计育种[J]. 中国科学: 生命科学, 2019, 49(10): 1185-1212.
	Guo T, Yu H, Qiu J, Li J Y, Han B, Lin H X. Advances in rice genetics and breeding by molecular design in China[J]. Scientia Sinica: Vitae, 2019, 49(10): 1185-1212. (in Chinese with English abstract)
[2]	邓兴旺, 王海洋, 唐晓艳, 周君莉, 陈浩东, 何光明, 陈良碧, 许智宏. 杂交水稻育种将迎来新时代[J]. 中国科学:生命科学, 2013, 43(10): 864-868.
	Deng X W, Wang H Y, Tang X Y, Zhou J L, Chen H D, He G M, Chen L B, Xu Z H. Hybrid rice breeding welcomes a new era of molecular crop design[J]. Scientia Sinica: Vitae, 2013, 43(10): 864-868. (in Chinese with English abstract)
[3]	Song S, Wang T, Li Y, Hu J, Kan R, Qiu M, Deng Y, Liu P, Zhang L, Dong H, Li C, Yu D, Li X, Yuan D, Yuan L, Li L. A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene[J]. Plant Biotechnology Journal, 2021, 19(2): 251-260.
[4]	李翔, 杜双林, 朱文平, 张毅. 水稻隐性雄性不育突变体osnp3的败育特征及基因定位[J]. 江苏农业科学, 2023, 51(11): 106-112.
	Li X, Du S L, Zhu W P, Zhang Y. Sterile characterization and gene mapping of a recessive male sterile mutant osnp3 in rice[J]. Jiangsu Agricultural Sciences, 2023, 51(11): 106-112. (in Chinese with English abstract)
[5]	Yajima I, Yanai T, Nakamura M, Sakakibara H, Hayashi K. Volatile flavor components of cooked Kaorimai (scented rice, O. sativa japonica)[J]. Agricultural and Biological Chemistry, 1979, 43(12): 2425-2429.
[6]	Buttery R G, Ling L C, Juliano B O, Turnbaugh J G. Cooked rice aroma and 2-acetyl-1-pyrroline[J]. Journal of Agricultural and Food Chemistry, 1983, 31(4): 823-826.
[7]	Bradbury L M T, Fitzgerald T L, Henry R J, Jin Q, Waters D L E. The gene for fragrance in rice[J]. Plant Biotechnology Journal, 2005, 3(3): 363-370.
[8]	Tian Y, Zhou Y, Gao G, Zhang Q, Li Y, Lou G, He Y. Creation of two-line fragrant glutinous hybrid rice by editing the W_x and OsBADH2 genes via the CRISPR/Cas9 system[J]. International Journal of Molecular Sciences, 2023, 24(1): 849.
[9]	李景芳, 温舒越, 赵利君, 陈庭木, 周振玲. 基于CRISPR/Cas9技术创制耐盐香稻[J]. 中国水稻科学, 2023, 37 (5): 478-485.
	Li J F, Wen S Y, Zhao L J, Chen T M, Zhou Z L. Development of aromatic salt-tolerant rice based on CRISPR/Cas9 technology[J]. Chinese Journal of Rice Science, 2023, 37(5): 478-485.
[10]	胡景涛, 黄文章, 严明建. 几种杂交水稻种子纯度鉴定的方法及其应用前景[J]. 安徽农业科学, 2009, 37(4): 1493-1495+1536.
	Hu J T, Huang W Z, Yan M J. Methods for identifying the purity of hybrid rice seeds and their application prospects[J]. Journal of Anhui Agricultural Sciences, 2009, 37(4): 1493-1495, 1536. (in Chinese)
[11]	张毅, 李云峰, 刘晓锋, 林茂祥, 沈福成, 何光华, 杨正林, 杨光伟. 水稻芽鞘紫线遗传分析[J]. 中国农业科学, 2004(11): 1693-1698.
	Zhang Y, Li Y F, Liu X F, Lin M X, Shen F C, He G H, Yang Z L, Yang G W. Analysis on the inheritance of coleoptile purple line in rice[J]. Scientia Agricultura Sinica, 2004(11): 1693-1698. (in Chinese with English abstract)
[12]	Du S, Wang Z, Chen Y, Tan Y, Li X, Zhu W, He G, Lei K, Guo L, Zhang Y. Coleoptile purple line regulated by A-P gene system is a valuable marker trait for seed purity identification in hybrid rice[J]. Rice Science, 2022, 29(5): 451-461.
[13]	张毅. 水稻两个芽鞘紫线相关基因的图位克隆与应用研究[D]. 重庆: 西南大学, 2009.
	Zhang Y. Mapping cloning and application of genes related to purple line in two bud sheaths of rice[D]. Chongqing: Southwest University, 2009. (in Chinese with English abstract)
[14]	任光俊, 颜龙安, 谢华安. 三系杂交水稻育种研究的回顾与展望[J]. 科学通报, 2016, 61(35): 3748-3760.
	Ren G J, Yan L A, Xie H A. Retrospective and perspective on indica three-line hybrid rice breeding research in China[J]. Chinese Science Bulletin, 2016, 61(35): 3748-3760. (in Chinese with English abstract)
[15]	Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(11): 3570-3575.
[16]	李新奇, 黄群策. 第3代杂交水稻育种技术策略探讨[J]. 杂交水稻, 2020, 35(1): 1-5.
	Li X Q, Huang Q C. Discussion on the breeding technical strategy of the third-generation hybrid rice breeding[J]. Hybrid Rice, 2020, 35(1): 1-5. (in Chinese with English abstract)
[17]	李雅礼, 唐华园, 黄群策, 李新奇. 第三代杂交水稻研究与实践[J]. 分子植物育种, 2024, 22(15): 5124-5131.
	Li Y L, Tang H Y, Huang Q C, Li X Q. Research and application of the third-generation hybrid rice[J]. Molecular Plant Breeding, 2024, 22(15): 5124-5131. (in Chinese with English abstract)
[18]	Chang Z, Chen Z, Wang N, Xie G, Lu J, Yan W, Zhou J, Tang X, Deng X W. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(49): 14145-14150.
[19]	宋丰顺, 倪大虎, 倪金龙, 李莉, 杨剑波. 杂交水稻种子纯度检测方法综述[J]. 江苏农业科学, 2016, 44(6): 6-11.
	Song F S, Ni D H, Ni J L, Li L, Yang J B. A review of methods for detecting the purity of hybrid rice seeds[J]. Jiangsu Agricultural Sciences, 2016, 44(6): 6-11. (in Chinese)
[20]	张倩倩, 殷春渊, 刘贺梅, 胡秀明, 孟利红. 48份水稻骨干材料香味及Badh2变异类型的鉴定[J]. 种子, 2024, 43(1): 107-113.
	Zhang Q Q, Yin C Y, Liu H M, Hu X M, Meng L H. Identification of fragrance and Badh2 variation types in 48 Oryza sativa L. backbone materials[J]. Seed, 2024, 43(1): 107-113. (in Chinese)

靶点名称 Target name	引物序列(5΄-3΄) Sequence(5΄-3΄)
OsbHLH141 target 1	CTGGTGGAACAGAAGAGGCA TGG
OsbHLH141 target 2	TGGGAAACAACCCATAATTT TGG
OsBadh2 target 1	TTTACTGGGAGTTATGAAAC TGG
OsMYB76 target 1	AGCACGCTCAGCCGCAAGAT CGG

靶点名称 Target name	引物序列(5΄-3΄) Sequence(5΄-3΄)
OsbHLH141 target 1	CTGGTGGAACAGAAGAGGCA TGG
OsbHLH141 target 2	TGGGAAACAACCCATAATTT TGG
OsBadh2 target 1	TTTACTGGGAGTTATGAAAC TGG
OsMYB76 target 1	AGCACGCTCAGCCGCAAGAT CGG

引物名称 Primer name	正向引物序列(5΄-3΄) Forward primer sequence(5΄-3΄)	反向引物序列(5΄-3΄) Reverse primer sequence(5΄-3΄)	目的 Purpose
Cas393F/R	TCGAGAACGGTCGTAAGAGGA	TGTAACGCTTCCTGTCGATGGT	转基因检测Transgenic detection
141F10/R10	CTCCAGTTAGGGTACCATGGTACT	CGGAATTCCTCACTGGCCTCATCGATGA	测序Sequencing
APCSF1/R1	CGGAATTCCAGGACTTGTTTGGAGCTTGC	GGGGTACCTGCTCGTCTGGGACCAGTAT	测序Sequencing
OsC1F4/R3	CAGTCTCACACCGCACAG	ACGGAAACCCGCAACTGC	测序Sequencing
141F8/R8	CCATGGTACTCAATCTCATC	GTCCTGAACTTCACATTTAG	分子标记检测Molecular marker detection
141F9/R9	GGTGATGCCATTGAGTATAT	GAGCTGATTGTCTTGATCAT	分子标记检测Molecular marker detection
APMF3/R3	CATGTATACCCCATCAATGG	GCTCAAAGTGTCTTGATCAC	分子标记检测Molecular marker detection
CMDF1/R1	CGAACAGACAATGAAATCAAG	CACGACGGAGCTGGACGAC	分子标记检测Molecular marker detection

引物名称 Primer name	正向引物序列(5΄-3΄) Forward primer sequence(5΄-3΄)	反向引物序列(5΄-3΄) Reverse primer sequence(5΄-3΄)	目的 Purpose
Cas393F/R	TCGAGAACGGTCGTAAGAGGA	TGTAACGCTTCCTGTCGATGGT	转基因检测Transgenic detection
141F10/R10	CTCCAGTTAGGGTACCATGGTACT	CGGAATTCCTCACTGGCCTCATCGATGA	测序Sequencing
APCSF1/R1	CGGAATTCCAGGACTTGTTTGGAGCTTGC	GGGGTACCTGCTCGTCTGGGACCAGTAT	测序Sequencing
OsC1F4/R3	CAGTCTCACACCGCACAG	ACGGAAACCCGCAACTGC	测序Sequencing
141F8/R8	CCATGGTACTCAATCTCATC	GTCCTGAACTTCACATTTAG	分子标记检测Molecular marker detection
141F9/R9	GGTGATGCCATTGAGTATAT	GAGCTGATTGTCTTGATCAT	分子标记检测Molecular marker detection
APMF3/R3	CATGTATACCCCATCAATGG	GCTCAAAGTGTCTTGATCAC	分子标记检测Molecular marker detection
CMDF1/R1	CGAACAGACAATGAAATCAAG	CACGACGGAGCTGGACGAC	分子标记检测Molecular marker detection

材料 Material	株高 Plant height (cm)	有效穗数 Effective panicle number	穗长 Panicle length (cm)	每穗粒数 No. of grains per panicle	千粒重 1000-grain weight (g)	结实率 Seed setting rate (%)
野生型 WT	98.04±2.25	14.80±1.48	23.22±0.55	201.20±16.44	22.89±0.26	94.49±1.28
突变体Mutant	90.77±1.48***	12.33±1.03*	22.20±0.66*	191.83±12.45	/	/