基于M1TDS靶向筛选技术的重离子束诱变定向改良杂交水稻卓两优1126性状的研究

doi:10.16819/j.1001-7216.2025.240106

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (5): 624-634.DOI: 10.16819/j.1001-7216.2025.240106

基于M₁TDS靶向筛选技术的重离子束诱变定向改良杂交水稻卓两优1126性状的研究

韶也¹^,²^,^#, 胡远艺¹^,²^,^#, 彭彦¹^,²^,^#, 毛毕刚¹^,², 刘慧敏¹^,², 唐婵娟¹, 雷斌¹^,², 唐丽¹^,², 余丽霞³, 李文建³, 罗武中³, 罗治斌¹^,², 袁远涛¹^,², 李曜魁¹^,², 张丹¹^,², 周利斌³, 柏连阳²^,⁴^,^*(), 唐文帮¹^,²^,^*(), 赵炳然¹^,²^,^*()

¹湖南杂交水稻研究中心，长沙 410125
²杂交水稻全国重点实验室，长沙 410125
³中国科学院近代物理研究所重离子加速器国家实验室，兰州 730000
⁴湖南省农业科学院，长沙 410125

收稿日期:2024-01-09 修回日期:2024-04-17 出版日期:2025-09-10 发布日期:2025-09-10
通讯作者: *email：lybai@hunaas.cn，email：tangwenbang@163.com，email：brzhao652@hhrrc.ac.cn
作者简介:^#共同第一作者
基金资助:
长沙市“揭榜挂帅”重大科技攻关项目(kq2301001);现代农业产业技术体系资助项目(CARS-01-01);湖南省 2023 年度十大技术攻关项目(2023NK1010)

Directed Improvement of Hybrid Rice Zhuoliangyou 1126 by Heavy Ion Beam Mutagenesis Based on M₁TDS Targeted Screening Technology

SHAO Ye¹^,²^,^#, HU Yuanyi¹^,²^,^#, PENG Yan¹^,²^,^#, MAO Bigang¹^,², LIU Huimin¹^,², TANG Chanjuan¹, LEI Bin¹^,², TANG Li¹^,², YU Lixia³, LI Wenjian³, LUO Wuzhong³, LUO Zhibin¹^,², YUAN Yuantao¹^,², LI Yaokui¹^,², ZHANG Dan¹^,², ZHOU Libin³, BAI Lianyang²^,⁴^,^*(), TANG Wenbang¹^,²^,^*(), ZHAO Bingran¹^,²^,^*()

¹Hunan Hybrid Rice Research Center, Changsha 410125, China
²National Key Laboratory of Hybrid Rice, Changsha 410125, China
³National Laboratory of Heavy Ion Accelerator, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China
⁴Hunan Academy of Agricultural Sciences, Changsha 410125, China

Received:2024-01-09 Revised:2024-04-17 Online:2025-09-10 Published:2025-09-10
Contact: *email：lybai@hunaas.cn，email：tangwenbang@163.com，email：brzhao652@hhrrc.ac.cn
About author:^#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】以两系杂交水稻卓两优1126为“底盘品种”，快速创制具有镉低积累、香味、耐储藏、耐淹、低升糖指数等性状的改良品种。【方法】¹²C⁶⁺碳离子束诱变卓两优1126亲本卓234S和湘农恢1126，利用M₁TDS技术从诱变M₁群体中鉴定OsNRAMP5、OsBADH2、OsLOX3、OsPAO5、OsSSIIIa和OsBEIIb基因嵌合突变株，通过KASP分型从诱变M₂群体中鉴定分离目标基因纯合(杂合)突变株，利用OsNRAMP5突变亲本配制OsNRAMP5突变组合。【结果】从重离子束诱变M₁群体中鉴定到13株为5个目标基因突变的嵌合突变株，其中7个M₂株系中鉴定到目标基因的纯合或杂合突变；OsNRAMP5纯合突变株镉含量较原始亲本显著降低，OsBADH2纯合突变株香味特征化合物2-乙酰基吡咯啉(2-AP)含量较原始亲本显著上升；OsNRAMP5基因突变亲本配制的组合，在镉污染土(土壤有效镉含量0.677 mg/kg，pH 5.6)盆栽种植，其籽粒镉含量为0.05 mg/kg，而原始组合为0.91 mg/kg。【结论】利用“重离子束诱变+M₁TDS技术”实现了卓两优1126镉低积累等性状的快速改良，为生物传统诱变育种进化到定向诱变育种提拱了成功案例和共性技术参考。

关键词: 水稻, 重离子束诱变育种, 镉低积累, 香味

Abstract:

【Objective】Using the two-line hybrid rice Zhuoliangyou 1126 as the base variety, we aimed to rapidly develop upgraded varieties with traits such as low cadmium accumulation, fragrance, storage tolerance, submergence tolerance, and a low glycemic index.【Method】Zhuo 234S and Xiangnonghui 1126, the parental lines of Zhuoliangyou 1126, were mutagenized using heavy-ion beams. Chimeric mutants of the OsNRAMP5, OsBADH2, OsLOX3, OsPAO5, OsSSIIIa, and OsBEIIb genes were identified in the M₁ generation via M1TDS technology. Mutations in the target genes were further detected and isolated in the M₂ generation using Kompetitive Allele-Specific PCR (KASP) genotyping. Hybrid rice with low cadmium accumulation was developed by crossing parents carrying OsNRAMP5 mutations.【Results】A total of 13 chimeric mutants involving six target genes were identified in the M₁ generation. Among these, seven were confirmed to carry homozygous or heterozygous mutations in the M₂ generation. The cadmium content in OsNRAMP5 homozygous mutants was significantly lower than in the wild type, while the content of the aromatic compound 2-acetylpyrroline (2-AP) was significantly higher in OsBADH2 homozygous mutants. When the low-cadmium hybrid rice derived from OsNRAMP5-mutated parents was pot-grown in cadmium-contaminated soil (available Cd: 0.677 mg/kg, pH 5.6), the cadmium content in grains remained consistently below 0.05 mg/kg, compared to 0.91 mg/kg in the wild-type control.【Conclusion】The combination of heavy-ion beam mutagenesis and M1TDS technology enabled the rapid improvement of traits such as low cadmium accumulation in Zhuoliangyou 1126. This study provides a successful example and a general technical reference for transitioning from traditional mutagenesis breeding to directed mutagenesis breeding.

Key words: rice, heavy ion beam mutation breeding, low cadmium accumulation, fragrance

韶也, 胡远艺, 彭彦, 毛毕刚, 刘慧敏, 唐婵娟, 雷斌, 唐丽, 余丽霞, 李文建, 罗武中, 罗治斌, 袁远涛, 李曜魁, 张丹, 周利斌, 柏连阳, 唐文帮, 赵炳然. 基于M₁TDS靶向筛选技术的重离子束诱变定向改良杂交水稻卓两优1126性状的研究[J]. 中国水稻科学, 2025, 39(5): 624-634.

SHAO Ye, HU Yuanyi, PENG Yan, MAO Bigang, LIU Huimin, TANG Chanjuan, LEI Bin, TANG Li, YU Lixia, LI Wenjian, LUO Wuzhong, LUO Zhibin, YUAN Yuantao, LI Yaokui, ZHANG Dan, ZHOU Libin, BAI Lianyang, TANG Wenbang, ZHAO Bingran. Directed Improvement of Hybrid Rice Zhuoliangyou 1126 by Heavy Ion Beam Mutagenesis Based on M₁TDS Targeted Screening Technology[J]. Chinese Journal OF Rice Science, 2025, 39(5): 624-634.

图/表 11

参考文献 34

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名称 Name	序列 Sequence
OsNRAMP5-246-8-F	GAAGGTGACCAAGTTCATGCTTGTGGTTGGCTCCGGCTTAA
OsNRAMP5-246-8-H	GAAGGTCGGAGTCAACGGATTTGTGGTTGGCTCCGGCTTGC
OsNRAMP5-246-8-C	CACAAAATGAAACAGTGGAAACCGATC
OsNRAMP5-234-6-F	GAAGGTGACCAAGTTCATGCTCACAAAATGAAACAGTGGAAACCGAT
OsNRAMP5-234-6-H	GAAGGTCGGAGTCAACGGATTCACAAAATGAAACAGTGGAAACCGAC
OsNRAMP5-234-6-C	CGTACCTCATATCTGTGGTTGGCTC
OsNRAMP5-204-6-F	GAAGGTGACCAAGTTCATGCTCACAAAATGAAACAGTGGAAACCGA
OsNRAMP5-204-6-H	GAAGGTCGGAGTCAACGGATTCACAAAATGAAACAGTGGAAACCGC
OsNRAMP5-204-6-C	CGTACCTCATATCTGTGGTTGGCTC
OsNRAMP5-37-7-F	GAAGGTGACCAAGTTCATGCTTGAAGAAGCTAAGAGAGGAAGCAACAA
OsNRAMP5-37-7-H	GAAGGTCGGAGTCAACGGATTTGAAGAAGCTAAGAGAGGAAGCAACAG
OsNRAMP5-37-7-C	ACTAGCTCTCCAGCTGATGCTC
OsBADH2-168-1-F	GAAGGTGACCAAGTTCATGCTGTGTAGTTGGGTTGATCACACCTT
OsBADH2-168-1-H	GAAGGTCGGAGTCAACGGATTGTGTAGTTGGGTTGATCACACCTC
OsBADH2-168-1-C	TGGGTCATAAATAAATATAAGCGCAGG
OsBADH2-224-8-F	GAAGGTGACCAAGTTCATGCTTTCGTCTTTTCTTGACAGCCTGTT
OsBADH2-224-8-H	GAAGGTCGGAGTCAACGGATTTTCGTCTTTTCTTGACAGCCTGTC
OsBADH2-224-8-C	AGGACTTTTTCCACCAAGTTCCAG
OsLOX3-760-7-F	GAAGGTGACCAAGTTCATGCTTATCTTCACCTATGCCACAAGGC
OsLOX3-760-7-H	GAAGGTCGGAGTCAACGGATTTATCTTCACCTATGCCACAAGGA
OsLOX3-760-7-C	CAGGGTATCATCATCACGTAAGAAC
OsLOX3-115-5-F	GAAGGTGACCAAGTTCATGCTGAGAGCAAAGTGTTGAACATGAAC
OsLOX3-115-5-H	GAAGGTCGGAGTCAACGGATTGAGAGCAAAGTGTTGAACATGAAG
OsLOX3-115-5-C	GGACCGACCCGGTTCTTGAG
OsPAO5-549-5-F	GAAGGTGACCAAGTTCATGCTTGCTTGACAGGAATCCACACCTA
OsPAO5-549-5-H	GAAGGTCGGAGTCAACGGATTTGCTTGACAGGAATCCACACCTG
OsPAO5-549-5-C	GCACCACCATTCCAGATATAGGTG
OsPAO5-1028-4-F	GAAGGTGACCAAGTTCATGCTAACTTTCCTCCAGGGTTACCAAAAT
OsPAO5-1028-4-H	GAAGGTCGGAGTCAACGGATTAACTTTCCTCCAGGGTTACCAAATC
OsPAO5-1028-4-C	GCTTGTCCCATCTTCAACACATAC
OsPAO5-84-2-F	GAAGGTGACCAAGTTCATGCTCTCAGCTCAAGAAAATGCTACCAGG
OsPAO5-84-2-H	GAAGGTCGGAGTCAACGGATTCTCAGCTCAAGAAAATGCTACCACT
OsPAO5-84-2-C	GACAGTACTCAACTGTACATGGTCTG
OsSSIIIa-598-7-F	GAAGGTGACCAAGTTCATGCTCCATTTGGTTCAAGGCCTAGAACTC
OsSSIIIa-598-7-H	GAAGGTCGGAGTCAACGGATTCCATTTGGTTCAAGGCCTAGAACTG
OsSSIIIa-598-7-C	CACTGTTTTCGACGTAGACCATG
OsBEIIb-931-5-F	GAAGGTGACCAAGTTCATGCTGTGTGATTGTTATTAAATCTCACCAGGA
OsBEIIb-931-5-H	GAAGGTCGGAGTCAACGGATTGTGTGATTGTTATTAAATCTCACCAGGC
OsBEIIb-931-5-C	GGCTATCTTAACTTTATGGGAAATGAGTTC

名称 Name	序列 Sequence
OsNRAMP5-246-8-F	GAAGGTGACCAAGTTCATGCTTGTGGTTGGCTCCGGCTTAA
OsNRAMP5-246-8-H	GAAGGTCGGAGTCAACGGATTTGTGGTTGGCTCCGGCTTGC
OsNRAMP5-246-8-C	CACAAAATGAAACAGTGGAAACCGATC
OsNRAMP5-234-6-F	GAAGGTGACCAAGTTCATGCTCACAAAATGAAACAGTGGAAACCGAT
OsNRAMP5-234-6-H	GAAGGTCGGAGTCAACGGATTCACAAAATGAAACAGTGGAAACCGAC
OsNRAMP5-234-6-C	CGTACCTCATATCTGTGGTTGGCTC
OsNRAMP5-204-6-F	GAAGGTGACCAAGTTCATGCTCACAAAATGAAACAGTGGAAACCGA
OsNRAMP5-204-6-H	GAAGGTCGGAGTCAACGGATTCACAAAATGAAACAGTGGAAACCGC
OsNRAMP5-204-6-C	CGTACCTCATATCTGTGGTTGGCTC
OsNRAMP5-37-7-F	GAAGGTGACCAAGTTCATGCTTGAAGAAGCTAAGAGAGGAAGCAACAA
OsNRAMP5-37-7-H	GAAGGTCGGAGTCAACGGATTTGAAGAAGCTAAGAGAGGAAGCAACAG
OsNRAMP5-37-7-C	ACTAGCTCTCCAGCTGATGCTC
OsBADH2-168-1-F	GAAGGTGACCAAGTTCATGCTGTGTAGTTGGGTTGATCACACCTT
OsBADH2-168-1-H	GAAGGTCGGAGTCAACGGATTGTGTAGTTGGGTTGATCACACCTC
OsBADH2-168-1-C	TGGGTCATAAATAAATATAAGCGCAGG
OsBADH2-224-8-F	GAAGGTGACCAAGTTCATGCTTTCGTCTTTTCTTGACAGCCTGTT
OsBADH2-224-8-H	GAAGGTCGGAGTCAACGGATTTTCGTCTTTTCTTGACAGCCTGTC
OsBADH2-224-8-C	AGGACTTTTTCCACCAAGTTCCAG
OsLOX3-760-7-F	GAAGGTGACCAAGTTCATGCTTATCTTCACCTATGCCACAAGGC
OsLOX3-760-7-H	GAAGGTCGGAGTCAACGGATTTATCTTCACCTATGCCACAAGGA
OsLOX3-760-7-C	CAGGGTATCATCATCACGTAAGAAC
OsLOX3-115-5-F	GAAGGTGACCAAGTTCATGCTGAGAGCAAAGTGTTGAACATGAAC
OsLOX3-115-5-H	GAAGGTCGGAGTCAACGGATTGAGAGCAAAGTGTTGAACATGAAG
OsLOX3-115-5-C	GGACCGACCCGGTTCTTGAG
OsPAO5-549-5-F	GAAGGTGACCAAGTTCATGCTTGCTTGACAGGAATCCACACCTA
OsPAO5-549-5-H	GAAGGTCGGAGTCAACGGATTTGCTTGACAGGAATCCACACCTG
OsPAO5-549-5-C	GCACCACCATTCCAGATATAGGTG
OsPAO5-1028-4-F	GAAGGTGACCAAGTTCATGCTAACTTTCCTCCAGGGTTACCAAAAT
OsPAO5-1028-4-H	GAAGGTCGGAGTCAACGGATTAACTTTCCTCCAGGGTTACCAAATC
OsPAO5-1028-4-C	GCTTGTCCCATCTTCAACACATAC
OsPAO5-84-2-F	GAAGGTGACCAAGTTCATGCTCTCAGCTCAAGAAAATGCTACCAGG
OsPAO5-84-2-H	GAAGGTCGGAGTCAACGGATTCTCAGCTCAAGAAAATGCTACCACT
OsPAO5-84-2-C	GACAGTACTCAACTGTACATGGTCTG
OsSSIIIa-598-7-F	GAAGGTGACCAAGTTCATGCTCCATTTGGTTCAAGGCCTAGAACTC
OsSSIIIa-598-7-H	GAAGGTCGGAGTCAACGGATTCCATTTGGTTCAAGGCCTAGAACTG
OsSSIIIa-598-7-C	CACTGTTTTCGACGTAGACCATG
OsBEIIb-931-5-F	GAAGGTGACCAAGTTCATGCTGTGTGATTGTTATTAAATCTCACCAGGA
OsBEIIb-931-5-H	GAAGGTCGGAGTCAACGGATTGTGTGATTGTTATTAAATCTCACCAGGC
OsBEIIb-931-5-C	GGCTATCTTAACTTTATGGGAAATGAGTTC

名称 Name	序列 Sequence
OsNRAMP5-3E-540F	GTTGGTCCTGGATTCATGGTGTC
OsNRAMP5-3E-540R	TCCAATCAGAATCACCCAGAGCAG
OsNRAMP5-1E-265F	TCTTCGTCTACTTCCAGCTAGCC
OsNRAMP5-1E-265R	ACAGTGAACAACTGTCCATGGTG
OsBADH-4-471F	TTGATGAAGCAGCATGGGACATG
OsBADH-4-471R	GCTGCTAGGTACAATTTGTGAGAC
OsBADH-8-357F	CTTCAGCTGCTCCTATGGTTAAGG
OsBADH-8-357R	TCTAGCATCCAGCTCAGTTAAGTGC
OsLOX3-8-535F	GAGAAGAACCTCGAAGGCCTCAG
OsLOX3-8-535R	CCATCACAGCATGTGTGTTGAGC
OsPAO-6/7-563F	GCTCTCTTTGATAAGGATGGTCGTC
OsPAO-6/7-563R	ATGGCCACCAGTAAGAACATGTTC
OsBEIIb-17-359F	GGCATGAGAACCTCACATACTG
OsBEIIb-17-359R	GGAAGTACTTGTGGAGCTCTTGG

名称 Name	序列 Sequence
OsNRAMP5-3E-540F	GTTGGTCCTGGATTCATGGTGTC
OsNRAMP5-3E-540R	TCCAATCAGAATCACCCAGAGCAG
OsNRAMP5-1E-265F	TCTTCGTCTACTTCCAGCTAGCC
OsNRAMP5-1E-265R	ACAGTGAACAACTGTCCATGGTG
OsBADH-4-471F	TTGATGAAGCAGCATGGGACATG
OsBADH-4-471R	GCTGCTAGGTACAATTTGTGAGAC
OsBADH-8-357F	CTTCAGCTGCTCCTATGGTTAAGG
OsBADH-8-357R	TCTAGCATCCAGCTCAGTTAAGTGC
OsLOX3-8-535F	GAGAAGAACCTCGAAGGCCTCAG
OsLOX3-8-535R	CCATCACAGCATGTGTGTTGAGC
OsPAO-6/7-563F	GCTCTCTTTGATAAGGATGGTCGTC
OsPAO-6/7-563R	ATGGCCACCAGTAAGAACATGTTC
OsBEIIb-17-359F	GGCATGAGAACCTCACATACTG
OsBEIIb-17-359R	GGAAGTACTTGTGGAGCTCTTGG

目标基因 Target gene	基因编号 Gene ID	目标表型 Target phenotype	外显子数 No. of exons	目标区域覆盖度 Coverage of target area(%)	平均测序深度 Average sequencing depth
OsNRAMP5	Os07g0257200	镉低积累 Low cadmium accumulation	13	100	53398×
OsBADH2	Os08g0424500	香味Aroma	15	100	53086×
OsLOX3	Os03g0699700	耐储藏Storage-tolerance	9	100	50202×
OsPAO5	Os04g0671300	耐淹 Submergence-tolerance	10	100	54263×
OsSSIIIa	Os08g0191433	低升糖指数 Low glycemic index (GI)	14	100	54510×
OsBEIIb	Os02g0528200	低升糖指数 Low glycemic index (GI)	22	100	52192×

基于M₁TDS靶向筛选技术的重离子束诱变定向改良杂交水稻卓两优1126性状的研究

Directed Improvement of Hybrid Rice Zhuoliangyou 1126 by Heavy Ion Beam Mutagenesis Based on M₁TDS Targeted Screening Technology

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目标基因 Target gene	亲本 Parent	混池编号 No. of the pool	突变位置 Mutation site	野生/突变基因型 Wild/mutant genotype
OsNRAMP5	卓234S Zhuo 234S	217-288E	第3外显子Exon 3	C/CTTAA
	卓234S Zhuo 234S	217-288W	第3外显子Exon 3	C/CTTAA
	卓234S Zhuo 234S	217-288E	第3外显子Exon 3	GA/G
	湘农恢1126 Xiangnonghui 1126	145-216S	第3外显子Exon 3	GCAGAT/G
	湘农恢1126 Xiangnonghui 1126	1-72N	第1外显子Exon 1	CTCAATCTCCAT/C
OsBADH2	卓234S Zhuo 234S	145-216E	第4外显子和第4内含子Exon 4/Intron 4	CCTTGGTATTTCACATTTTTCT/C
OsBADH2	湘农恢1126 Xiangnonghui 1126	217-238S	第8外显子Exon 8	GTT/G
OsLOX3	卓234S Zhuo 234S	718-789W	第7外显子Exon 7	G/GC
	湘农恢1126 Xiangnonghui 1126	73-144S	第9外显子Exon 9	GAAC/G
	湘农恢1126 Xiangnonghui 1126	73-144E	第9外显子Exon 9	GAAC/G
OsPAO5	卓234S Zhuo 234S	504-575W	第6外显子和第6内含子Exon 6/Intron 6	CACTTACTTT/C
	卓234S Zhuo 234S	1004-1040E	第9外显子Exon 9	ATGAT/A
	卓234S Zhuo 234S	991-1040W	第9外显子Exon 9	ATGAT/A
	湘农恢1126 Xiangnonghui 1126	73-144E	第9外显子Exon 9	GTAGCTCC/G
OsSSIIIa	卓234S Zhuo 234S	576-647E	第13外显子Exon 13	CTCG/C
OsBEIIb	卓234S Zhuo 234S	863-932W	第17外显子Exon 17	GATGT/G

基因 Gene	品种 Variety	突变株定位（行-株） Location of mutants (row-plant)	突变分蘖占比 Proportion of tillers containing mutations
OsNRAMP5	卓234S Zhuo 234S	246-8	18/18
OsNRAMP5	卓234S Zhuo 234S	234-6	4/18
OsNRAMP5	湘农恢1126 Xiangnonghui 1126	204-6	3/6
OsNRAMP5	湘农恢1126 Xiangnonghui 1126	37-7	1/8
OsBADH2	卓234S Zhuo 234S	168-1	3/18
OsBADH2	湘农恢1126 Xiangnonghui 1126	224-8	1/6
OsLOX3	卓234S Zhuo 234S	760-7	1/18
OsLOX3	湘农恢1126 Xiangnonghui 1126	115-5	2/6
OsPAO5	卓234S Zhuo 234S	549-5	1/18
OsPAO5	卓234S Zhuo 234S	1028-4	5/18
OsPAO5	湘农恢1126 Xiangnonghui 1126	84-2	1/6
OsSSIIIa	卓234S Zhuo 234S	598-7	1/18
OsBEIIb	卓234S Zhuo 234S	931-5	4/18

材料 Material	株高 Plant height (cm)	有效分蘖数 No. of effective tillers	主穗长 Panicle length(cm)	一次枝梗数 No. of primary branches	二次枝梗数 No. of secondary branches
卓两优1126（对照） Zhuoliangyou 1126（CK）	124.6±3.3 a	13.2±1.3 a	28.9±1.4 a	18.2±1.3 a	99.4±6.4 a
镉低积累卓两优1126 Zhuoliangyou 1126 with low cadmium accumulation	124.8±2.7 a	13.6±1.1 a	27.9±0.9 a	19.0±0.7 a	104.2±5.7 a
材料 Material	总粒数 Spikelets per panicle	实粒数 Grains per panicle	结实率 Seed setting rate(%)	千粒重 1000-grain weight(g)
卓两优1126（对照） Zhuoliangyou 1126（CK）	347.4±39.1 a	301.6±28.9 a	87.0±2.0 a	22.9±0.4 a
镉低积累卓两优1126 Zhuoliangyou 1126 with low cadmium accumulation	360.8±24.1 a	316.8±19.6 a	88.0±2.0 a	22.9±0.5 a

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