Analysis The Level And Pattern On Genomic DNA Methylation In Different Ploidy And Parent-offsprings For Loaches

In order to investigate the genomic DNA methylation levels and patterns of different ploidy and parentoffspring loaches which analysis their epigenetic differences. In this research, we selected natural diploid, natural triploid and natural tetraploid and hybridized triploid(4n♀ × 2n♂) as subjects.We used orthogonal experiment to established the best systems for MSAP and used this method to analysis genomic DNA methylation level and pattern. The results were as follows:(1) Establishment and optimization of MSAP reaction system for loachWe established the best MSAP reaction system for loaches which included 10 U Eco R I and 10 U Hpa II/Msp I for 8 h at 37 ℃; the best pre-selective amplification mixture contained d NTPs 0.2 mmol/L, Mg2+ 1.5 mmol/L, Taq 1 U in total 20 μL; the best selective amplification mixture contained d NTPs 0.375 mmol/L, Mg 2+ 1.25 mmol/L, Taq 1 U and selective primers 1.5 μL in total 20 μL.The system was stable,reliable and well repeatability.It also laid a foundation for MSAP research.(2)Analyzing the levels and patterns of DNA Methylation in difference of ploidy loaches.We selected natural diploid, natural triploid and natural tetraploid as subjects. There are 1438 sites in 6 pairs of selective amplification primers from 25 pairs of selective amplification primers. The atlas showed that the total methylation rates of diploid,triploid and tetraploid was 39.98%、33.72% and 32.36%. Analyzing of DNA methylation patterns showed that the percentage of the same methylation patterns among the autopolyploid is 24.1%,and the different patterns is 15.1%. The rate that one autopolyploid methylation patterns is identical to another is 60.9%. Comparing the same methylation patterns among the polyploid showed that the same rate is 38.6% between diploid and triploid, respectively; the same rate is 41.8% between diploid and tetraploid. The research showed that the total methylation rate decreased in the process of chromosome doubling. There was a little difference among of the different ploidy loaches in the methylation expression. But there is a big difference between the pairwise comparison. Compared with the diploid, the tetraploid’s epigenetic expression is closed to diploid.(3) Analyzing the levels and patterns of DNA Methylation in parent-offspring loaches.We selected female tetraploid, male diploid and their offsprings of hybridized loaches as subjects and selected 6 pairs of selective amplification primers from 25 pairs of selective amplification primers. The atlas showed that the total methylation rates of the male diploid, female tetraploid, and their offsprings were 38.27%, 24.21% and 27.21%. The methylation patterns have changed a lot in the process of hybridization, including Atype, monomorphism 480(29.0%); B-type, demethylation 285(17.2%); C-type, hypermethylation 628(37.9%); D-type, hypomethylation 263(15.9%).The research showed that the methylation level of offsprings between the parents in the process of hybridization. The main variation methylation pattern was hypermethylation.In conclusion, we believed that the changes of genomic DNA methylation levels and patterns in a certain degree occurred in loaches’ polyploidy and hybridization process, this article is stydied in a perspective of molecular biology.It’s aim to explore the epigenetic mechanisms of polyploidy and hybridization process. It also provide a scientific basis for the loaches industry, as well as laid a foundation for high efficiency.