Analysis On The Genetic Influence Of Heterologous Sperm In The Genome Of Gynogenetic Grass Carp

By inducing the gynogenetic development of mature grass carp eggs with UV-irradiated common carp sperm and then inhibiting the first cleavage or the second polar body release of the gynogenetic eggs, absolute pure diploid individual or very highly pure diploid individual could be obtained. Modern artificial gynogenetic technology provides a rapid and economic way to establish pure line or highly pure inbreeding line of grass carp. The genetic and developmental impact of the UV-irradiated heterologous sperm on artificial gynogenetic fish, however, has remained a subject of debate in the fish artificial gynogenetic studies. In order to determine the genetic influence of heterologous sperm in the genome of artificial gynogenetic grass carp, genomes of artificially induced gynogenetic grass carp and their parents were comparatively analyzed with the techniques of random amplification polymorphism DNA (RAPD) and microsatellite analysis. The 14 gynogenetic grass carp individuals used in this examination is from a two-generation artificially induced gynogenetic grass carp group (meio-gynogenetic-2 group), in which all the individuals share an identical gene type. The mother of this meio-gynogenetic-2 group is an individual from a one-generation artificially induced gynogenetic grass carp group (meio-gynogenetic-1 group).The main contents and results were as follows:1. Chromosome analysis indicated that chromosome number of the meio-gynogenetic-1 grass carp group, in which the grass carp mother was selected in this studying, was 2N=48, and the karyo formula is 2N =18m+24sm+6st. No chromosome or chromosomal fragments from the common carp sperm in the metaphase the meio-gynogenetic-1 grass carp group, were detected. Pure mother could simplify ascertaining the heritable origin of gene loci in the gynogenetic grass carp.2. With 10 pair of polymorphic random primers, total 104 RAPD loci were detected in both the genomes of the meio-gynogenetic-2 group and in their grass carp mother, and 103 loci in the paternal common carp. There were 104 similar bands in the meio-gynogenetic-2 group and their grass carp mother. The number of bands amplified from the 10 pair of polymorphic random primers was from 6 to 13, and their length was from 0.2Kb to 3Kb. On one RAPD locus, through the PCR products of the meio-gynogenetic-2 grass carp group could not be distinguished from that of the paternal common carp by their length, significant difference were observed in their nucleotide sequences. There were no similar bands amplified from other primers in the meio-gynogenetic-2 group and their paternal common carp.3. In microsatellite survey with 7 pairs of microsatellite primers, 4 microsatellite loci in both of the meio-gynogenetic-2 grass carp group and their grass carp mother, and 22 in their paternal common carp were detected respectively. Their size was from 124bp to 297bp. All loci were identical in the meio-gynogenetic-2 grass carp group and their grass carp mother and no locus was identical in the meio-gynogenetic-2 group and the common carp.4. According to the RAPD and microsatellite results, Genetic similarity analysis was 0.9903 to 1.000 between the meio-gynogenetic-2 grass carp group and the meio-gynogenetic-1 grass carp mother, but 0.000 between the meio-gynogenetic-2 grass carp group and the paternal common carp. These results indicated that no locus of the detected loci was identical in the gynogenetic grass carp and their paternal common carp, the genetic materials of the heterologous sperm did not contaminate the genome of the meio-gynogenetic-2 grass carp group and the genetic materials of common carp sperm could be completely destroyed by properly UV-irradiation.