| Oreochromis niloticus,GIFT is a genetically improved tilapia,which belongs to a new strain formed by breeding.It has fast growth rate,large individual size and high meat yield.At present,the development of tilapia aquaculture industry has suffered from serious species degradation and impurity of mixed germplasm due to the factors such as interspecific hybridization and instability of various traits,which has affected the economic benefits of aquaculture.Therefore,it is necessary to carry out genetic improvement and breeding of new varieties of O.niloticus,GIFT.At present,the main methods of fish genetic breeding include cross breeding,gynogenesis,androgenesis,polyploid breeding and so on.Many kinds of fish have developed new varieties with high economic value through these methods,which has produced great economic benefits currently.The present study was conducted to explore the process of distant hybridization between O.niloticus,GIFT(?)and Parachromis managuensis(?),Heterotilapia buttikoferi(?),Siniperca chuatsi(?)separately to explore the possible process of distant hybridization to obtain the hybrids with good traits of parents for enrich the basic data of distant hybridization of fish.Further,the cytogenetic and molecular genetic analysis of the well developed hybrids was carried out,researching the genetic relationship among the father,mother and their progeny,the homozygosity of the hybrids population,genetic relationship between hybrids population and two other tilapia strains,in order to provide reference for the development and utilization of distant hybrid F1;Otherwise,screening suitable gynogenetic sperm,the sperm of P.managuensis was used to induce eggs of O.niloticus,GIFT for development of meiotic gynogenesis,further analysis of embryonic development,cytogenetics and molecular genetics of gynogenetic offspring,which provides technical method for further study on the theory of gynogenesis of O.niloticus,GIFT.The main findings are as follows:1.Study on embryonic development between of hybrids of Oreochromis niloticus,GIFT(?)and Parachromis managuensis(?),Heterotilapia buttikoferi(?),and Siniperca chuatsi(?)The embryonic development of O.niloticus,GIFT(?)and P.managuensis(?),H.buttikoferi(?),S.chuatsi(?)in the blastula period was basically the same as that in the control group.When the embryos of the control group developed to the gastrula period,the embryos of the three distant hybridization groups showed the peak of development stagnation.When the embryos in the control group developed to the early segmentation period,the embryonic development of the three distant hybridization groups was significantly slower than that in the control group,and a large number of abnormal embryos appeared.In the hatching period,the abnormal embryos died continuously.At first feeding,some of the abnormal individuals survive,but they can't eat,and then all die.In addition,there are some deaths from the hatching period to the first feeding in the normal phenotype of each group of embryos.The fertilization rate of embryo of O.niloticus,GIFT(?)and P.managuensis(?),H.buttikoferi(?),S.chuatsi(?)were 96.18± 1.74%,94.78 ± 1.68% and 95.61 ± 1.19%,respectively.The hatching rate of embryo of O.niloticus,GIFT(?)and P.managuensis(?),H.buttikoferi(?),S.chuatsi(?)were1.41 ± 0.29%,2.02 ± 0.16% and 16.25 ± 1.64%,the survival rate of embryo of O.niloticus,GIFT(?)and P.managuensis(?),H.buttikoferi(?),S.chuatsi(?)were 0%,0% and 0.34 ± 0.13%,respectively.The fertilization rate of the three distant hybridization groups was significantly lower than that of the control group(P < 0.05),the hatchability rate and the survival rate of the three distant hybridization groups were all significantly lower than that of the control group(P < 0.01).Fertilization rate of the hybrids of O.niloticus,GIFT(?)and P.managuensis(?)was 96.18 ± 1.74%,fertilization rate of the hybrids of O.niloticus,GIFT(?)and H.buttikoferi(?)was 94.78± 1.68%,hatching rate and survival rate of two hybridization groups were 0.Fertilization rate of the hybrids of O.niloticus,GIFT(?)and S.chuatsi(?)was 95.61 ±1.19%,the embryo development of hybrids had a peak of development stagnation and a large number of abnormal embryos appeared,the abnormal embryos died in the development process constantly.The hatching rate and survival rate of embryos were 16.26 ± 1.64% and 0.34 ± 0.13% merely.2.Study on the cytogenetics and molecular genetics of the hybrids of Oreochromis niloticus,GIFT(?)and Siniperca chuatsi(?)The relative DNA content of the surviving individuals was analyzed by flow cytometry.The results showed that there was no significant difference between the DNA content of the well developed hybrids and that of the O.niloticus,GIFT.The chromosome karyotype analysis results showed that the chromosome number of well developed hybrids is 2n = 44,karyotype is 6sm + 24 st + 14 t,numbers of chromosome arms(NF)=50,the chromosome number of the maternal O.niloticus,GIFT(?)is 2n =44,karyotype is 6sm + 26 st + 12 t,NF=50,the chromosome number of the faternal S.chuatsi is 2n = 48,karyotype is 8sm + 10 st + 30 t,NF = 56.The hybrids of O.niloticus,GIFT(?)and S.chuatsi(?)were diploid and contained the same number of chromosome as the female parent O.niloticus,GIFT,and their karyotypes are similar.Chromosome number and karyotype of hybrids is different from the male parent S.chuatsi.10 microsatellite markers were selected to amplify the microsatellites of S.chuatsi,which could not produce bands in O.niloticus,GIFT population but had high polymorphism in the S.chuatsi population,the amplification results of 10 microsatellite markers of S.chuatsi in the male parent and the group of hybrids of the whole sib family showed that the microsatellite loci did not amplify the bands in the hybrids population,but in the male parent.The results showed that the hybrids has not inherited the genetic material of the male parent.Ten microsatellite markers of tilapia were amplified and the size of the bands was the same in the female parent and hybrids of the whole sib family.SSR analysis showed that 69 alleles were amplified with 10 SSR markers of tilapia in the group of O.niloticus,GIFT,hybrids,and O.niloticus in Wuxi and the number of alleles at each site was 1 or 2,the amplification bandwidth of these primers ranged from 100 ~ 300 bp.The results showed that the average observed homozygosity and average expected homozygosity of hybrids were highest.The result of cluster analysis revealed two clustering groups.One cluster included hybrids and O.niloticus,GIFT,and the other cluster was composed of O.niloticus in Wuxi.Based on the analysis of relative DNA content,chromosome number and karyotype analysis,microsatellite genetic analysis,it can be considered that hybrids of O.niloticus,GIFT(?)× S.chuatsi(?)belongs to the gynogenetic individual.3.Study on the gynogenesis of Oreochromis niloticus,GIFTMeiotic gynogenesis was induced in O.niloticus,GIFT eggs using UV-irradiated spermatozoa from P.managuensis,and then the female parent DNA was duplicated with cold shock at 10? ~ 12?,the duration of cold shock is 30 ~ 45 min,the population of O.niloticus,GIFT was successfully obtained.Fertilization rate of embryos of gynogenesis was 96.34 ± 1.18%,hatching rate of embryos were 15.36 ± 1.86%ultimately and survival rate of embryos were 2.12 ± 0.42% merely.There was no significant difference in the fertilization rate between the gynogenetic group and the control group(P > 0.05).The hatching rate and survival rate of embryos in the gynogenetic group were significantly lower than those in the control group(P < 0.01).Two kinds of individuals,normal individuals and abnormal tortuous individuals,were hatched.Their DNA content was measured by flow cytometry.The normal individuals were identified as diploid,whereas the abnormal tortuous individuals were haploid.The chromosome number of the gynogenesis O.niloticus,GIFT is 2n = 44,karyotype of which is 6sm + 22 st + 16 t,NF = 50,the chromosome number of the maternal O.niloticus,GIFT is 2n = 44,karyotype of which is 6sm + 26 st + 12 T,NF =50,the chromosome number of the faternal P.managuensis is 2n = 48,karyotype of which is 8sm + 20 st + 20 t,NF = 56.SSR analysis showed that 66 alleles were amplified with 10 microsatellite markers in three group,and the number of alleles at each site was1 or 2,the amplification bandwidth of these markers ranged from 100 ~ 300 bp.The results showed that the average observed homozygosity and average expected homozygosity of gynogenetic O.niloticus,GIFT were highest.The result of cluster analysis revealed two clustering groups.One cluster included gynogenetic O.niloticus,GIFT and O.niloticus,GIFT,and the other cluster was composed of O.niloticus in Gaozhou.The results showed that the genetic consistency of gynogenetic population was quite high. |
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