Morphologic And Genetic Characteristics Of Clones And Temperature Influence On Growth And Development Of Heterozygous Clones Of Japanese Flounder (Paralichthys Olivaceus)

Clone fishes are high quality genetic breeding materials, which havescientific value in genetic, developmental biological, physiological andenvironmental toxicological studies. Clone line can be used to eliminate thegenetic differences in order to improve the accuracy of the experiment forits homozygosity and isogenicity between individuals. Heterozygous clonesare produced by cross between male and female double haploids or clones.The genetic background of the heterozygous clones is identical but theindividuals are heterozygous. So far, homozygous and heterozygous fishclones have been successfully produced in few fish species, such aszebrafish (Brachydanio rerio), Medaka (Oryzias latipes), Red sea bream(Pagrus major), etc.In this research, clones of Japanese flounder (Paralichthys olivaceus)were produced by the method of meiotic gynogenesis using the eggs fromone female doubled haploid. Then, morphological and geneticcharacteristics of clones were studied. Heterozygous clones of Japaneseflounder (Paralichthys olivaceus) were produced by the cross of male andfemale doubled haploids, and the influence of sex differentiation, sex ratiosand growth differences by water temperature were studied.The main results are as follows:(1) Clones were highly similar in morphology. The coefficient variationvalues of total length/body length, total length/head length, totallength/caudal peduncle length, total length/body depth, caudal pedunclelength/caudal peduncle depth and body depth/caudal peduncle depth were 0.51%,1.67%,1.96%,1.67%,2.14%and2.00%, respectively. Thesecoefficient variation values were significantly smaller than those inordinary groups (P <0.05).(2) The Shape discriminant functions of clones and ordinary group (controlgroup) were established severally at the age of120days. The functionswere as follows.Y(Clone1)=21370.264X1+448.802X2+243.273X3-2035.533X4+4715.655X5-1087.834X6-12212.519Y(Control)=21196.452X1+464.518X2+246.094X3-2064.598X4+4745.065X5-1103.811X6-11989.084The results showed that the F test had a good test effect (P <0.01).The shape discriminant functions of Clones were also apply to clones at theage of150days, while functions of the common group changed as the agechanged.(3) Twenty-four microsatellite markers with high recombination rate werechosen to identify the genetic status of three clone groups. The resultsshowed that the homozygosity reached100%and genetic similarity indexeswere1.0between maternal parents and their progenies also among progenyindividuals.(4) The heterozygous clones were grouped and reared at different watertemperature of16℃,19℃,22℃,25℃,28℃and31℃from35days to110days after hatch and the sex differentiation, sex ratios and growthdifferences were studied. The results showed that there was significantcorrelation between sex differentiation and water temperature. From16℃to28℃, the higher the temperature was, the faster sex differentiated. whilethe gonad development stopped in31℃. Heterozygous clones in differentwater temperatures were all female by histological observation, whichsuggested that high water temperature had no effects on sex-reverse. Theresult was significantly different from the former study ontemperature-induced sex reversal, so the genetic mechanism was stillunknow which need further research. (5) Temperature made significant effect on the growth rate of heterozygousclones (P <0.01). From16℃to28℃, heterozygous clones grew fasterwhen the temperature rises. In the meanwhile, the larva grew fastest in25℃, which provided references for future breeding.