Development Of SSR Markers And Construction Of Genetic Linkage Maps Of Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole(Cynoglossus semilaevis) is an important cultured marine fish species in the northern China. Half-smooth tongue sole which is the native fish species of China, mainly distributed in the north China coast. At present the number of wild individuals of the fish species is little, and the fisheries resource is rather critical.In aquaculture, It has been the most popular fish species together with turbot (Scophthalmus maximus), olive flounder (Paralichthys olivaceus), Spotted halibut (Verasper variegatus), and barfin flounder(Verasper moseri) in the northern China. In order to benefit the molecular breeding of fish species, the four items were studied in this paper including.1.Isolation and characterization of polymorphic microsatellite markersFive repeat-enriched genomic libraries (the repeat units were GATA, GA, ATC, ACTC and AAAC, respectively) were constructed by FIASCO (fast isolation by AFLP of sequences containing repeats) in Cynoglossus semilaevis.500 sequences were obtained, of which 412 contained microsatellite repeats.273 pairs of primers were designed successfully, and 111 microsatellite loci were proved to be polymorphic. These markers will be benefit for the evaluation of genetic resource and the construction of genetic linkage map in half-smooth tongue sole.2.Parentage determination of Cynoglossus semilaevis based on microsatellite DNA markers and its application to family management.This study was initiated to assess the feasibility of parentage determination in aquaculture populations for Cynoglossus semilaevsis.Under different scenarios both simulation and reality, we used microsatellite markers to assign parentage to offspring. Simulations based on allele frequency data from the population of Cynoglossus semilaevis demonstrated when marker data from five loci to ten loci were combined, the assignment success of progeny to their true parental couple from 96.45% increased to 99.71% in parentage identification with unknown parental and filial information. Out of 473 offspring,95.35%(451/473)was assigned to their true parental couple in mixed families groups. Based on these results, we conclude that the use of a number of microsatellite markers represents a realistic and effective alternative to physical tagging in a selection program and it allows the identification of parental effects on offspring performances from early life stages.3.Development of two microsatellite multiplex PCR systems for Cynoglossus semilaevis and its application to family management.We developed two microsatellite multiplex PCR systems,9 microsatellite loci,for Cynoglossus semilaevis, one of the most important aquaculture species in China. The multiplex PCR microsatellite assay was used to estimate the kinship and the genetic diversity of sixteen Cynoglossus semilaevis families.The average polymorphic information content (PIC) was 0.6333.The number of the alleles at each locus ranged from three to eight.The number of the average alleles was 6.333.Based on the genotypes of either known parent or none of them and offspring all the absent parental genotypes of the sixteen families were deduced.According to the unique alleles among the families,each family could be distinguished from the others.Cervus2.0 analysis showed that the combined exclusion probability was 95.66% if parents were both unknown and the combined exclusion probability was 99.65% when only one of the parental genotype was known. A dendrogram was constructed using UPGMA based on genetic similarity matrix.The UPGAM dendrogram of the 160 individuals indicated that 93.13% the individuals from each family could be fully clustered and the classification results accord with the source of genealogy.In fact 90% correct assignment was performed with two microsatellite multiplex PCR. The results showed that microsatellite marker is useful tool for general logical identification of Hat-Smooth Tongue Sole.4. The construction of genetic linkage mapsTwo genetic linkage maps of Cynoglossus semilaevis.(♀) and Cynoglossus semilaevis (♂) were constructed using microsatellite markers. A total of 289 SSR markers and 2 female-specific AFLP markers were mapped in genetic linkage maps of Cynoglossus semilaevis.The genetic linkage maps of Cynoglossus semilaevis (♀) map consisted of 237 SSR markers and 2 female-specific AFLP markers, and it was grouped in 23 linkage groups.The linkage between the loci was identified by LOD score of≥3.0. The number of markers per group ranged from 2 to 20, with an average of 10.4. The length of groups ranged from 28.2cM-116.2cM (Kosambi), with an average distance of 6.98cM between markers. The observed consensus map length and the estimated consensus map length were 1668.2cM and 2082.9cM, respectively, with the coverage of 80.09%.The genetic linkage maps of Cynoglossus semilaevis (♂) consisted of 218 SSR markers and it grouped 20 linkage groups.The number of markers per group ranged from 2 to 32,with an average of 10.9.With an average resolution of 7.21 cM.The observed consensus map length and the estimated consensus map length were 1571.7cM and 1935.0cM, respectively, with the coverage of 81.23%.186 microsatellite markers were common to both maps. It is the first time to construct SSR genetic linkage maps for Cynoglossus semilaevis which could be useful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding) and construction of high density linkage map of this species fish.