DNA Methylation Sensitive Amplification Polymorphism Analysis And The Effects Of Endosymbiotic Wolbachia On DNA Methylation In Hylyphantes Graminicola(Araneae:Linyphiidae)

DNA methylation is an important epigenetic modification in eukaryotic organisms.It is considered as an environmental response mediator between genotypes and phenotypes,and plays an important role in regulating gene expression and maintaining genome stability.However,the research level of epigenetics of arthropods is far behind that of vertebrates and higher plants,and DNA methylation of spider genome has not been reported.Wolbachia is a common matrilineal endosymbiotic bacteria in arthropods,which plays an important role in reproductive manipulation and RNA virus resistance.Previous studies have shown that the maintenance of Wolbachia and its complex effects on their host are related to the DNA methylation.Hylyphantes graminicola is the most common spider specie in farmland of China.It can prey on many crop pests,including aphids,leafhoppers,planthoppers,diamondback moths and corn borers.It is considered to be one of the potential biological control agent,and has the characteristics of easy breeding and rapid reproduction.In this study,the methylation detection method of spider genome DNA was established for the first time,and the effect of endosymbiotic bacteria Wolbachia on the methylation of spiders’genome were studied.This study will fill the gaps in the researches of spider genome methylation,and can provide basic information for the study of spider epigenetics.At the same time,it is helpful to understand the complex symbiotic relationship between Wolbachia and spider hosts,and to provide theoretical guidance for the protection of spiders in farmland and the use of Wolbachia for pest biological control.The main contents and results of this paper are as follows:1.The establishment of DNA methylation sensitive amplification polymorphism analysis:MSAP analysis was established by using F₀female spiders,which proved for the first time that DNA methylation existed in the spider genome.Different selective primers in MSAP analysis had obvious different amplification effects,so nine pairs of primers with high polymorphism and good repeatability were selected from 36 pairs of selective primers as follow-up experimental primers,so as to establish a more suitable MSAP analysis system for genomic methylation research in H.graminicola.2.The effects of endosymbiotic Wolbachia infection on DNA methylation in Hylyphantes graminicola:DNA methylation levels and patterns of DNA methylation were compared among the different Wolbachia infection groups by optimized MSAP analysis method.Sequence analysis of partial methylation bands was also carried out.The results showed that the spider had low level of DNA methylation,and the total methylation rate was significantly higher than the hemi-methylation rate（p<0.01）.Female and male spiders without Wolbachia had similar methylation rates（8.67%for male and 8.72%for female;P=0.84）.But their methylation pattern were significantly different,which might be related to the differentiation of male and female spiders.The DNA methylation rate of female and male spiders infected with Wolbachia was significantly lower than that of no-infected female and male spiders（7.18%for infected female and 6.31%for infected male,p<0.01）,resulting in demethylation of the genome.Moreover,the DNA methylation rate of male spiders infected with Wolbachia was significantly lower than that of female spiders（P=0.001）.It is speculated that Wolbachia infection may lead to some important physiological and biochemical changes in male spiders by lower methylation rate.The methylation band sequence analysis showed that the methylation sites in the genome of the spider were homologous with the genes sequences of gene expression regulation and physiological and biochemical reactions.3:The effect of Wolbachia infection on Dnmt1 expression in Hylyphantes graminicola:By analysing the transcriptome data of the H.graminicola,the Dnmt1 related sequence was obtained,and its bioinformatics and phylogenetic analysis were carried out.Then the expression of Dnmt1 gene in different Wolbachia infection combinations were detected by q RT-PCR.The results showed that the Dnmt1 of the spider had a typical conserved domain of DNA methyltransferase,and had nearly 55%homology with Dnmt1 of other species.Phylogenetic analysis showed that it had the closest relationship with insect Dnmt1 and was conservative in evolution.The results of qRT-PCR showed that Wolbachia infection significantly inhibited the expression of Dnmt1 gene（p<0.01）.Combined with genomic MSAP analysis,the decrease of Dnmt1 gene expression was an important reason for the demethylation of the genome.