Analysis Differences Of DNA Methylation Of Different Ginkgo Biloba L.var.epiphylla Mak.Trees

Ginkgo biloba L. is the only relict seed plant that has survived since the quaternaryglaciation.During the long evolution Process, there were morphological variations happenedto the leaves and the seeds of Ginkgo biloba L. In1891, the first Ginkgo biloba L. var.epiphylla Mak. tree of the world was discovered by Shirai in Japan. The Ginkgo biloba L. var.epiphylla Mak.which great feature was the ontogeny and structure of epiphyllous femalereproductive organ (EFRO), is a special germplasm in Ginkgo biloba L. family, it had greatacademic and ornamental values. DNA methylation is an important epigenetic modifiy that isassociated with gene express, cell differentiation, gender expression and Germ celldevelopment and so on. In the experiment, MSAP（Methylation-sensitive amplificationpolymorphism, MSAP）was applied on Ginkgo biloba L. var. epiphylla Mak.with samplesderived from leaves, to investigate the DNA methylated level and patterns of different Ginkgobiloba L. var. epiphylla Mak. trees. Cloning and sequencing the DNA methylation modifysites, then, sequence alignment in the NCBI. The main results as follows:(1) In this study, efficient MSAP system was established thought selecting the DNAtemplate, Taq, Mg2+and so on. By using14pairs of selective primers from the36pairs ofMSAP selective primers,22different Ginkgo biloba L. var. epiphylla Mak. trees and oneGinkgo biloba L. tree of MSAP fingerprints obtained from amplification.(2)The results showed a total of5386legible and repeatable amplified DNA bands weredetected. There are2161DNA methylation bands in the total amplified bands. The average ofDNA methylation is40.71%in Ginkgo biloba L. var. Epiphylla. The DNA half-methylationand full methylation were14.59%and26.11%, respectively.30.71%were methylated inGinkgo biloba L.. The DNAhalf-methylation and full methylation were9.06%and21.65%,respectively. DNA methylation level had significant differences among the different plants.DNA methylation level is26.98%-61.71%in different individuals. DNA methylation level ofGinkgo biloba L. var. Epiphylla is higher than Ginkgo biloba.(3) To detected the DNA methylated modification levels and patterns of22differentGinkgo biloba L. var. epiphylla and a Ginkgo biloba at the genome-wide level, appear4typesbands in the polyacrylamide gel,by the double digestion of EcoR Ⅰa ndHpaⅡ/Msp Ⅰ. Theseare non-methylated, hemimethylated, permethylated and special bands which has no digest site. According to the four types of mode, the patterns can be divided10methylation patterns.Compared the Ginkgo biloba L., there were1004methylated sites re-methylated in Ginkgobiloba L.var. epiphylla, accounting51.04%of total variation sites. There were706methylated sites de-methylatod in Ginkgo biloba L.var. epiphylla, accounting35.86%%oftotal variation sites. re-methylated played dominant role in Ginkgo biloba L.var. Epiphylla.(4) According to the methylation-sensitive methylation polymorphism and methylationinsensitive polymorphism produced “0”,“1” matrix of different individuals, the clusteranalysis and principal components analysis by NTSYSpc software. The results showed WYand TC3were closed in genetic relatives, second the NS2and Oha. have a closer relationship.Cluster analysis and principal component analysis. Others has further distance relationship,there are not cluster into one group in different individual plants which lie in the nearer region.Cluster analysis using NTSYSpc was consistent with principal components analysis.Theprincipal components analysis for different individuals using data of HpaⅡand MspⅠ,Axis-dim1extracted60.37%and69.34%of the variance, respectively. Axis-dim2extracted5.48%and4.65%of the variance, respectively. The contribution rate of axis-dim1is greathigher than the axis-dim2.(5)Modify sites of Ginkgo biloba L.var. Epiphylla Were recovered, cloned andsequenced and homology analyzed. DNA methylation modified sites were existed many DNAsequence, example hypothetical protein, repetitive sequence and transposon factors and so on.DNA methylated modification levels, patterns and DNA methylation diversity ofGinkgo biloba L. var. epiphylla were detected in this paper. Laid the foundation ofepigenetics research in Ginkgo biloba L.var. Epiphylla, and played an important role inrevealing the mutation mechanism.