Construction Of Genetic Linkage Map And Preliminary Study Of Chromosome Mapping In Half Smooth Tongue Sole (Cynoglossus Semilaevis)

Half smooth tongue sole {Cynoglossus semilaevis) is a kind of large farmed ifsh inYellow Sea and Bohai Sea. The wild resources of this species are dwindling and thesituation of ifshery resources is grim. The present factory farming fries are mainlygenerated from captured and domesticated wild broodstock，which is very detrimentalto the long-term development of the aquaculture industry. Three parts of work aroundthis situation were carried out in this study: development of microsatellite molecularmarkers; construction of genetic linkage map; preliminary study of chromosome map.Our results laid the foundation for molecular marker-assisted breeding work in halfsmooth tongue sole.1. Development of microsatellite molecular markersWe developed microsatellite molecular markers of half smooth tongue sole in threeways: enriched library+colony in situ hybridization; screening in cDNA librarysequences; screening in Fosmid library sequences. Using the way of enriched library+colony in situ hybridization, we screened1718positive clones and sequenced1192clones. A total of784pairs of primers were designed，474of which were polymorphicdetected in six wild individuals. Using the way of screening in cDNA librarysequences,71pairs of primers were designed in908cDNA library sequences,12ofwhich were polymorphic. Using the way of screeing in Fosmid library sequences,117pairs of primers were designed in768Fosmid library sequences,74of which werepolymorphic. By comparing the eiffciency of the three methods, we found that markerobtaining rate by way of enriched library+colony in situ hybridization (65.8%) washigher than that of the other ways (8.5%and15.2); polymorphic primers rate by screening in Fosmid library sequences (63.2%) was slightly higher than that by theway of enriched library+colony in situ hybridization (60.5%) and was much higherthan that of screening in cDNA library sequences (15.6%).2.Construction of genetic linkage mapWe constructed the ifrst microsatellite genetic linkage map of half smooth tonguesole with polymorphic markers developed in this research and published by ourlaboratory. Mapping population employed a full-sib F1family. The female map wascomposed of21linkage groups with the total length of1041cM. There were193markers in this map and174loci were located. Among the21linkage groups, theshortest was2cM and the longest was88cM. The minimum average interval was2.0cM and the maximum was17.0cM，with an average resolution of6.8cM. There were8.3markers per linkage group contained, and the expected length was1356.9cM. Thecoverage rate was reckoned to be approximately76.7%. The male map was alsocomposed by21linkage groups with the total length of1154cM. There were195markers in this map and181loci were located. Among the21linkage groups, theshortest was6cM and the longest was102cM. The minimum average interval was3.5cM and the maximum was16.5cM，with an average resolution of7.2cM. Therewere8.6markers per linkage group contained. The expected length was1477.3cMand the coverage rate was approximately78.1%. The recombination ratio offemale/male was about1:1.02estimated in the sex-speciifc frame map.3.Preliminary study of chromosome mapWe located3Fosmid clones containing microsatellite markers and2Fosmid clonesfrom library walking by Fluorescence in situ hybridization (FISH). We veriifed thepossibility of integrating the microsatellite genetic linkage map and the chromosomemap using Fosmid-FISH technology. Three Fosmid clones were found, which couldlocated in W chromosome and a pair of autosomes simultaneously. The chromosomallocalization of both18S rDNA and5S rDNA were studied through designing PCR probes. The results showed the positioning pattern of18S rDNA was slightly differentin females and males. Seven signals of the major rDNA (18S rDNA) were mapped tothe telomeric region of the long arm on W chromosome and the centromeric regionsin three pairs of autosomes in females. While there were only six signals mapping tothe centromeric regions of three pairs of autosomes in males. As to the minor rDNA(5S rDNA), twelve signals were found mapped to the centromeric regions of six pairsof autosomes in females and males. Double FISH further certiifed that two pairs ofthe18S rDNA signals and two pairs of the5S rDNA signals were correspondinglylocated at the same positions in the same autosomes. Only18S DNA signal could beidentiifed on the W chromosome in female genome. Signals of18S rDNA were alsolocated on another pair of autosomes, while those of5S rDNA were observed on other4pairs of autosomes. It was found that there were at least two repeat fragment richregions on W chromosome through locating clone B106-62and the other two Fosmidlibrary walking clones. We speculated that original sex chromosomes of half smoothtongue sole might undergo chromosome fusion, repeat fragment accumulation andnon-homologous chromosome translocation during evolutionary process, and theformation of W chromosome was a gradual transition process.