Establishment And Genetic Analysis Of Japanese Flounder (Paralichthys Olivaceus) Mitotic Gynogenesis And F3Family

Japanese flounder (Paralichthys olivaceus) is an important marine fish in China,which is distributed in China, Japan, South Korea, Russia and other regions. In China,it is mainly distributed in the BoHai sea, yellow sea and south China sea. Japaneseflounder is of high economic value, however, under the conditions of environmentand other factors, the catches of Japanese flounder decreased gradually. Therefore, theraising quantity increased sharply in the vast coastal areas in our country. However,the Japanese flounder breeding industry is lack of excellent variety. It is extremelyurgent to cultivate a fast-growing, hardly and well-taste Japanese flounder variety.There are two aspects of the research in this study, on one hand, hydrostaticpressure method was used to establish the lines of mitotic gynogenesis of Japaneseflounder, and genetic analyses of the inbred family and related gynogenesis familieswere done. On the other hand, Japanese flounder F3family was established andgenetic analyses were done. According to the genetic analyses of different familycombinations, seed-parents of high heritability and high breeding value were selected,and excellent cross combinations were selected. In addition, sperm cryopreservationof Epinephelus moara was carried out, aiming to provide technical support to the theestablishment of the Japanese flounder cleavage gynogenetic pure line.Bass sperm cryopreserved in Yellow Sea Fisheries Research Institute fish spermfreezer and good family built in2007were used as experimental materials,establishment and related experiments of mitotic gynogenesis Japanese flounderfamily were done. The results on mitotic gynogenesis show that, under the conditionof the water temperature17℃,58min after fertilization, disposed for6min byhydrostatic pressure590kg/cm2, with inactivate sea bass sperm, a higher success rateof induction (32.30±3.34%) was obtained. The typical haploid syndrome emerged atthe late gastrul stage of Japanese flounder, which were hard to survive. There was nosignificant difference between embryos of mitotic gynogenesis and normal diploid.Detection by flow Cytometer on three types of embryos shows that, the relativeamount of DNA was about27in embryos of mitotic gynogenesis and normal diploid, however, it was about17in the haploid. It indicates that by cleavage inducedgynogenesis, the chromosomes doubled and the embryos turned into normal diploid.Contrast the average weight of the two families, the results show that the differencebetween the two is extremely significant. The average weight of Inbreeding line wasabout50g growth at6-month, while the gynogenetic family was about22g.Identification and analysis on the0719Inbreeding line and correspondingcleavage of gynogenesis, by the31microsatellite sites of the highly polymorphic, theresults indicate that, all of the31microsatellite sites show heterozygous genepolymorphism, it shows homozygous singleton on site1、2、15、16、19and21, theother sites except24were in lines with the genetic law of segregation. Calculated bypopgene software, genetic similarity between selfing group and gynogenesis group is0.8139, while the genetic distance between which is0.2059Incomplete diallel cross was done on the parents from18families,51of F3families were established. Additive–dominant model of QGA software was used tocompare the genetic parameter estimates of F3families on5traits: the total length,body width, body weight, daily length incremental and daily weight increment. Ofall the selected families,1202、1226、1211、1206、1221and1219family showexcellent performance in4traits of total length, body width, body weight and dailyweight incremental. By additive effect analysis, NO. KS、F0917and F0905parentsshow a significant positive effect on5traits, the F09125shows a significant positiveeffect on the total length, body width, daily length incremental and daily weightincrement. The F0751×F0915、F0751×F09125、KS×F0750、F0917×F09121andF0917×F09104hybridized combinations show a significant positive effect (P＜0.01)on at least4traits. The research achievements provide theoretical basis for thefollowing analysis and family establishment.To study the sperm cryopreservation of Epinephelus moara, mature E. moarasemen was used as experimental material, suitable concentration of variety semendiluent, cryoprotectant and cryopreservation solution were selected. We use the60Lliquid nitrogen jar and2ml freezing tube to preserve sperm by “three-step coolingprocedure”. The result shows that, EM1-2semen diluent prepared with9g/L NaCl, 10g/L KHCO3and10%FBS(fetal bovine serum) was better than TS-2, ES1-3andother EM semen diluent, it could preserve56.67±5.77%sperm motility after unfrozen.Using EM1-2as liquid foundation to prepare cryoprotectant, no significant differencewas found in sperm motility by10%~20%DMSO and PG cryopreservation(P>0.05).The best of all are15%DMSO and10%PG, which could preserve54.52±7.81%and57.24±3.69%sperm motility after unfrozen separately. Using1-year cryopreservedsemen to fertilize E. moara eggs, the rate of fertilization and hatchability could reach80%or more, without significant difference by fresh sperm(P>0.05). Our study showsthat using EM1-2as liquid foundation to prepare15%DMSO or10%PG couldcryopreserve E. moara semen. We established the frozen sperm library based on thisstudy and provide a foundation for establishment and study of mitotic gynogenesisfamilyof Japanese flounder.