DNA Methylation Analysis For Different Tissues And Individuals In Weight In Beijing You Chicken

ABSTRACT:In eukaryotes, DNA methylation is one of the important epigenetic modification patterns by regulating the gene expression. Many studies have shown that DNA methylation plays an important role in the growth and differentiation of the animals. In this study, using methylation sensitive amplified polymorphism (MSAP), the DNA methylation levels and patterns of CCGG sites in genomes were analyzed in muscle and ovary tissues from the17week-old Beijing You chicken which were assigned into two groups with high body weight and low body weight. As a result, we optimized experimental conditions by capillary electrophoresis result, developed an automated data analysis software for MSAP fluorescence detection, analyzed genomic DNA methylation levels of muscle and ovary tissues or indivious with different weight from Beijing You chicken. The results were as follows:MSAP selective amplification products were analyzed using fluorescence labeling and capillary electrophoresis technology. We optimized the MSAP experimental conditions including the genomic DNA concentration, dilution of pre-amplification product, the concentrations of selective primers, Mg2+and dNTPs, DNA loading dosage, the internal standard content and injection time. Repeated experiments showed that this system can automatically detect the global DNA methylation states in Beijing You chicken.Genomic DNA methylation levels were detected with fluorescence labeled MSAP in muscles. The initial data of MSAP was analyzed by direct integral method, whole interval method and half interval method respectively. The results showed the whole interval method and half interval method were more accurate than direct integral method for analyzing the MSAP data. Based on half interval method, the new online software named MSAP Analyst was built for initial data processing of MSAP.With methylation sensitive amplified polymorphism, the DNA methylation levels and patterns in genomes were analyzed in muscle and ovary tissues from the Beijing You chicken. Total53027clear bands were obtained with12pairs of selective amplification primers, about150bands per sample per pair of primer combination. The degree of methylation was approximate54.04%in muscle and57.06%in ovary in detected individuals, and a significant difference existed in the methylation levels between two tissues (P<0.05). These DNA methylation differences were mainly reflected in the full methylated sites. Five tissue-specific MSAP fragments were isolated, sequenced and characterized, one located in3’ downstream while others in the introns.With methylation sensitive amplified polymorphism, the DNA methylation levels and patterns in genomes were analyzed in high and low weight groups from the Beijing You chicken. Total50776clear bands were obtained with12pairs of selective amplification primers. The degree of methylation was approximate53.70%in high weight group chickens and55.86%in low weight group chickens in detected individuals, and a significant difference existed with ANOVA in the methylation levels in different groups (P<0.05), but no interaction between body weight and gender factors. Pearson rank correlation analysis showed that:there was a significant negative correlation (P<0.05) between genomic DNA methylation levels and body weight. We isolated and identified14possible weight-related specific methylated fragments, two of which were located in the5’ upstream, two across introns and exons, six in introns, one at3’ downstream.