MSAP and SSR
SSR data were extracted from published SSR experimental results ofC. chinensis in DHS (Wang et al ., 2014). Methylation sensitive amplified polymorphism (MSAP) is widely used in studies of population epigenetic variation for its being simple and relatively inexpensive (Latiff, 2009). MSAP in this paper follows the way described by Schulzet al . (2014). A set gradient parameters method and orthogonal experiment method was used to optimise the optimal reaction system and adapter sequences (see supporting information: The optimal reaction system, Table S1). First, 128 primers were randomly combined based on the EcoR I and Hap II/Msp I universal primers. After polymorphic amplifying, the products were electrophoresed and screened by ABI 3730XL DNA. Based on the visualised peak figure, six primers that could amplify clear, complete and high polymorphism bands were selected (Table S2). GS500 was used as the DNA standard. To ensure that the data were credible, using GeneMarker version 2.2.0 (SoftGenetics), 25–30 well-defined sites were selected from ~100–500 bp fragments for each primer in later research. The fragment statistic also followed Schulz’s way (Schulz et al ., 2014).
A controversial defect in MSAP is that scoring of MSAP data may lead to ambiguous interpretation of MSAP data because a cytosine in any sequence context can be methylated with decreased frequency for CG, CHG and CHH motifs (H = A or T or C) (Fulneček & Kovařík, 2014). In addition, full methylation of external cytosines (mCCGG) cannot be detected by MSAP (Xia, Zou, Zhang, Feng & Wang, 2014). However, a screening of literature revealed that, for quantification of genome-wide cytosine methylation, methylation sites assayed by MSAP was greater than those obtained by high-performance liquid chromatography, which is recommended because of its global assessment, accuracy and reproducibility (Alonso, Pérez, Bazaga, Medrano & Herrera, 2016). Furthermore, full-methylation and hemi-methylation show clear distinction in MSAP analysis.