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Alternative TitleDevelopment of Type I genetic markers and analysis of population genetic structure in common carp and bighead carp
Thesis Advisor童金苟
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword Est 瘦素受体基因 数据挖掘 Ssr Snp 遗传结构 经济性状
Abstract鲤(Cyprinus carpio)和鳙(Aristichthys nobilis)广泛分布于我国各大水系,是重要的养殖鱼类。由于过度捕捞和生境破坏等问题,我国淡水鱼类的天然资源显著减少;再加上缺乏科学的人工繁育管理体系,鲤科经济鱼类的种质状况令人堪忧。本研究研发了鲤和鳙的I型遗传标记即功能基因标记,用于分析其群体遗传结构,探讨不同基因型与生长性状的关联性,从而为鲤和鳙的种质资源保护、分子标记辅助育种等提供理论依据。主要研究结果和结论如下: 1、利用生物信息学方法从公共数据库中下载并分析鲤的18840条EST(Expressed Sequence Tag)序列,其中微卫星(Simple Sequence Repeat,SSR)的EST占总数的5.55%,聚类分析表明903条是唯一的EST。包含SSR的EST序列(EST-SSR)主要由二、三、四和五碱基组成,它们占所分析的鲤EST序列的百分比分别为37.2%、30.8%、20. %和11.7%。部分EST-SSR引物在鲫和鳙的跨种扩增多态比率分别为17%和5%。共设计合成470对EST-SSR引物。本文所研发的鲤EST-SSR标记适合于群体遗传学和基因组作图的研究。 2、利用10个基因功能已知的多态EST-SSR位点,分析了11个鲤鱼群体(10个野生群体,1个养殖群体)的遗传结构。总体而言,鲤鱼野生群体的遗传变异较高,黑龙江水系的遗传多样性明显低于长江水系,西江群体的等位基因最为丰富。NJ(Neighbor-Joining Clustering)聚类和SOM(Self-Organized Map)人工神经网络结果显示,遗传距离和地理距离相关性不显著,鲤鱼群体间有一定的基因交流,分化程度为中等。本研究证实了EST-SSR属非中性选择标记的结论,运用这类功能性遗传标记“扫描”物种的基因组有助于探讨鲤鱼群体对不同生境的适应性及其与经济性状的相关性。这方面的研究有待进一步加强。 3、利用4个基因功能已知的多态EST-SSR位点,探讨鲤鱼EST-SSR标记的基因型与鲤鱼外观性状的关系。结果表明4个EST-SSR位点均有与体长、体重和肥满度显著性相关的基因型(P<0.05),推测它们与鲤鱼经济性状有重要的关联性。 4、克隆并分析了鳙瘦素受体(Leptin receptor, Lepr)基因序列及其内含的微卫星和SNP(Single Nucleotide Polymorphisms)位点的变异。比对结果显示鳙的Lepr基因较保守,与哺乳动物的同源性较高。运用PCR-SSCP技术和DNA测序方法,对鳙家系的Lepr基因进行SSR和SNP多态位点的查找及关联性分析,得到一个SSR和两个SNP多态位点,并利用得到的SSR位点比较鳙养殖群体和野生群体的遗传多样性,结果表明两者差异不显著。通过广义线性模型分析SSR、SNP与外观性状的关联性,表明微卫星座位不同的基因型与外观性状不相关;SNP位点的基因型Aa的体高显著大于AA、aa(P<0.05),Bb型的体高和头长显著大于bb型(P<0.05);等位基因G的体长、体高、头长、体重和肥满度大于等位基因A(P<0.05),为群体中的优势等位基因。本研究表明Lepr基因的SNP位点与鳙的某些生长性状有显著相关性,可作为鳙经济性状的潜在候选基因,用于分子标记辅助育种的进一步研究。
Other AbstractThe common carp (Cyprinus carpio) and bighead carp (Aristichthys nobilis) are two widely distributed fish species and have been intensively cultured in China. In the past a few decades, fish resources in Chinese inland waters have been severely influenced by over-fishing and environmental deterioration. Furthermore, lack of scientific management system of artificial breeding has led to the decline of germplasm resources of cyprinid fishes including common carp and bighead carp. In the present study, we developed Type I markers and analyzed population genetic in the two carps, and compared various genotypes associated with growth traits. We aimed to provide theoretical and applied bases for further studies on genetic resource conservation and marker-assisted selective breeding in common carp and bighead carp. The main results of the dissertation are shown as follows: 1. A total of 18840 ESTs (Expressed Sequence Tag) of common carp were downloaded from public databases and analyzed by bioinformatics methods. In total, about 5.5% of these ESTs had SSRs (Simple Sequence Repeats) inside. After clustering and assemblying, 903 unique ESTs were identified. The abundance of di-, tri-, tetra-, and penta-nucleotide motifs among the ESTs is 37.18%, 30.81%, 20.36%, and 11.71%, respectively. The rates of polymorphic EST-SSRs were 17% in crucian carp (Carassius auratus), and 5% in silver carp (Hypophthalmichthys molitrix), respectively when cross-species amplificatons were tested. 470 pairs of EST-SSR primers were designed for these novel EST-SSRs in order to provide sufficient polymorphic markers for population genetic studies and genome mapping in common carp and its closely related fishes. 2. Population genetic diversity of the common carp (ten wild populations and one cultivated population) was investigated based on 10 polymorphic EST-SSR loci. On the whole, the genetic diversity of wild common carp population is high, and the genetic diversity in Heilongjiang population is lower than in Yangzi River populations. The Fst value (0.10) showed that common carp had moderate genetic differentiation, and the gene flow analyses indicated that the genetic exchange among populations is not difficult. There were no significant relations between genetic distances and geographical distributions of the eleven populations based on NJ (Neighbor-Joining) clustering and SOM (Self-Organized Map). These results indicate that EST-SSRs in common carp are non-neutral molecular markers, and they would be helpful to investigate the functional diversity of genes and adaptability of common carp populations from major waters in China. 3. Four polymorphic EST-SSR loci were used to explore the relationship between genotypes and growth traits of common carp. As the results, the effects of the four EST-SSR loci on one or more the growth traits (body length, weight, and relative fatness) were evident (P<0.05). These results suggest these EST-SSRs loci may be associated with the growth traits of common carp, and is necessary to be further studied. 4. Gene cloning and searching for genic markers were performed for the Leptin receptor gene (Lepr) in bighead carp. Homology comparisons showed that compared with mammals, the Lepr gene in fish is relatively conserved. One SSR and two SNP (single nucleotide polymorphism) loci were identified in bighead carp population by PCR-SSCP (single strand conformation polymorphism) analysis and DNA direct sequencing. At the SSR locus, the difference in genetic diversity between breeding populations and wild population was not significant. The associations between Lepr gene polymorphisms and growth traits were estimated with the general linear model. The result showed that there were no significant effects on the growth trait at the genic SSR locus in Lepr. At the SNP loci, the individuals with genotype Aa had a larger body height than those with genotype AA and aa (P<0.05); the individuals with genotype Bb had a larger body height and head length than genotype bb (P<0.05); as a dominant allele in the population, the individuals with allele G had a larger body length, body height, head length, body weight and relative fatness than those with allele A (P<0.05). These data showed that the genotypes of SNP loci in Lepr gene were significantly associated with some of the growth traits in bighead carp and could serve as potential candidate gene in further studies of marker-assisted breeding.
Document Type学位论文
Recommended Citation
GB/T 7714
王丹. 鲤和鳙I型遗传标记的研发和群体遗传结构分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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