一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证

doi:10.3864/j.issn.0578-1752.2025.11.002

中国农业科学 ›› 2025, Vol. 58 ›› Issue (11): 2062-2080.doi: 10.3864/j.issn.0578-1752.2025.11.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证

曾跃辉¹^,²(), 邹文广²^,³(), 赵福铭¹^,², 肖长春¹^,², 黄建鸿¹^,², 马彬林²^,³, 杨旺兴²^,³, 韦新宇¹^,²(), 许旭明²^,³()

¹ 三明市农业科学研究院生物技术研究所，福建三明 365500
² 福建省（山区）作物遗传改良与创新利用重点实验室，福建三明 365500
³ 三明市农业科学研究院水稻研究所，福建三明 365500

收稿日期:2024-11-20 接受日期:2024-12-19 出版日期:2025-06-01 发布日期:2025-06-09
通信作者:
韦新宇，E-mail：wxy1209@163.com。
许旭明，E-mail：fj63xxm@sina.com
联系方式: 曾跃辉，E-mail：1_zengyuehui_1@163.com。邹文广，E-mail：52587656@qq.com。曾跃辉和邹文广为同等贡献作者。
基金资助:
财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-01); 福建省自然科学基金(2021J01535); 福建省自然科学基金(2021J01536); 三明市科技计划(2022-N-7); 三明市科技计划(2024-N-3)

Map-Based Cloning and Functional Verification of A Novel Split Glume Gene OsSG2 in Rice (Oryza sativa L.)

ZENG YueHui¹^,²(), ZOU WenGuang²^,³(), ZHAO FuMing¹^,², XIAO ChangChun¹^,², HUANG JianHong¹^,², MA BinLin²^,³, YANG WangXing²^,³, WEI XinYu¹^,²(), XU XuMing²^,³()

¹ Biotechnology Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian
² Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountainous Areas, Sanming 365500, Fujian
³ Rice Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian

Received:2024-11-20 Accepted:2024-12-19 Published:2025-06-01 Online:2025-06-09

摘要/Abstract

摘要：

【目的】水稻颖花开裂突变体sg2（split glume 2）来源于三明市农业科学研究院自主选育的恢复系GK1327高世代育种群体的自然突变，通过对其颖花开裂基因OsSG2（Oryza sativa Split Glume 2）进行遗传学分析、图位克隆和功能验证，为进一步开展OsSG2在水稻花器官发育中的功能研究奠定基础和提供重要种质资源。【方法】通过对突变体sg2颖花进行形态特征解剖观察，分析其花器官发育突变特点。通过对野生型WT（GK1327）与突变体sg2表型和农艺性状进行调查，以及花粉在I₂-KI溶液中的染色观察，分析该突变表型对主要产量性状和花粉育性的影响。利用突变体sg2分别与野生型WT和9311杂交，构建多个F₂分离群体，对OsSG2进行遗传学分析和物理定位。采用集群分离分析法（bulked segregant analysis，BSA）和二代测序（next-generation sequencing，NGS）相结合的BSA-seq技术，以及半定量PCR和实时荧光定量PCR对候选基因进行筛选和表达模式分析。结合PCR扩增和测序技术对候选基因进行克隆和序列分析。通过CRISPR-Cas9基因编辑技术和转基因互补试验对OsSG2候选基因进行功能验证。【结果】该突变体在生殖生长时期花器官发育出现明显异常，其颖花内外稃片瘪弱，表现出不同程度的扭曲变形，且开裂不抱合表型，穗部颖花发育不正常的比例达到100%。相比野生型，突变体sg2结实率和千粒重显著降低，种子萌发率显著下降，花粉育性未出现明显异常。遗传分析表明，sg2突变表型由单个细胞核隐性基因控制。通过图位克隆技术，将OsSG2精细定位在水稻第3染色体短臂端，位于2个InDel（insertion/deletion）标记InDel-12083和InDel-17610之间，物理距离为919.85 kb，该区间共注释75个开放阅读框（open read frames，ORFs）。利用BSA-seq技术，鉴定到一个潜在的OsSG2候选基因LOC_Os03g11614，该基因编码一个定位于细胞核的OsMADS1转录因子。表达和序列分析表明，相比野生型，LOC_Os03g11614在突变体sg2抽穗期幼穗中的表达水平极显著下调，且该基因编码序列（coding sequence，CDS）在突变体中发生单个核苷酸多态性（single nucleotide polymorphism，SNP）突变，即LOC_Os03g11614第1外显子第3个碱基由G突变为A，导致其起始密码子由ATG突变为ATA，进而影响该基因的转录表达和相应蛋白OsMADS1的结构和功能。CRISPR-Cas9基因编辑技术和功能互补试验进一步证实候选基因LOC_Os03g11614即为OsSG2。【结论】鉴定并克隆获得的水稻颖花开裂基因OsSG2为一个新的OsMADS1等位突变基因。

关键词: 水稻, 花器官, OsSG2, 图位克隆, 基因编辑, OsMADS1

Abstract:

【Objective】 The sg2 (split glume 2) is a naturally occurring rice mutant derived from an advanced-generation breeding population of the restorer line GK1327 originating from the Sanming Academy of Agricultural Sciences (SAAS). In this work, the OsSG2 (Oryza sativa Split Glume 2) gene in the mutant was performed to conduct genetic analysis, map-based cloning and functional verification, which will provide a theoretical basis and an important germplasm resource for further study on the function of OsSG2 in the development of rice floral organs.【Method】 The morphological characteristics of the sg2 mutant florets were anatomically observed to analyze the features of floral organ development. By investigating the phenotypic and agronomic characteristics of wild-type GK1327 and sg2 mutant, and observing the pollen staining in I₂-KI, the effects of the mutant phenotype on yield-related traits and pollen fertility were analyzed. The F₂ populations developed by crossing sg2 mutant with wild-type GK1327 and 9311, respectively, were used to perform genetic analysis and physical mapping of the OsSG2. Bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) technology (BSA-seq) was used to screen the candidate genes of OsSG2, and a combination of RT-PCR (reverse-transcription PCR) and qRT-PCR (quantitative real-time PCR) was performed to analyze their expression patterns. The OsSG2 candidate genes were cloned and sequenced by PCR amplification and sequencing approaches, and the functional verification was conducted by knockout using CRISPR-cas9 and a complementation test.【Result】 The sg2 mutant exhibited an abnormal spikelet phenotype at the reproductive stage, the palea and lemma of florets were weak, distorted, and dehiscent, and with the proportion of abnormal florets in the panicles were 100%. Compared with the wild-type GK1327, the seed setting rate, the 1000-grain weight, and the seed germination rate of sg2 mutant were markedly lower, whereas the pollen fertility was normal. Genetic analysis demonstrated that a recessive nuclear gene was responsible for the mutant phenotype of sg2. A map-based cloning strategy delimited OsSG2 to a 919.85 kb physical interval on the short arm of chromosome 3, flanked by two InDel (insertion/deletion) markers, InDel-12083 and InDel-17610, that included 75 putative ORFs (open read frames). Moreover, a BSA-seq technology identified a potential candidate gene LOC_Os03g11614 as the OsSG2 gene, which encodes an OsMADS1 transcription factor localized to the nucleus. Expression and sequence analyses revealed that the transcript levels of LOC_Os03g11614 were markedly lower in young panicles of the sg2 mutant compared with those in the wild-type GK1327 during the flowering stage, and a SNP (single nucleotide polymorphism) mutation (G-A) was identified at the third nucleotide position of exon 1 in the LOC_Os03g11614, resulting in the change of the start codon from ATG to ATA, which caused a transcriptional suppression of LOC_Os03g11614 and a functional loss of the OsMADS1 protein. These results further supported the tentative identification of LOC_Os03g11614 as the candidate OsSG2 gene, which was confirmed by knockout using CRISPR-cas9 and a functional complementation test.【Conclusion】 The split glume gene OsSG2, which was successfully isolated from sg2 mutant in this work, was identified as a new allele of OsMADS1 in rice.

Key words: rice (Oryza sativa L.), floral organ, OsSG2, map-based cloning, gene editing, OsMADS1

曾跃辉, 邹文广, 赵福铭, 肖长春, 黄建鸿, 马彬林, 杨旺兴, 韦新宇, 许旭明. 一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证[J]. 中国农业科学, 2025, 58(11): 2062-2080.

ZENG YueHui, ZOU WenGuang, ZHAO FuMing, XIAO ChangChun, HUANG JianHong, MA BinLin, YANG WangXing, WEI XinYu, XU XuMing. Map-Based Cloning and Functional Verification of A Novel Split Glume Gene OsSG2 in Rice (Oryza sativa L.)[J]. Scientia Agricultura Sinica, 2025, 58(11): 2062-2080.

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杂交组合 Cross combinations	F₂表型F₂ phenotype			χ²	χ²_0.05	P
杂交组合 Cross combinations	分离群体 Segregation population	野生型 WT	突变型 Mutant type	χ²	χ²_0.05	P
sg2×WT	1136	863	273	0.565	3.84	0.8513
sg2×9311	1448	1091	357	0.092	3.84	0.9741
9311×sg2	1040	798	243	1.661	3.84	0.7175

引物名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Usage
Rm14462	TCTGTATTCAAGGAGGGCTAGATGC	GCTGGAGAGATGCATTGACTGG	OsSG2初步定位 Preliminary mapping for OsSG2
Rm3392	AGCAACCAACCCAGTAGTTAGCC	GCTCATTTGCATGCTGTGTTAGC
Rm14722	ACACACCACATTGTTGGCATTGG	TGGTGGGAGGAAGATTTCAGTGG
Rm14728	CTCAGCGGACTAGAATGCAAGC	AAACGGATACATCTTCGCAGAACG
Rm7	TTCGCCATGAAGTCTCTCG	CCTCCCATCATTTCGTTGTT
InDel-2923	GCCTAAATTTGCAGTGATGTG	TACCAGTGCCCAATAATCGT	OsSG2精细定位 Fine mapping for OsSG2
InDel-12083	GCAATCAGGTCGCGTATCAG	GGTTGGGTGTTCGACGTATC
InDel-17610	ACGAATGTAACTTTGGCATGTCA	ATGCTCTGAGGTGATTACTTTT
InDel-31068	AAGCCCCGTTTGACTTAGGT	AAGCCCCGTTTGACTTAGGT
InDel-31355	TTAGGCCCAGTTTTGCTCTC	CTCCCTCCGTCCCGAAAA
OsMADS1-Q	TTGGAGAGGTACCGCAGC	TCTGGTTCTCAAGCTGCTCC	OsMADS1的qRT-PCR检测 qRT-PCR detection for OsMADS1
OsMADS1-Seq01	CGACGTGACAGCAGTTTGAT	AGCACTAAAAGCACGCGAAA	OsMADS1的测序 Sequencing for OsMADS1
OsMADS1-Seq02	TGCGCAAATCAAAACTGTACAG	ATTGCGGCCCATCTAAACTG
OsMADS1-Seq03	TAGGGCGCATTGTGACTTTG	GCCTGTGTTAAAAGCCATGC
OsMADS1-Seq04	AGCAAGCACTTCACACCATG	AGAACAGTGCTAGAACCGCT
OsMADS1-Seq05	AGCCATCGATCACCCTGAAA	GTCTGCTGCTTCATTGCTCA
OsMADS1-Seq06	CCATCAGGGTCTTCTCCACC	TGAGATAACAGCTGCCGTCT
OsActin-F/R	ACCTTCAACACCCCTGCTAT	CACCATCACCAGAGTCCAAC	OsActin的qRT-PCR检测 qRT-PCR detection for OsActin (as internal standard)
FMHPT-F/R	ATTTGTGTACGCCCGACAGT	GTGCTTGACATTGGGGAGTT	PCR鉴定HPT PCR detection for HPT
FMCas9-F/R	AGAACCTCTCCGATGCTATCC	AGCAAGAGGACCAACGTAG	PCR鉴定Cas9 PCR detection for Cas9

样本 Samples	原始数据量 Raw base (Gb)	有效数据量 Clean base (Gb)	有效数据占比 Clean base percent (%)	>Q20 (%)	>Q30 (%)	GC含量 GC content (%)
9311	26.84	26.28	97.93	97.67	93.43	42.80
sg2	27.78	27.20	97.92	97.65	93.39	42.92
WT-Pool	29.36	28.68	97.71	97.84	93.86	42.81
MT-Pool	30.73	30.03	97.73	97.81	93.79	42.82

样本 Samples	有效序列数 Total reads	比对序列数 Mapped reads	比对率 Mapped rate (%)	平均覆盖深度 Average coverage (×)	1×覆盖度 1×coverage (%)	10×覆盖度 10×coverage (%)
9311	176181050	136126271	98.23	52.7028	93.72	88.85
sg2	182366852	140044150	98.16	54.2271	93.16	88.38
WT-Pool	192599862	145760573	98.13	56.2530	94.71	90.18
MT-Pool	201645008	152345579	98.11	58.7479	94.74	90.45

一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证

Map-Based Cloning and Functional Verification of A Novel Split Glume Gene OsSG2 in Rice (Oryza sativa L.)

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