Analysis of differentially methylated regions of genomic DNA in maize (Zea mays L.) exposed to salt stress

Figures & data

Table 1. Adaptors and primers used in MSAP analysis.

Adaptors/primers	Type	Sequences
Adaptors	EcoRI		5′-CTCGTAGACTGCGTACC-3 3′-CTGACGCATGGTTAA-5ʹ
	HpaII-MspI		5′-GACGATGAGTCTAGAA-3ʹ 3′-CTACTCAGATCTTGC-5ʹ
Pre-amplification primers	EcoRI		5′-GACTGCGTACCAATTCA-3ʹ
	HpaII-MspI		5′-GATGAGTCTAGAACGGT-3ʹ
Selective amplification primers	E1		5′-GACTGCGTACCAATTCAAC-3ʹ
	E2		5′-GACTGCGTACCAATTCAAG-3ʹ
	E3		5′-GACTGCGTACCAATTCACA-3ʹ
	E4		5′-GACTGCGTACCAATTCACT-3ʹ
	HM1		5′-GATGAGTCTAGAACGGTAG-3ʹ
	HM2		5′-GATGAGTCTAGAACGGTAC-3ʹ
	HM3		5′-GATGAGTCTAGAACGGTTG-3ʹ
	HM4		5′-GATGAGTCTAGAACGGTTC-3ʹ
	HM5		5′-GATGAGTCTAGAACGGTGT-3ʹ
	HM6		5′-GATGAGTCTAGAACGGTGC-3ʹ
	HM7		5′-GATGAGTCTAGAACGGTCT-3ʹ
	HM8		5′-GATGAGTCTAGAACGGTCG-3ʹ

Table 2. Primers for real-time quantitative PCR.

Sequencing No./Gene name	GenBank Accession	Primer Sequence(5ʹ → 3ʹ)
HXC6-2	AC185486.5	-F-	GAGCGATTGAACACGAGGTC
		-R-	TACTTCTAAAATGCCGGCGC
HXC7-2	AC196472.3	-F-	AAGATGGAGTTGGTGGAGGG
		-R-	GAACTTCTTCGGGCGACATC
HXC8-1	AC226723.4	-F-	CATGTAAAGCTCCACGGTCG
		-R-	ATAGGTTCGGGCCACTTTGA
HXC27-2	BT062751.1	-F-	AGGAAACCGAGGAGCAATGA
		-R-	TGCTTGGCGTCATCACAAAA
HXC30-2	AC165178.2	-F-	CTTCTTCGGGCGACATCTTG
		-R-	AAGATGGAGTTGGTGGAGGG
HXC36-2	AC216070.4	-F-	TGATTGATGGGGCTGTGTCT
		-R-	CCCTCCACCAACTCCATCTT
HXC40-2	AC225222.3	-F-	CTCGGCTCGACATTCACAAG
		-R-	GTTGAGCTCCTCTTGCATCG
GAPDH	X07156.1	-F-	CCATCACTGCCACACAGAAAAC
		-R-	AGGAACACGGAAGGACATACCAG

Table 3. MSAP-based cytosine methylation levels at different time-points under salt stress.

				Salt stress and control time-point
	Enzyme digestion^a			0 h	1 h		10 h		12h
Type	H	M	Methylation pattern^b	CK	Salt	CK	Salt	CK	Salt	CK
I	0	1	CCGG	1267	1488	1697	1723 *	1190	1249	1424
			GGCC
II	1	0	CCGG，CCGG	324	819 **	152	541 *	1023	764	520
			GGCC，GGCC
III	0	0	CCGG，CCGG，CCGG，CCGG，CCGG	5677	4620 *	5582	4476	4514	5180	5037
			GGCC，GGCC，GGCC，GGCC，GGCC
IV	1	1	CCGG，CCGG	415	756	252	943	956	490	702
			GGCC，GGCC
Total amplified loci ((I+II+III+IV）				7683	7683	7683	7683	7683	7683	7683
Total methylation loci(I+II+III)				7268	6927	7431	6740	6727	7193	6981
Type I loci no./primer combination				39.594	46.500	53.031	53.844*	37.188	39.031	44.500
Type II loci no./primer combination				10.125	25.594 **	4.750	16.906 *	31.969	23.875	16.250
Type III loci no./primer combination				177.406	144.375 *	174.438	139.875	141.063	161.875	157.406
Methylation loci no./primer combination				227.125	216.469 **	232.219	210.625	210.219	224.781	218.156
Total methylation loci (I+II+III)/ratio to total loci				94.6%	90.2%	96.7%	87.7%	87.6%	93.6%	90.9%
Non-methylation loci (IV)/ratio to total loci				5.4%	9.8%	3.3%	12.3%	12.4%	6.4%	9.1%

^aH and M represent fragments produced by digestion with HpaΙ Ι/EcoRΙ and Msp Ι/EcoRΙ respectively; 1: band present; 0: band absent; the same below.

^bC represents methylated cytosine; the 10 methylation patterns were divided into four types according to the enzyme digestion results.

*P < 0.05; ** P < 0.01.

Table 4. Analysis of DNA methylation patterns under salt stress with respect to control conditions in maize seedlings.

		Banding pattern				Different treatment
		Control		Salt
Description of pattern	Class	HpaII	MspI	HpaII	MspI	1 h	10 h	12 h
	A	1	1	1	1	84	373	185
	B	1	0	1	0	23	180	114
No change	C	0	1	0	1	447	684	474
	D	0	0	0	0	4088	3673	4190
	Total					4442	4910	4963
						57.82%	63.91%	64.60%
	E	1	0	1	1	31	192	42
	F	0	1	1	1	378	199	193
Hypomethylation	G	0	0	1	1	263	179	70
	H	0	0	1	0	526	124	375
	I	0	0	0	1	905	538	402
	Total					2103	1232	1082
						27.37%**	16.04%	14.08%**
	J	1	1	1	0	31	168	70
	K	1	1	0	1	99	300	294
Hypermethylation	L	1	1	0	0	38	115	153
	M	1	0	0	0	61	450	285
	N	0	1	0	0	633	238	552
	Total					862	1271	1354
						11.22%	16.54%	17.62%
	O	0	1	1	0	239	69	205
Pattern changed	P	1	0	0	1	37	201	79
	Total					276	270	284
						3.59%	3.51%	3.70%

**P < 0.01 at the level of difference, the hypomethylation pattern was significantly higher than the hypermethylation band.

Table 5. The BLAST results of differentially methylated sequences.

Sequencing No.	Description	E value	Max ident	GenBank Accession
HXC6-2	Zea mays zinc finger protein CONSTANS-LIKE 16-like	2.00E-15	98%	AC185486.5
HXC7-2	Zea mays NOD26-like membrane integral protein ZmNIP2-1	2.00E-15	98%	AC196472.3
HXC8-1	Zea mays probable non-specific lipid-transfer protein 3	1.00E-18	90%	AC226723.4
HXC27-2	Zea mays protein kinase APK1B	5.00E-33	99%	BT062751.1
HXC30-2	Zea mays myb-related protein 306	5.00E-29	87%	AC165178.2
HXC36-2	Zea mays protein NRT1/PTR FAMILY 7.2-like	7.00E-48	95%	AC216070.4
HXC40-2	Zea mays putative receptor protein kinase ZmPK1	2.00E-10	96%	AC225222.3

Figure 1. The expression of selected genes detected by RT-qPCR in maize inbred line LH196 at different time points. The transcript levels were normalized to that of GAPDH, and the level of each gene in the control samples was set at 1.0. Error bars represent the SEM for three independent experiments. The small letters a, b, c and d represent differences at level of P < 0.05 between different processing times.