DNA methylation variation and growth in the clonal Duchesnea indica is regulated by both past and present lead environments

Figures & data

Figure 1. Schematic of the experiment. The design consisted of two past environments (past env.) (control and Pb contaminated) and three Pb contaminated levels of present environments (present env.) (control, heterogeneous Pb, and homogeneous Pb) for the newborn ramets. The past environment is represented inside the red square, present environment is represented outside the red square. See the text and table 1 for experimental design details.

Table 1. Different treatments conducted in the study.

Treatments (present env. past env.)	Interpretation
Control−Control	All the patches were uncontaminated soil, contain the past environment.
Heter−Control	Half of patches of the present environment were contaminated and half were uncontaminated soil, and the contaminated patches were arranged randomly. Ramets were past under control patches.
Homo−Control	All the patches were Pb contaminated soil except past ramets were under control patches.
Control−Pb	All the patches were uncontaminated soil except past ramets were under Pb-contaminated patch.
Heter−Pb	Half of patches of the present environment were contaminated and half were uncontaminated soil, and the contaminated patches were arranged randomly. Ramets were past under Pb-contaminated patch.
Homo−Pb	All the patches were Pb contaminated soil, contain the past environment.

Figure 2. Past environment (control versus Pb) and present environment (control, heterogeneous Pb, and homogeneous Pb) on morphological and growth performance of Duchesnea indica. total biomass (a); leaf biomass(b); stolon biomass(c); root biomass(d); stolon length(e); specific stolon length(f); R/S(g); branch number(h) and node number(i) of wholegenet. Means and SE are shown. Columns sharing the same letter are not significantly different from each other at p < 0.05.

Table 2. ANOVA result for effects of past environment (control versus Pb), present environment (control, heterogeneous Pb versus homogeneous Pb), and their interaction on the morphological and growth performance of Duchesnea indica.

	Past Env. (Pa)		Present Env. (Pr)		Pa × Pr
	Dev./F1,28	P	Dev./F1,28	P	Dev./F1,28	P
Total biomass^a	0.01	0.938	4.02	0.036	1.76	0.201
Leaf biomass	0.27	0.613	1.80	0.198	0.93	0.413
Stolon biomass^a	0.98	0.334	4.19	0.030	1.55	0.236
Root biomass	2.04	0.169	0.90	0.422	0.42	0.665
Stolon length	4.30	0.049	1.46	0.253	1.84	0.180
Specific stolon length^a	0.03	0.875	7.36	0.004	2.34	0.122
R : S^a	1.38	0.257	3.80	0.043	4.10	0.035
Branch number	0.09	0.763	0.25	0.879	2.49	0.288
Node number	0.10	0.758	3.01	0.222	3.35	0.188

Note: ^aLog transformed following Gaussian distribution.

Figure 3. CCA analysis of offspring loci composition.

Table 3. ANOVA/CCA analysis results for effects of the past environment (control vs. Pb), and present environment (control, heterogeneous Pb, and homogeneous Pb) on loci composition, Shannon’s diversity index, and total DNA methylation level of offspring Duchesnea indica.

			Loci composition		Shannon’s diversity index		DNA methylation level
		Df	F	P	F	P	F	P
Past environment (Pa)		1,18	1.91	0.002	1.71	0.207	1.72	0.207
Present environment (Pr)		2,18	1.62	0.002	3.48	0.053	4.10	0.034
Pa × Pr		2,18	1.70	0.002	0.86	0.439	0.72	0.502

Degrees of freedom (df), F, and p values are given. Values for p < 0.05 are in bold.

Figure 4. Relationship between CCA1 score and CCA2 score in loci composition of offspring ramets at different past environments.

Figure 5. Relationship between CCA1 score and CCA2 score in loci composition of offspring ramets at three present environments.

Table

Adapters/primers	EcoRI	HpaII/MspI
Adapter	5’-CTCGTAGACTGCGTACC-3’	5’-GATCATGAGTCCTGCT-3’
	5’-AATTGGTACGCAGTC-3’	5’-CGAGCAGGACTCATGA-3’
Pre-amplification primers	5’-GACTGCGTACCAATTCA-3’	5’-ATCATGAGTCCTGCTCGGT-3’
Selective primers	5’-GACTGCGTACCAATTC ACA-3’	5’-ATCATGAGTCCTGCTCGG TCC-3’
	5’-GACTGCGTACCAATTC AAG-3’	5’-ATCATGAGTCCTGCTCGG TTG-3’
	5’-GACTGCGTACCAATTC ACG-3’	5’-ATCATGAGTCCTGCTCGG TTC-3’
	5’- GACTGCGTACCAATTC AGC-3’	5’-ATCATGAGTCCTGCTCGG TGA-3’
		5’-ATCATGAGTCCTGCTCGG TCT-3’
		5’-ATCATGAGTCCTGCTCGG TCG-3’
		5’-ATCATGAGTCCTGCTCGG TAA-3’
		5’-ATCATGAGTCCTGCTCGG TAC-3’

Note: A selective amplification step was carried out with 12 pairs of selective primer combinations.

Including the following: EcoRI-AGC/Hpall-TTG, EcoRI-AAG/Hpall-TAC, EcoRI-AAG/Hpall-TGA, EcoRI-AAG/Hpall-TTC, EcoRI-AAG/Hpall-TCG, EcoRI-ACG/Hpall-TAA, EcoRI-ACG/Hpall-TCT, EcoRI-ACA/Hpall-TTG, EcoRI-ACG/Hpall-TCT, EcoRI-ACA/Hpall-TCT, EcoRI-AGC/Hpall-TTG, EcoRI-AGC/Hpall-TCC

Data availability statement

The data that support the findings of this study are available from the corresponding author.