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.