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The phenotype of mammals is influenced by environmental factors and can be inherited transgenerationally.Sperm is the only medium carrying transgenerational paternal information,and DNA methylation,chromatin proteins,and RNAs in sperm are candidates that carry such transgenerational epigenetic information.Of these methylome is a useful candidate for the study of the environmental-dependent phenotype.We analyzed the relationship among the sperm methylome,genome and gene expression patterns related to the testes phenotypes of two wild brown rat (Rattus norvegicus) populations with different reproductive strategies.Our results indicated that methylation variation of a series of genes in the sperm genomes might determine expression patterns of another series of genes related to the testes phenotype by cascade regulation of these genes.Comparison analysis of nucleotide diversities showed significantly higher average π value in Different Methylated Regions (DMRs) than Non-DMRs.It indicated that DNA mutation rate might be driven by phenotype-related methylation variation.Expression pattern differentiation of key DMGs between two populations and its relationship with gene expression patterns directly determining the testes developmental characters after birth indicated that sperm DNA methylome might associate with programming of the spatial and temporal gene expression patterns related to phenotype determination.