Bioinformatics Analysis, Development and Application of Molecular Markers for Seed Dormancy Gene Sdr4 in Rice

doi:10.16819/j.1001-7216.2026.250211

Abstract

Abstract:

【Objective】Pre-harvest sprouting (PHS) severely compromises rice yield and grain quality, which is strongly influenced by seed dormancy genes. This study aims to elucidate the biological function of the seed dormancy gene Sdr4 and develop molecular markers for germplasm improvement, providing theoretical and practical insights for PHS-resistant rice breeding.【Method】A combination of bioinformatics analysis, molecular marker development, and marker-assisted selection was employed to systematically investigate Sdr4 functionality. Allelic variations between the strong-dormancy accession Kasalath and the weak-dormancy accession Nipponbare were analyzed to design genotype-specific markers.【Result】The Sdr4 promoter harbors core regulatory elements (e.g., TATA-box) and ABA-responsive cis-elements. The encoded protein is hydrophobic, exhibits low stability, and is extensively phosphorylated (25.93% within amino acids 101-150). Tertiary structure prediction indicated a composition of 15.50% α-helices, 15.50% β-sheets, and 66.37% random coils. Haplotype analysis of the 3K Rice Genome panel combined with AlphaFold modeling revealed that Sdr4 comprises six functional mutation-associated haplotypes. Furthermore, multiple amino acid sequence differences were identified in the Sdr4 protein between Kasalath and Nipponbare. Phylogenetic analysis demonstrated high conservation with Oryza glaberrima and Zizania palustris. Two functional markers (Sdr4-KF/KR and Sdr4-PF/PR) were developed to discriminate dormancy strength, and their efficacy was validated across 25 rice cultivars. Application of these markers in improving Zhongzhu 18 successfully generated novel lines with enhanced seed dormancy.【Conclusion】Sdr4 plays a critical role in regulating PHS resistance through the modulation of seed dormancy. Molecular screening confirmed weak dormancy phenotypes in 20 commercial varieties. The improved lines K17 and K88, developed via Sdr4-specific markers, exhibited significantly enhanced PHS resistance. This study establishes a framework for Sdr4 functional analysis and accelerates the molecular breeding of PHS-resistant rice germplasm.

Key words: pre-harvest sprouting, Sdr 4, rice, bioinformatics analysis, molecular marker-assisted selection

摘要：

【目的】水稻收获前穗发芽严重制约产量与品质形成，该性状主要受种子休眠基因调控。解析种子休眠分子机制对改良水稻穗萌抗性具有重要理论价值与育种意义。【方法】综合运用生物信息学分析、分子标记开发及标记辅助选择技术，系统解析种子休眠基因Sdr4(Seed dormancy 4)的生物学功能，探究其分子标记在多基因型种质改良中的应用潜力。【结果】Sdr4启动子区含TATA-box等核心元件及ABA响应元件等多种顺式作用元件，其编码蛋白为低稳定性疏水蛋白(101~150氨基酸区段磷酸化修饰占比25.93%)，三级结构以α-螺旋(15.50%)、β-折叠(15.50%)和无规则卷曲(66.37%)为主。3K单倍型数据库与AlphaFold分析表明Sdr4基因具有6种功能性突变的单倍型，且在强休眠种质Kasalath与弱休眠种质日本晴(Nipponbare)的Sdr4氨基酸序列存在多个位点差异。系统发育分析显示Sdr4在非洲栽培稻(Oryza glaberrima)与沼生菰(Zizania palustris)中具有高度保守性。通过Kasalath与日本晴的Sdr4等位变异，开发了Sdr4-KF/KR与Sdr4-PF/PR两对功能标记，可精准区分种子休眠强度。基于25个水稻品种的分子检测筛选出休眠性显著差异的种质，并利用上述标记成功创制了强休眠型中组18改良系。【结论】Sdr4通过调控种子休眠深度影响水稻穗萌抗性。分子标记筛选表明20个主栽品种均呈种子弱休眠表型，利用Sdr4特异性标记选育的K17与K88新品系具有显著穗萌抗性。本研究为Sdr4功能解析及水稻抗穗萌分子育种提供了理论支撑与技术储备。

关键词: 穗发芽, Sdr4, 水稻, 生物信息学, 分子标记辅助选择

HUANG Qina, JIANG Hongrui, YANG Jie, YU Kunyu, YANG Changdeng, LIANG Yan. Bioinformatics Analysis, Development and Application of Molecular Markers for Seed Dormancy Gene Sdr4 in Rice[J]. Chinese Journal OF Rice Science, 2026, 40(1): 61-71.

黄奇娜, 姜鸿瑞, 杨婕, 于坤宇, 杨长登, 梁燕. 种子休眠基因Sdr4的生物信息学分析与分子标记开发和应用[J]. 中国水稻科学, 2026, 40(1): 61-71.

Figures/Tables 8

Table 1. Whole-genome amplification primers for Sdr4 and functionally validated molecular markers

引物名称 Primer	引物序列(5’-3’) Sequence(5’-3’)	片段长度 Length(bp)	基因组位点 Location
Sdr4-F	CTTCTTAACCCCACCACCCC	1161	引物5’端距离ATG上游102 bp 102 bp upstream of ATG
Sdr4-R	TTTGCTCCGGCTTGATGCATC	1161	引物3’端距离TGA下游27 bp 27 bp downstream of TGA
Sdr4-KF	AAGGTCATCTCGCCGCGCG	197	距离ATG 493~511 bp编码区 Coding region 493-511 bp from ATG
Sdr4-KR	GTTGCTGGAGTCCGAGACC	197	距离ATG 702~720 bp编码区 Coding region 702-720 bp from ATG
Sdr4-PF	AAGCTGCTGGAGCCG TGG	227	距离ATG 493~509 bp编码区(画横线为引入的错配碱基) Coding region 493-509 bp from ATG (underlined bases are introduced mismatches)
Sdr4-PR	CTTGTACGCGTCGTTCACC	227	距离ATG 672~690 bp编码区 Coding region 672-690 bp from ATG

Table 2. cis-acting elements in the promoter region of Sdr4

类别 Type	顺式作用元件 cis-acting element	序列 Sequence	数量 Number	功能 Function
激素相关作用元件Hormone-related response elements	ABRE	ACGTG/CACGTG	11	脱落酸响应元件 Abscisic acid response element
	TCA-element	CCATCTTTTT	1	水杨酸途径响应元件 Salicylic acid pathway response element
	CGTCA/TGACG-motif	CGTCA	10	茉莉酸甲酯调控元件 Methyl jasmonate regulatory element
逆境相关作用元件 Stress-related response elements	ARE	AAACCA	3	厌氧诱导调控元件 Anaerobic induction regulatory element
逆境相关作用元件 Stress-related response elements	MYB/MYC	CAACCA/CATGTG	4	干旱诱导响应元件 Drought-induced response element
生长发育相关作用元件Growth and development- related response elements	RY-element	CATGCATG	4	种子特异性调控元件 Seed-specific regulatory element
生长发育相关作用元件Growth and development- related response elements	Sp1	GGGCGG	7	光照响应元件 Light-responsive element

Fig. 1. Phylogenetic tree (A), protein phosphorylation (B) and haplotype analysis (C, D) with 3K of Sdr4

Fig. 2. Sequence alignment and protein structure prediction of Sdr4 A, Alignment of DNA sequences at different sites and the location of the forward primer F (highlighted in green) for molecular marker development; B, Alignment of the Sdr4 amino acid sequences in Kasalath and Nipponbare; C, Prediction of the secondary structure of Sdr4 in Kasalath and Nipponbare; D, AlphaFold analysis of the three-dimensional structure of Sdr4 in Kasalath and Nipponbare.

Fig. 3. Analysis of detection results of molecular markers Sdr4-KF/KR (A) and Sdr4-PF/PR (B) Ka, Kasalath; Nip, Nipponbare; Mar, Easy 2K DNA marker.

Fig. 4. Analysis of molecular marker detection results in diverse rice varieties 1, Kasalath; 2, Nip; 3, IR24; 4, IR36; 5, IR64; 6, Zhongzu 1; 7, Zhongzu 3; 8, Zhongzu 9; 9, Zhongzao 22; 10, Zhongzao 35; 11, Zhongzao 39; 12, Zhongzu 18; 13, Zhongleng 23; 14, Zhongjiazao 10; 15, Zhe 106; 16, Zhenong 131; 17, Zhenong 135; 18, Yongxian 15; 19, Yongxian 57; 20, Yongxian 378; 21, Wen 723; 22, Taizao 502; 23, Taizao 816; 24, Jinzao 47; 25, Zaoxian 310; M, Easy 2K DNA marker.

Fig. 5. Seed germination phenotypes of different rice varieties

Fig. 6. Breeding process of new materials resistant to panicle sprouting (A), molecular marker detection results and their phenotypes (B) Genomic DNA from the rice varieties ZZ18, IR36, K17, and K88 was amplified using the Sdr4-KF/KR (197 bp) and Sdr4-PF/PR (227 bp) primers, respectively. Seeds of ZZ18, K17, and K88 were soaked for 48 at room temperature and subsequently incubated at 37℃ for 48 h to promote germination. Following germination, phenotypic characterization was performed, and images were captured for documentation. M, Marker.

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