摘要
用紫外照射遗传失活的异源精子人工诱导成熟草鱼卵子发育,再抑制被激活卵第一次卵裂或者第二极体排出可以得到人工雌核发育草鱼。人工雌核发育技术可加快草鱼纯化过程,是快速经济地建立遗传背景一致纯系的有效方法。然而,在鱼类人工雌核发育中,有关紫外照射遗传失活的异源种精子是否对人工雌核发育鱼有着遗传和发育方面的影响仍然存在争论。为分析外源精子对人工诱导雌核发育草鱼基因组的影响,本研究利用RAPD方法和微卫星技术对14尾二代雌核发育草鱼及其母本一代雌核草鱼和父本鲤鱼的基因组DNA进行了比较分析。
本研究的内容和结果如下:
1.通过对一代雌核发育草鱼中期分裂相的观察统计结果表明:一代雌核发育草鱼具24对染色体,染色体核型为2N=48=18m+24sm+6st,核型与野生草鱼完全一致,没有发现任何额外的染色体片断。故可以肯定所用的雌核发育一代亲本中没有异源鲤鱼精子染色体片段的存在。
2.RAPD分析表明,10条随机引物在二代雌核发育草鱼,母本雌核草鱼和父本鲤鱼中分别检测到104,104,103条扩增带,二代雌核发育草鱼中共扩增出104条片段与母本雌核草鱼相同。单个引物检测到的扩增片段数目在6-13之间,大小在0.2Kb-3Kb之间。10条引物中仅S66在二代雌核发育草鱼,母本雌核草鱼和父本鲤鱼中扩增出1条大小相同的条带,但经过序列分析,它们的核苷酸序列明显不一样,其它9条引物均未扩增出与父本鲤鱼片段大小相同的条带。
3.采用微卫星技术对二代雌核发育草鱼及其亲本基因组DNA进行统计分析。结果表明7对鲤鱼微卫星引物在雌核发育草鱼中共扩增出4个微卫星位点,在鲤鱼中检测到22个位点,大小在124-297bp之间。且7对引物在二代雌核发育草鱼和其母本一代雌核草鱼中的扩增条带完全相同,而与鲤鱼的完全不同。
4.遗传相似度分析:根据RAPD和微卫星实验数据进行的遗传相似度分析表明二代人工雌核发育草鱼群体与其一代人工诱导雌核发育草鱼母本的遗传相似度从0.9903到1.000,而与父本鲤鱼的遗传相似度为0.000。
以上这些实验结果显示在二代雌核发育草鱼、母本雌核草鱼和父本鲤鱼之间没有检测到一致的位点,说明在适当的紫外线处理强度下,鲤鱼精子的遗传物质能够被完全破坏,不会对雌核发育草鱼的基因组造成遗传污染。
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.
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