Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica

doi:10.3969/j.issn.1004-1524.20240997

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (8): 1648-1657.DOI: 10.3969/j.issn.1004-1524.20240997

• Crop Science • Previous Articles Next Articles

Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica

CONG Jianmin¹(), HUANG Weijian¹, YIN Xinxing¹, LIU Jinping¹, ZHAO Yong²^,^*()

1. Crop Research Institute, School of Agricultural and Biological Engineering, Taizhou Vocational College of Science and Technology, Taizhou 318020, Zhejiang, China
2. College of Life Sciences, Baicheng Normal University, Baicheng 137000, Jilin, China

Received:2024-11-19 Online:2025-08-25 Published:2025-09-03
Contact: ZHAO Yong

Abstract

Abstract:

To investigate the role of 5-azacytidine (5-AzaC) in the epigenetic regulation of genomic DNA methylation during somatic embryogenesis in Fraxinus mandsharica Rupr., somatic embryos were induced by adding 20.0 μmol·L^-1 5-azacytidine to the medium, with untreated samples as the control, methylation-sensitive amplification polymorphism (MSAP) was used to analyze the effects of cytosine methylation levels, patterns, and variations in the genomic DNA of explants on somatic embryogenesis. The results showed that, the browning rate of the explants was reduced by 5-AzaC treatment, which led to alterations in the genomic DNA methylation status, specifically an increase in fully methylated cytosines and a decrease in hemimethylated cytosines. 5-AzaC inhibited cytosine methylation and induced DNA demethylation variation in explant genomic DNA, thereby reducing the induction rate of somatic embryos. These findings demonstrated that epigenetic mechanisms participate in regulating somatic embryogenesis in trees, which provided theoretical guidance for the study of tree somatic embryogenesis.

Key words: Fraxinus mandsharica, somatic embryo, DNA methylation, 5-azacytidine, epigenetics

CLC Number:

S718.3

CONG Jianmin, HUANG Weijian, YIN Xinxing, LIU Jinping, ZHAO Yong. Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica[J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1648-1657.

Figures/Tables 8

References 22

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外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化的CCGG位点数(占比/%) Methylation sites of CCGG(ratio/%)
外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化总位点 Methylation total sites	全甲基化位点 Holomethylation sites	半甲基化位点 Semimethylation site
HT	381	268(70.34)	113(29.66)	68(17.85)	45(11.79)
HY	390	276(70.77)	114(29.23)	68(17.44)	46(11.79)
HD	390	276(70.77)	114(29.23)	71(18.21)	43(11.02)
HW	388	272(70.10)	116(29.90)	75(19.33)	41(10.57)
WD	389	274(70.44)	115(29.56)	75(19.28)	40(10.28)
WW	390	276(70.77)	115(29.23)	69(17.69)	45(11.54)
5-HT	388	272(70.10)	116(29.90)	68(17.53)	48(12.37)
5-HY	389	274(70.44)	115(29.56)	63(16.20)	52(13.36)
5-HD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-HW	389	274(70.44)	115(29.56)	64(16.45)	51(13.11)
5-WD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-WW	389	274(70.44)	115(29.56)	68(17.48)	47(12.08)

外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化的CCGG位点数(占比/%) Methylation sites of CCGG(ratio/%)
外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化总位点 Methylation total sites	全甲基化位点 Holomethylation sites	半甲基化位点 Semimethylation site
HT	381	268(70.34)	113(29.66)	68(17.85)	45(11.79)
HY	390	276(70.77)	114(29.23)	68(17.44)	46(11.79)
HD	390	276(70.77)	114(29.23)	71(18.21)	43(11.02)
HW	388	272(70.10)	116(29.90)	75(19.33)	41(10.57)
WD	389	274(70.44)	115(29.56)	75(19.28)	40(10.28)
WW	390	276(70.77)	115(29.23)	69(17.69)	45(11.54)
5-HT	388	272(70.10)	116(29.90)	68(17.53)	48(12.37)
5-HY	389	274(70.44)	115(29.56)	63(16.20)	52(13.36)
5-HD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-HW	389	274(70.44)	115(29.56)	64(16.45)	51(13.11)
5-WD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-WW	389	274(70.44)	115(29.56)	68(17.48)	47(12.08)

模式类型 Type of Patterns	位点类型Type of sites				HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	未褐化未长大WW		其他类型 Other types
	H	M	H	M
1	-	-	-	-	23	21	15	21	24	21	17	18	16	19	19
2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
3	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
4	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
5	+	-	+	-	44	44	42	41	40	45	45	42	44	43	42
6	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
7	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
8	-	+	-	+	43	44	44	45	45	45	44	43	43	43	43
9	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
10	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
11	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
12	+	+	+	+	274	272	272	270	270	272	274	272	272	272	272

模式类型 Type of Patterns	位点类型Type of sites				HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	未褐化未长大WW		其他类型 Other types
	H	M	H	M
1	-	-	-	-	23	21	15	21	24	21	17	18	16	19	19
2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
3	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
4	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
5	+	-	+	-	44	44	42	41	40	45	45	42	44	43	42
6	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
7	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
8	-	+	-	+	43	44	44	45	45	45	44	43	43	43	43
9	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
10	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
11	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
12	+	+	+	+	274	272	272	270	270	272	274	272	272	272	272

变异类型 Mutation types		带型Types				数量Number
		未褐化未长大WW		其他类型 Other types		HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
		H	M	H	M	HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
A	A1	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
	A2	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
B	B1	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
	B2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
C	C1	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
	C2	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
D	D1	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
	D2	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
总计Total						6	9	17	13	11	7	10	15	15	13	14

Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica

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模式类型 Pattern types	位点类型Site types				数量Number
	对照Control		其他类型Other types		5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	H	M	H	M	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
1	-	-	-	-	21	16	11	16	21	19
2	+	-	-	-	2	5	6	7	6	5
3	-	+	-	-	1	0	0	1	0	0
4	-	-	+	-	0	0	2	0	0	3
5	+	-	+	-	45	46	41	41	40	42
6	-	-	-	+	0	2	9	2	1	1
7	+	-	-	+	0	0	0	2	0	0
8	-	+	-	+	44	44	44	45	45	43
9	+	+	-	+	0	1	1	2	2	1
10	+	-	+	+	1	1	1	1	2	0
11	-	+	+	+	2	1	1	0	0	2
12	+	+	+	+	272	272	272	270	270	272

甲基化变异类型 Methylation mutation types		数量Number
甲基化变异类型 Methylation mutation types		5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
甲基化上升位点	CNG位点升高CNG increase sites	0	0	0	0	0	0
Methylation increase site	CG位点升高CG increase sites	0	1	1	2	2	1
	CG/CNG位点升高CG/CNG increase sites	0	0	0	0	0	0
甲基化位点上升总数(比例)Total methylation increase sites(rate)		0(0)	1(20%)	1(7.15%)	2(40%)	2(40%)	1(40%)
甲基化下降的位点	CNG位点下降CNG decreased site	1	3	12	3	3	1
Decreased methylation site	CG位点下降CG decreased site	2	1	1	0	0	2
	CG/CNG下降位点CG/CNG decreased site	0	0	0	0	0	0
甲基化位点下降总数(比例)Total methylation decreased sites(rate)		3(100%)	4(80%)	13(92.85%)	3(60%)	3(60%)	3(75%)
甲基化总变异位点Total methylation mutation sites		3	5	14	5	5	4

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