| In this thesis, the method of artifical induction of gynogenesis for Nibea albiflora was established and optimized, the embryonic development and early growth were compared in different groups, and the genetic analysis to both gynogenetic diploid and normal diploid has been conducted by using microsatellite(SSR) and AFLP markers.The main results were as follows:1. The gynogenetic diploids of Nibea albiflora were successfully induced by activating egg’s development with UV-irradiated sperms combined with cold shock to prevent extrusion of the second polar body. Relative DNA content in newly hatched larvae of candidate haploid gynogens, candidate diploid gynogens and the control groups from normal fetilization were measurated, and paternity test was taken by using SSR markers for confirmation of gynogenesis. The gynogenetic diploid was successfully induced by activating egg’s development with UV-irradiated sperm combined with cold shock to prevent extrusion of the second polar body. UV irradiation time range was preliminarily determined by an insemination trial with the sperms irradiated by different doses of UV. The fertilized egg hatchability exhibited typical Hertwig effect when sperm irradiation time was 0~100 seconds at a UV intensity of 3800μw/(cm2·s). All newly hatched larvae exhibited typical haploid syndrome when the UV irradiation time was over 60 seconds. In addition, we design an orthogonal experiment to further optimize UV irradiation time, cold shock initiation time and cold shock duration time. The orthogonal experiment results showed that the hatching rate was highest(16%) in the following conditions: sperm irradiation time was 60 seconds, cold shock temperature was 3~4℃, cold shock began at 2 minutes after fertilization and persisted for 10 minutes. The larvae of the optimal group had the same morphological characteristics and cellular DNA content with normal diploids. A further paternity test by using five compatible SSR primers proved that the larvae of the optimal group were all gynogenetic diploids for without paternal specific alleles.2. The gynogenesis was induced by the former method. The embryonic developments of gynogenetic diploid(G),haploid(H) and normal diploid(N) of Nibea albiflora were observed, and their early growth were compared. The results were as follows:(1) Fertilization rate was N>H>G, Hatching rate was N>G>H, Deformity rate was H>G>N, while survival rate of 72 hours larvae was N>G>H.(2) It took about 21 hours 10 minutes to hatch for the normal diploid, while it took 23 hours 10 minutes and 23 hours 30 minutes to hatch for gynogenetic diploid and haploid, respectively when the water temperature was(24±0.5)℃ and the salinity was 26~27. The embryonic development lag phenomenon were mainly appeared in early gastrula stage and hatching stage for gynogenetic diploid, in late gastrula stage for haploid. Morphologically, the haploid embryos showed typical haploid syndrome, while the gynogenetic diploid and normal diploid embryos had normal morphology and no significant difference between them. The death peak appeared in late gastrula stage for each group, all haploid larvae dead before mouth opening.(3) Before 60 days age, the growth rate of larvae of gynogenetic diploid was obviously lower than that of normal diploid,with larger individual different within the former group.3. Microsatellite and AFLP markers were applied to identificate and analyse the induction of Nibea albiflora gynogenetic diploid offspring, as well as the transmission and separation of these molecular markers in the gynogenetic diplpid family and the normal family, and their homozygous genetic relationship. The results showed that:(1) none of paternal allele was found in 8 microsatellite loci and 5 pairs of AFLP primer’s amplification loci of gynogenetic family, indicated that the gynogenesis induction rate was 100%.(2) none of completely homozygous loci was found in 7 pairs of SSR primer in the two strains. Compared to normal family, average homozygosity of the gynogenetic family enhanced, homozygosity of several sites declined; the highest homozygosity site was LYC0267, of which the pure degree was 0.825; while the lowest homozygosity loci was LYC0012, of which the homozygosity was 0; the average homozygosity was 0.382, and it was 2.37 times of that of the normal group(0.161). Except the LYC0012 site, the separation ratio of the remaining alleles in gynogenetic family were in line with Mendel’s Law(P>0.05). Homozygous loci gynogenesis pedigree was 0~6, and the homozygous ratio was 0~0.857.(3) 5 pairs of AFLP primers had amplified 182 bands, 21 specific bands of male parent, 16 female specific bands.16 female specific bands had 10 separate, as heterozygous loci, and 7 loci in the gynogenetic family suggested significant partial separation(P<0.05); 2 female specific bands in the gynogenetic progenies appeared and in normal group generation partly appeared, which might closely linked to the recessive lethal gene.(4) Microsatellite and AFLP statistics on the genetic similarities and differences got the same results: gynogenetic family and female had higher similarity than normal family; the latter had the roughly same genetic distance with parents, they were amphimixis individuals. |
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