蒺藜苜蓿开花过程中的全基因组DNA甲基化分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0366

摘要/Abstract

摘要：

目的探索在长日照条件下，蒺藜苜蓿开花过程中的DNA甲基化变化，探究光周期相关基因与DNA甲基化的可能关系。方法利用全基因组亚硫酸氢盐测序（WGBS）检测营养期和开花期的蒺藜苜蓿叶片，分析DNA甲基化差异。结果（1）甲基化类型主要为CG型（69.74%），其次是CHG（35.22%）和CHH（21.81%）；基因上游的CHG甲基化，以及基因上下游及基因体区域的CHH甲基化水平均是营养期比开花期的高。（2）差异甲基化区域（DMR）共有20 647个，其中CHH型最多（11 247个），且68%为低甲基化区域。（3）基于基因本体（GO）和信号通路（KEGG）对全部DMR关联基因（DMG）进行分析，发现DMG主要参与在高亲和力寡肽跨膜转运蛋白活性、FAD结合核苷三磷酸酶活性、ATP酶活性、水解酶活性，作用于酸酐四氢叶酸生物合成过程等通路中。（4）参与光周期开花途径关键基因CRY1、CRY2、FKF1、PHYA、ELF3、COL2、FT、LHY、ZTL都发生了显著的甲基化水平改变。结论 DNA甲基化可能参与调控蒺藜苜蓿开花，探索了蒺藜苜蓿光周期诱导成花过程中DNA甲基化的可能角色，为蒺藜苜蓿育种研究提供新见解。

关键词: 蒺藜苜蓿, 开花, DNA甲基化, 转录因子, 光周期

Abstract:

Objective To investigate DNA methylation variations during flowering in Medicago ruthenica under long-day conditions and explore the potential relationship between photoperiod-related genes and DNA methylation. Method Whole-genome bisulfite sequencing (WGBS) was performed on M. ruthenica leaves at the vegetative and flowering stages to analyze differences in DNA methylation. Result 1) The predominant type of methylation was CG (69.74%), followed by CHG (35.22%) and CHH (21.81%). CHG methylation upstream of genes and CHH methylation in upstream, downstream, and gene body regions were all higher during the vegetative stage than in the flowering stage. 2) A total of 20 647 differentially methylated regions (DMRs) were identified, with CHH-type DMRs accounting for the majority (11 247), and 68% of them showing hypomethylation. 3) Gene Ontology (GO) and KEGG pathway analyses of all DMR-associated genes (DMGs) revealed that these genes were mainly involved in high-affinity oligopeptide transmembrane transporter activity, FAD-binding nucleoside triphosphatase activity, ATPase activity, and hydrolase activity acting on acid anhydrides, as well as in pathways such as tetrahydrofolate biosynthesis. 4) Key genes in the photoperiodic flowering pathway, including CRY1, CRY2, FKF1, PHYA, ELF3, COL2, FT, LHY, and ZTL,presented significant changes in DNA methylation levels. Conclusion DNA methylation may be involved in the regulation of flowering in M. ruthenica. This study provides new insights into the potential role of DNA methylation in photoperiod-induced flowering, offering valuable information for M. ruthenica breeding research.

Key words: Medicago truncatula, flowering, DNA methylation, transcription factors, photoperiod

蒋天威, 李亚娇, 马培杰, 陈才俊, 刘晓霞, 陈莹, 王小利. 蒺藜苜蓿开花过程中的全基因组DNA甲基化分析[J]. 生物技术通报, 2025, 41(11): 301-310.

JIANG Tian-wei, LI Ya-jiao, MA Pei-jie, CHEN Cai-jun, LIU Xiao-xia, CHEN Ying, WANG Xiao-li. Whole-genome DNA Methylation Analysis during the Flowering Processof Medicago truncatula[J]. Biotechnology Bulletin, 2025, 41(11): 301-310.

图/表 7

表1 蒺藜苜蓿全基因组甲基化测序数据处理

Table 1 Processing of whole-genome methylation sequencing data of Medicago truncatula

样本 Sample	总读取数 Total reads	比对读取数 Mapped reads	唯一比对读取数 Uniquely reads	测序深度 Seqencing depth	唯一比对率 Uniquely mapped reads （%）	比对率 Mapping rate （%）	Q30 （%）	转换率 Conversion rate （%）
FP_1	107573870	62923962	43437098	34.38	40.38	58.49	90.98	99.69
FP_2	93350734	52038408	34920690	29.86	37.41	55.75	91.34	99.67
FP_3	114432912	66064890	45284390	36.65	39.57	57.73	91.97	99.65
NP_1	103921110	60296822	44356076	33.25	42.68	58.02	91.44	99.67
NP_2	120328788	72294208	50808854	38.52	42.23	60.08	91.87	99.59
NP_3	129107620	74594082	51931090	41.32	40.22	57.78	91.59	99.61

图1 蒺藜苜蓿从营养期到开花期DNA甲基化的变化A：样品相关性分析；B：3种序列下甲基化水平统计；C：基因体区域及其侧翼区域内3种序列的甲基化的分布。FP为开花期，NP为营养期。下同

Fig. 1 Variations in DNA methylation from the vegetative stage to the flowering stage in M. truncatulaA: Sample correlation analysis. B: Methylation level statistics for the three sequences. C: Methylation distribution in gene body regions and their flanking regions for the three sequences. FP refers to the flowering stage, and NP refers to the vegetative stage. The same below

图2 FP vs NP中差异甲基化区域分析A：3种序列下差异甲基化区域聚类热图；B：差异甲基化区域数量统计图；C：差异甲基化区域在基因组的分布比率图。图B、C中Hyper为高甲基化，Hypo为低甲基化；图C中图例展示了基因间区（Intergenic）、内含子（Intron）、外显子（Exon）、3'非翻译区（3' UTR）、启动子（Promoter）和5'非翻译区（5' UTR）

Fig. 2 Differentially methylated region analysis between FP and NPA: Clustering heatmap of differentially methylated regions (DMRs) in the three sequences. B: Statistical chart of the number of DMRs. C: Proportional distribution of DMRs in the genome. In figure B and C, Hyper indicates hypermethylation, and Hypo indicates hypomethylation

图3 FP vs NP的CG 、CHG和CHH类型DMR 关联基因的 GO（A、B、C）、KEGG（D、E、F）富集分析PPAH： hydrolase activity， acting on acid anhydrides， in phosphorus-containing anhydrides

Fig. 3 GO (A, B, C) and KEGG (D, E, F) enrichment analysis of genes associated with CG, CHG, and CHH types of DMRs in FP vs NP

图4 DMRs关联的转录因子分析A： FP vs NP中DMR关联基因中转录因子编码基因的比例；B、C、D：3种序列背景下DMR关联转录因子家族丰度词云图。图B对应CG、图C对应CHG、图D对应CHH；图B、C、D中文字越大丰度越高

Fig. 4 Transcription factor analysis associated with DMRsA: Proportion of transcription factor-encoding genes among DMR-associated genes in FP vs NP. B, C, D: Word clouds showing the abundance of DMR-associated transcription factor families under three sequence contexts. Figure B corresponds to CG, Figure C to CHG, and Figure D to CHH; the larger the text in Figure B, C, and D, the higher the abundance

图5 转录因子编码基因的蛋白互作网络图A：CG序列下蛋白互作网络；B：CHG序列下蛋白互作网络；C：CHH序列下蛋白互作网络。节点代表蛋白，线代表互作关系，节点旁红色文字为关键节点基因编号

Fig. 5 Protein-protein interaction network of transcription factor-encoding genesA: Protein-protein interaction network under CG context. B: Protein-protein interaction network under CHG context. C: Protein-protein interaction network under CHH context. Nodes indicate proteins, edges indicate interaction relationships, and red text next to the nodes indicates the gene IDs of key nodes

表2 与光周期相关的DMR关联基因

Table 2 DMR-associated genes related to photoperiod

序列类型 Type	基因 Gene	去甲基化 Hypo-DMR	超甲基化 Hyper-DMR
CG	ACTR4	-	Up
	GA20ox1	-	Up
	CRY1	-	Up
	UBOX13	-	Up
	NFYB3	-	Up
	CRY2	-	Up
	MYB1	-	Up
	CHS	-	Up
	SRO1	Down	-
	BHLH130	Down	-
CHG	CCX5	-	Up
	VOZ1	-	Up
	ACTR4	-	Up
	ATX2	-	Up
	FKF1	-	Up
	MYB75	-	Up
	PHYA	-	Up
	ELF3	-	Up
	UPF2	Down	-
	ELF6	Down	-
	BRN1	Down	-
	CHS	Down	-
	COL 2	Down	-
CHH	EZA1	-	Up
	PEPPER	-	Up
	MYB75	-	Up
	CSNK2A	-	Up
	CHS9	-	Up
	CHS2	Down	-
	DMR6-LIKE OXYGENASE 2	Down	-
	RENT3	Down	-
	FD	Down	-
	CCA1	Down	-
	DMR6-LIKE OXYGENASE 2	Down	-
	bHLH122	Down	-
	MYST family 2	Down	-
	COL 9	Down	-
	EZA1	Down	-
	BRN1	Down	-
	AGL18	Down	-
	FT	Down	-
	MYB114	Down	-
	LHY	Down	-
	MYB1	Down	-
	CHS	Down	-
	ZTL	Down	-
	COL2	Down	-

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