An Optimizing Experiment To Artificially Induce Gynogenesis And Genetic Marker Analysis In The Breeding Population Of Culter Alburnus

In order to efficiently establish Culter alburnus breeding population, this paper used a quadratic orthogonal rotation combined design to determine the optimal combination of factors to induce gynogenesis in Culter alburnus, then used the polymorphism microsatellite makers analysis of genetic purity variations among Culter alburnus of breeding pupulation and successive generation gynogenesis populations(Meio-gynogenesis), and paternity identified for multiple families of Culter alburnus mixed breeding population by screening the specific parent polymorphism microsatellite makers. The results were as follows:1) Culter alburnus gynogenetic diploids were created by activating eggs with ultraviolet-irradiated common carp sperm and preventing extrusion of the second polar body using cold shock to induce chromosome duplication. Three-factor five-level quadratic orthogonal rotation combined design was used to determine the optimal combination of three experimental factors, including time after fertilization(x1), treatment duration(x2), and shock temperature(x3) to induce artificial gynogenesis in Culter alburnus, using gynogenesis rate(y1) and hatching rate(y2) as indicators in the quadratic regression model. Furthermore, the response surface method was used to determine the effect of single factors and their interactive effect on the response value.We found that, the optimal to induce gynogenesis(18.00%) was cold shock for 18 min at 7℃ applied 6min right after fertilization, as for maximal egg hatch(33.00%) was cold shock for 23 min at 5℃ applied 6min right after fertilization. The relationship between the various factors on the response values was paraboloidal with a downward opening. According to the factor contribution rates, cold shock start time had a highly significant effect on cold shock,followed by shock temperature and treatment duration. This is the first study to use a quadratic orthogonal rotation combined design to determine the optimal combination of factors to induce gynogenesis in Culter alburnus. These results provide a technical support and theoretical guidance for Culter alburnus breeding.2) With the usage of the transcriptome data,we screened all of the Culter alburnus out and obtained 109 pairs of polymorphic primers from 337 pairs of microsatellite primers. Among them we selected 20 pairs of suitable primers with good reproducibility and polymorphism for the further experiments. Through the research and analysis of genetic structure and the degrees of homozygosity of gene loci which all of the meiosis gynogenesis were artificially induced including Meio-G1,G2,G3, Culter alburnus cultured stocks types were regarded as a control, we addressed the effect of induced gynogenesis for three generation on the gene purification. The results showed that,the amount of 20 pairs of microsatellite genes amplified to 103, 76, 51, 38 alleles, the average observed heterozygosity(H0) was 0.7724、0.4362、0.2203、0.1050 and the average homozygosity were 0.2276、0.5638、0.7797、0.8950 in the control group and three generations of Culter alburnus gynogenesis(Meio-G1,2,3) respectively. The cluster analysis showed that, Meio-G2 group and the control group were clustered in a clad, as same as the Meio-G2 group and Meio-G3 group. In addition, there were obvious and stable homozygous loci in the Meio-G2 and Meio-G3 groups. This study suggested that consecutive artificial induction of gynogenesis can enhance greatly the homozygosity of gene loci. Also, Culter alburnus gynogenetic groups in this study provide an excellent breeding material to establish Culter alburnus breeding population for the future research.3) In this study we screened out different specific markers for 3, 4, 5, 6 Culter alburnus gynogenetic mixed families paternity identification as a reference in practical breeding population. The combined probabilities of exclusion gradually increased to maximize and tende to be stable as the number of markers identified increased which were used as a paternity identification marking combination, this method decreased the aimless of selection Culter alburnus parentage markers. In the reference population, the combined probabilities of exclusion reached 100% with screened genetic markers. All offspring of Culter alburnus can be assigned to the corresponding maternity parents in 95% confidence. It is proved that those genetic narkers can be fully used in Culter alburnus gynogenesis paternity identification. These genetic markers of high parental exclusion can be used for parents’ selection at subsequent new strains multiplication population and designated as candidate markers to develop joint breeding with DNA molecular marker in future.