Development Of Microsatellite Markers From Half-smooth Tongue Sole (Cynoglossus Semilaevis) And The Application In The Gynogenesis Fish

Half-smooth tongue sole (Cynoglossus semilaevis) is a kind of special rare economic cultured marine fish species in the northern China. mainly distributed in the north China coast. It has been the most important fish species in the northern China.This paper consists of three sections:The first one is isolation and characterization of polymorphic microsatellite markers of half-smooth tongue sole; The second section is genetic analysis of cultured and gynogenetic stocks of half-smooth tongue sole using SSR markers; The last section is Assessment of Homozygosity in Gynogenetic Diploid Using Microsatellite Markers in half-smooth tongue sole. The main research results are as follows:Enriched genomic library of half-smooth tongue sole was constructed using the FIASCO method (fast isolation by AFLP of sequences containing repeats). After sequencing, 68 clones were found containing microsatellite repeat sequences. Finally, PCR primers were designed for 45 loci successfully, of which 22 loci showed polymorphic. By searching GenBank database of half-smooth tongue sole, 150 sequences were obtained from. PCR primers were designed for 106 loci successfully, of which 65 loci showed polymorphic. These markers will be benefit for the construction of genetic linkage map,the evaluation of genetic resource,genetic diversity,and marker-assisted selection in half-smooth tongue sole.The genetic structure and variation of the cultured stock and artificial meio-gynogenetic population of half-smooth tongue sole were analyzed using twenty-four microsatellite markers. The results showed that the average number of alleles and average effective alleles were 7.0 and 4.8, and 3.935 and 2.411 in the cultured stock and the artificial gynogenetic population, respectively. the mean observed heterozygosity(H_O)were 0.7135 and 0.5865, respectively. The results indicate that the genetic diversity of the cultured group was much higher than that of the gynogenetic population of half-smooth tongue sole. Thirteen loci significantly deviate from Hardy-Weinberg equilibrium (HWE) in the gynogenetic population,while one loci deviated from HWE in the cultured group. The coefficient of gene differentiation (G_ST) was 0.131 0, and the genetic distance was 0.4200 between the two populations, suggesting a significant genetic differentiation between the two populations.In the present study, a protocol for artificial induction of mito-gynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole. The optimal initiation time for pressure shock of mito-gynogenetic embryos was determined to be 22 min after fertilization when water temperature is at 23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min.Assessment of Homozygosity in Gynogenetic Diploid Using eight Microsatellite Markers in half-smooth tongue sole. The results showed that The high proportion of heterozygotes for eight loci demonstrates that it is not a practical method for producing homozygous inbred lines in the gynogenetic fish produced by retention the seeond polar body;and treatments of suppressing the first cell division were more promising for this purpose.