Genomic Microsatellite Polymorphism And DNA Methylation Analysis Of The White Apostichopus Japonicus

Sea cucumber(A. japonicus), with high nutritional value and medicinal value, is traditional tonic food in China. Color variation is a distinct characteristic of A. japonicus, and body color can affect market price of holothurian products. A. japonicus can generally be divided into three color morphs: black, red and green. Completely white morph of A. japonicus is rare in nature. Its price is expected to be more expensive for its rarity and higher nutritional value. White progeny produced by crossing white female and white male A. japonicus were successfully cultivated in the cooperative aquaculture base at Penglai City, China.In this study, on the one hand, we use the microsatellite DNA techniques to study the genetic diversities and genetic structures of A. japonicus and white A. japonicus populations; on the other hand, we use the technique of MSAP(Methylation-sensitive amplified polymorphism) to study the methylation pattern of genomic DNA of different tissues in two populations, and discuss the relationship between the albino of white A. japonicus and the genome-wide DNA methylation. The main results were as follows:1.Genetic diversitiey of A. japonicus and white A. japonicus populations were examined using 14 microsatellite makers. The number of alleles generated from each locus ranged from three to six. Average values of polymorphism information content(PIC) of these two populations were 0.6465 and 0.6212, respectively; the average values of observed heterozygosity(Ho) were 0.7619 and 0.8286, respectively; the average values of expected heterozygosity(He) were 0.7218 and 0.6895, respectively. The results showed that the heterozygosity and the genetic diversity of white A. japonicus population were lower, compared with the A. japonicus population. Significant deficits of deviation from Hardy-Weinberg expectations were tested in both populations. F-test data showed that there were varying degrees of heterozygosity deficiency of the two pupolations, and the genetic differentiation was weak between them, which illustrated that the genetic variation existed among individuals within population rather than between populations. Genetic distance and genetic similarity showed that the genetic differentiation between two populations has not yet reached the standard species differentiation.2.With the technique of MSAP(Methylation-sensitive amplified polymorphism), we analyzed methylation level and pattern of genomic DNA in the body wall, respiratory tree, and digestive tract of A. japonicus and white A. japonicus population, and preliminary discussed the influence of DNA methylation on gene specific expression in different tissues between A. japonicus and white A. japonicus populations. In this study, 9 pairs of primer combinations were selected out, which can be amplified to stable and clear fragments in two populations. A total of 5932 and 5208 sites were detected in normal A. japonicus and white A. japonicus populations respectively, and most of them were based on the none-methylated CCGG sites. There were 4317 and 3944 none-methylated sites in normal A. japonicus and white A. japonicus populations, accounting for 72.78% and 75.73% respectively. The methylation rates in the body wall, respiratory tree, and digestive tract of normal A. japonicus and white A. japonicus were about 31.07%, 23.36%, 26.34% and 29.88%, 23.25%, 19.45% respectively. These results demonstrated that there were differences in DNA methylation level and pattern among various tissues and between different populations. DNA methylation may play an important role in the process of tissue differentiation and development in A. japonicus.