Development Of Sex-Specific Markers And SSR Markers And Construction Of Genetic Linkage Maps Of Three Important Cultured Marine Fish Species

Spotted halibut (Verasper variegatus), barfin flounder (Verasper moseri) and half-smoothtongue sole (Cynoglossus semilaevis) are the three important cultured marine fish species inthe northern China. Spotted halibut mainly distributed in the Bohai Sea, the Yellow Sea, aswell as along Korean Peninsula, barfin flounder mainly distributed in cold sea basins aroundEast Hokkaido, Japan, as well as the Bohai Sea and the Yellow Sea, China, while half-smoothtongue sole which is the native fish species of China, mainly distributed in the north Chinacoast. At present the number of wild individuals of the three fish species is little, and thefisheries resource is rather critical. In aquaculture, they have been the most popular fishspecies together with turbot (Scophthalmus maximus) and olive flounder (Paralichthysolivaceus) in the northern China. In order to benefit the molecular breeding of fish species,the three items were studied in this paper including (1) Isolation, cloning sex-specific DNAmarkers and development genetic sex identification technique; (2) Isolation of polymorphicmicrosatellite markers (ⅠandⅡtypes); (3) Construction of genetic linkage maps. The mainlyresearch results are as follows:1. Isolation and cloning of sex-specific markers:(1) The differences of genomic DNA between females and males of spotted halibut (Veraspervariegatus) were studied by using AFLP technique. As a result, three female-specific AFLPmarkers (VevaF160, VevaF218 and VevaF533) were found to be only present in all femalesbut absent in any males. The three markers were all cloned and sequenced, and analysisindicates that they are different and novel markers for spotted halibut. Furthermore, thegenetic sex identification technique was developed which could be useful to shorten the breeding cycle, and improve the breeding process. The inheritance patterns of thefemale-specific marker VevaF218 in an interspecific hybrid family was examined, and theresult showed a female-specific inheritance patterns from mother to daughter. It may behelpful for better understanding the sex determination system in spotted halibut (Veraspervariegatus).(2) Based on the previous study, four female-specific AFLP markers (CseF783, CseF464,CseF305 and CseF136) were cloned in half-smooth tongue sole (Cynoglossus semilaevis), andsuccessfully converted into SCAR (sequence characterized amplified regions) markers.Subsequently, the simple and fast technique for genetic sex identification was developed. Atlast, this technique was applied in the genetic sex identification of three different derivedindividuals including the candidate neo-males (the genetic sex is female but the phenotypicsex is male), the progeny from the cross of the neo-male with the common female, theprogeny from the cross of common male with female.(3) The differences of genomic DNA between females and males of barfin flounder (Veraspermoseri) were studied by using AFLP and SSR markers. As a result, several candidatesex-linked AFLP markers were found but none of them were sex-specific.2. Isolation and characterization of polymorphic microsatellite markers(1) Three repeat-echriched genomic libraries (the repeat units were GT, GA and GATA,respectively) were constructed by FIASCO (fast isolation by AFLP of sequences containingrepeats) in spotted halibut (Verasper variegatus). 229 sequences were obtained, of which191 contained microsatellite repeats. 141 pairs of primers were designed successfully, and 40microsatellite loci were proved to be polymorphic. These markers will be benefit for theevaluation of genetic resource and the construction of genetic linkage map in spotted halibut.(2) A total of 31 polymorphic microsatellite loci were reported, of which, 28 loci wereisolated from two repeat-echriched genomic libraries (the repeat units were GT and GATA,respectively), and threeⅠtype loci were isolated from the function known genes. This studycould provide the candidate markers for the evaluation of genetic resource and theconstruction of genetic linkage map in barfin flounder.(3) Functional genes of half-smooth tongue sole (Cynoglossus semilaevis) were screened fromGenBank database. 19 microsatellites were identified and successfully designed 19 pairs of primers. After tested on 30 individuals, seven microsatellite loci showed polymorphic. It is thefirst time to reportⅠtype micrsatellites linked with functional genes in half-smooth tonguesole (Cynoglossus semilaevis), and will be benefit for evaluation of genetic resource,construction of genetic linkage map and molecular marker-assisted breeding (MAS) inhalf-smooth tongue sole (Cynoglossus semilaevis).3. The construction of genetic linkage maps(1) A first consensus AFLP-based genetic linkage map of half-smooth tongue sole(Cynoglossus semilaevis) was constructed. A total of 205 AFLP markders were mapped in 27linkage groups. The linkage between the loci was identified by an LOD score of≥3.5. Thenumber of markers per group ranged from 2 to 28, with an average of 7.6. The length ofgroups ranged from 4.4 cM to 87.2 cM (Kosambi), with an average distance between markersof 5.05cM. The observed consensus map length and the estimated consensus map length were899.4 cM and 1299.3 cM, respectively, with the coverage of 73.2%. This linkage map may behelpful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding)and construction of middle or high density linkage map of half-smooth tongue sole(Cynoglossus semilaevis).(2) Preliminary genetic linkage maps of barfin flounder (Verasper moseri) (♀) and spottedhalibut (Verasper variegatus) ((?)) were constructed using microsatellite and AFLP markers.The barfin flounder map consisted of 98 markers (40 SSR markers and 58 AFLP markers)grouped in 27 linkage groups, with an average resolution of 8.9 cM. The spotted halibu mapconsisted of 86 markers (38 SSR markers and 48 AFLP markers) grouped in 24 linkagegroups, with an average resolution of 10 cM. Five microsatellite markers were common toboth maps. More markers especially microsatellites should be mapped on the two maps, as theresolution of the two maps is too low. It is the first time to construct genetic linkage maps forbarfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus), and could beuseful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding)and construction of middle or high density linkage map of this two fish species.