| With the development of the study on determine factors of phenotype,epigenetics has attracted more and more attention.Epigenetics is defined as meiotically and mitotically heritable changes in gene expression that cannot be explained by changes in DNA sequence.Epigenetic marks can be influenced by environmental variation and are capable of influencing post embryonic development.Epigenetic inheritance plays a fundamentally important role in mediating gene regulation and phenotypic plasticity.As an important epigenetic mechanism,DNA methylation is implicated in modulating gene transcription and altering gene activity states without variation on gene sequence in a wide array of taxa.The close correlation between cytosine methylation and phenotype plasticity was proved in several individuals.The two-spotted spider mite,Tetranychus urticae Koch,as a cosmopolitan agricultural pest belongs to an assemblage of web-spinning mites and usual outbreak in greenhouse.Two forms existed in T.urticae:green form(TuG)and red form(TuR)which was also called as T.cinnabarinus(Boisduval).A great deal of recent studies revealed that the red form has similar genetic background to the green form,which raising an intriguing question that these two color forms shape with low genetic divergence may induced by epigenetic modifications,such as DNA methylation.Therefore,it is required to explore that how DNA methylation status varies during the maturity stages,whether DNA methylation plays a key role in these different stages,and what the correlation between DNA methylation and the diverse pigment phenotypes of these two color forms in T.urticae.Firstly,total genomic DNA was extracted from adults of TuR using the Wizard(?)SV Genomic DNA Purification System(Promega USA)after optimized to match the condition of MSAP.The condition of digestion reaction,ligation reaction,pre-amplification reaction and selective-amplification were modified to adjust to the two-spotted spider mites,separately.12 amplification primer combinations were selected from 32 amplification primer combinations taking advantage of the modified protocol.Secondly,we took advantage of MSAP technique to examine the divergence of gene wide DNA methylation between TuR and TuG A total of 585 stable and repeatable fragments(loci)were obtained from TuG and TuR by using the masp analysis with twelve selective primer combinations.Among the loci,421(72%)loci were methylation-susceptible loci,being polymorphic 338(80%)in spider mites.Epigenetic diversity of two color forms was assessed with the Shannon's diversity index(I).Patterns of individual and population differentiation were depicted by principal coordinates analysis(PCoA)in msap.The division of epigenetic variance among populations was estimated as epigenetic population differentiation((?)st)with an analysis of molecular variance(AMOVA).The main research results of this study are as follows:1.The results of AMOVA analyses and PCoA revealed significant difference between two forms and different level of epigenetic variation between them.The distributions of Shannon's diversity index(I)for TuG and TuR was almost equal(TuG I=0.64±0.06;TuR I=0.65±0.05),indicating that the similarly diverse was present among different developmental stages in each color form.2.It turned out that the tendency of global methylation levels in two forms were similar across maturation stages for eggs,larvae,nymphs and adults.The results of pairwise AMOVAs confirmed the most divergence between larva and adult in TuG((?)ST=0.27).But the outcome of AMOVA in TuR showed the most divergence between larva and nymph((?)ST=0.06).PCoA analyses were able to differentiate among developmental stages in TuQ but failed in TuR.3.In the research of the same developmental stage of two forms,AMOVA results from msap exhibited that epigenetic differentiation was significantly different between the stage of female adult in two color forms((?)ST=0.50 P=0.03).And the stage of female adult in TuR has higher differentiation than other developmental stages and statistical significant((?)ST=0.06 P=0.04).PCoA results of showed visualizing separations between two color forms in larva and adult.Finally,immunodetection of 5-methylcytosine(5mC)and microinjection of 5-azacytidine(5-azaC)were taken advantage of to confirm the existence of 5mC in two-spotted spider mite.Visual observation of the pattern of immunodetection of 5mC compared with DNA revealed that 5mC specific sites were located in the organization of ovary,oocyte and oviduct,which were relating to reproduction in two-spotted spider mite.The percentage of 5mC in wo-spotted spider mite was statistically significantly lower after the microinjection of 5-azaC indicating the important role of 5-azaC in demethylation. |
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