Abstract: A reaction system of MSAP was established by orthogonal experiment in order to obtain genomic DNA methylation level and pattern of different ploidy of loachMisgurnusanguillicaudatusand used for MSAP analysis of diploid loach. The results showed that the best double digestion systems for MSAP including 10 UEcoR I, 10 UHpaII, and 10 UMspI were observed for 8 h at 37 ℃; the best pre-selective amplification mixture contained 0.2 mmol/L dNTPs, 1.5 mmol/L Mg2+,Taq1 U and selective primers 0.8 μL in total 20 μL; the best selective amplification mixture contained 0.375 mmol/L dNTPs, 1.5 mmol/L Mg2+,Taq1 U and selective primers 1.5 μL in total 20 μL. The atlas showed that full methylation rate of three samples were 24.1%, 20.8% and 22.3%, respectively; hemi-methylation rates were 41.4%, 20.8% and 33.3%, respectively; and total methylation rate was 65.5%, 41.6% and 55.6%, respectively. The findings indicated that this MSAP reaction system was stable, reliable and reproducible, providing the foundation for the MSAP technology with loach-related research.
2014, Vol. 29 
