Analysis of DNA methylation in tissues and development stages of pearl oyster Pinctada fucata

Abstract

In this study, we developed a methylationsensitive amplification polymorphism technique to investigate DNA methylation profiles in different tissues and in the early-development stages of the pearl oyster Pinctada fucata (P. fucata). Methylation levels in adductor muscle, digestive gland, axe foot, heart, and gill ranged from 11.71 to 14.71 %, and significant differences (P \ 0.05) between methylation levels in different tissues were observed. The DNA methylation levels of sperm, egg cells, two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae were 13.51, 11.80, 12.14, 12.60, 14.65 and 13.18 %, respectively. Development stages of two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae indicated a higher number of identical DNA methylation status loci in the egg, compared to that in the sperm. It is probable that DNA methylation patterns of the progeny are mainly influenced by the egg, while the sperm may become increasingly important during the process of early embryo development. The observed differences in methylation levels in the tissues and the development stages of P. fucata suggest that DNA methylation may act as an epigenetic regulator during tissue differentiation, individual growth, and development.

Indication of family-specific DNA methylation patterns in developing oysters

“…These results are similar to what has been seen in zebrafish where embryos inherit the methylation profile of sperm rather than oocyte (Jiang et al 2013b, Potok et al 2013). In contrast, methylation ratios in Pearl oysters are mainly influenced by oocytes, rather than sperm (Li et al 2014), possibly due to maternal influences on DNA methylation patterns in early larvae, while later stages attain methylation patterns similar to sperm.…”

DNA methylation is associated with expression level changes of galectin gene in mantle wound healing process of pearl oyster, Pinctada fucata

“…The DNA methylation level was calculated using the following formula: DNA methylation level (%) ¼ total methylated loci/(sum of Types I, II, III and IV loci). The sum of types II, III and IV loci represents the total methylated loci [25].…”

“…Five primer pairs for selective PCR (E þ ATC and HM þ TTC, E þ ACT and HM þ TAA, E þ ATG and HM þ TTT, E þ AGT and HM þ TTT, E þ ATC and HM þ TCA) that could yield clear bands were used for selective amplification. The selective PCR primer was designed by adding two bases to the 3 0 end of the Eco þ A primer or HM þ T primer [25]. The total volume of selective PCR was 20 mL, including 10.5 mL Premix Ex Taq (Takara, Dalian, China), 0.5 mL 20 pmol/mL EcoRI primers, 2 mL 20 pmol/mL HpaII-MspI primers, 1 mL of the diluted pre-amplification product, and 6 mL ddH 2 O.…”

A comparative analysis of DNA methylation in diploid and tetraploid apple (Malus × domestica Borkh.)

“…The template represented genomic DNA restricted with either EcoRI and HpaII or EcoRI and MspI (enzymes obtained from New England Biolabsinc., Beverly, USA). The methods used for restriction digestion, adapter ligation, pre-amplification and selective amplification followed those given by Li et al (2015). The amplicons were resolved by electrophoresis through a 6% denaturing polyacrylamide gel and were visualized by silver staining (Dong et al 2006).…”

“…To characterize the DNA methylation status of CCGG tetranucleotides, comparisons were made between the MSAP profiles derived from EcoRI/HpaII and EcoRI/MspI digests. HpaII and MspI are isoschizomers which both recognize CCGG; however, while MspI cleaves either a non-methylated site, a hemi-methylated (mC in one DNA strand only) site or a fully methylated CmCGG site (but neither hemi-methylated nor fully methylated mCCGG nor mCmCGG site), in contrast, HpaII cleaves only nonmethylated (CCGG) and hemi-methylated (mCCGG) sites (Lu et al 2008;Li et al 2015). The resulting fragments therefore fall into four types (I through IV): type I fragments, which are shared by both digests, are formed when the CCGG is non-methylated; type II fragments, which are present in the EcoRI/HpaII but not in the EcoRI/MspI digest, are difficult to interpret in plant genomes, where methylation can occur at both CCG and CG motifs; type III fragments, which are present in the EcoRI/MspI but not in the EcoRI/ HpaII digest, are produced when a CmCGG site is present; finally type IV fragments (not present in either digest) are detected where the CCGG site varies between genetically distinct samples (here the diploid and tetraploid cultivar) (Fulneček & Kovařík 2014).…”