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银鲫遗传和生殖方式多样性的分子遗传标记研究
周莉
Subtype博士
Thesis Advisor桂建芳
2001
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword性别进化 雌核发育 两性生殖 分子标记 遗传多样性 生殖方式多样性 雌性特异dna片段
Abstract本研究在了解性别进化以及单性动物的进化遗传学及其相关研究进展的基础上,针对银鲫存在多个雌核发育克隆和雄性个体这两大问题,利用分子标记技术,将银鲫遗传多样性和生殖方式多样性联系起来进行研究。进展主要包括三个方面。一是运用RAPD和微卫星DNA分子标记,比较分析了银鲫5个雌核发育克隆间的遗传异质性及其相互关系,分别鉴定出特异于不同雌核发育克隆的88个RAPD分子标记和23个微卫星DNA标记。DNA扩增图谱及由此构建的分支图清晰表明了其克隆内的遗传同质性,克隆间的遗传异质性及其相互关系。接着从鉴定出的RAPD标记中选出5个标记,经回收纯化、克隆和测序,并依据所测序列,设计特异的SCAR引物,建立了5个可快速有效区分银鲫5个不同雌核发育克隆的SCAR标记。二是利用银鲫的克隆多样性和其种群中存在的雄性个体,设计大量的不同繁育组合,运用己鉴定的RAPD和SCAR分子标记,分析比较了不同繁育组合子代的DNA扩增情况,探讨了这些分子标记在各个交配子代中的重组分布规律。通过一系列研究,首次为银鲫同时具有两种生殖方式,即雌核发育和两性生殖提供了确凿的分子遗传证据。研究还表明,两性生殖方式为银鲫种群提供了丰富的遗传变异,创造了银鲫的遗传多样性。由此提出了银鲫两种生殖方式的基本模式图,该图为指导银鲫的育种和育苗实践提供了新的思路。三是在分析银鲫雌雄个体生长差异的基础上,采用RAPD引物对同一繁育组合中的雌雄个体进行扩增,试图寻找性别特异片段。经120个引物的大量筛选,从引物Oph-8的扩增产物中,检测到一大小为0.9kb的雌性特异DNA片段,并得到Southern blot验证。测序表明该片段为898bp, 在GeneBank中无同源序列。根据所测序列设计的一对引物经PCR检测也证实该片段为雌性特异。利用该引物还可在转性的雄性银鲫中检测到特异片段,表明该特异片段可用于鉴定正常雄鱼和转性雄鱼。
Other AbstractGenetic diversity, reproductive mode diversity and their relationships were studied by molecular marker techniques in natural gynogenetic silver crucian carp (Carassius auratus gibelio Bloch) based on understanding the research advances about sex evolution and evolutionary genetics of unisexual animals. The studies were mainly focused on two phenomena that there exist various gynogenetic clones and male individuals in the natural populations. Three major achievements were obtained in this dissertation. First, genetic heterogeneity and their relationships among five gynogenetic clones of silver crucian carp were analyzed by RAPD and microsatellite techniques. A total of 88 RAPD markers and 23 microsatellite markers were scored from 24 RAPD primers and 5 pairs of microsatellite primers. The amplified patterns and the constructed dendrograms clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and their phylogenetic relationships. Five typical RAPD markers were selected for molecular cloning and sequencing. According to the nucleotide sequences, five pairs of SCAR primers were designed and synthesized to identify the specific fragments in these gynogenetic clones. Five gynogenetic clones could be easily discriminated by the 5 SCAR markers. Second, a lot of artificial propagation experiments were performed between different gynogenetic clones to analyze whether or not genetic recombination occurs by utilizing the identified RAPD and SCAR markers. Direct molecular genetic evidence for gonochoristic reproduction was revealed in the gynogenetic silver crucian carp, and a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically was conformed. The responding mechanism of two different reproductive development modes might be the first discovery in vertebrates so far. On the other hand, gonochoristic reproduction produces abundant genetic variation, and creates genetic diversity. Based on the discovery, a preliminary model of reproductive modes was proposed, which can direct the breeding and propagation of silver crucian carp. Third, growth differences between females and males were analyzed, and sex-specific DNA fragments were attempted to screen by 120 RAPD primer amplification in the gonochoristic offspring. A 0.9 kb DNA fragment specific for females was screened in the RAPD pattern amplified by primer Oph-8, and was confirmed by Southern blot. The female-specific fragment was composed of 898 bp. According to the nucleotide sequences, one pair of PCR primers were designed and synthesized to detect the female-specific fragment in gonochoristic offspring. Additionally, the primers can be used to discriminate between the normal and the sex-revised males, because the revised males contain the female-specific fragment.
Pages158
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12584
Collection学位论文
Recommended Citation
GB/T 7714
周莉. 银鲫遗传和生殖方式多样性的分子遗传标记研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2001.
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