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题名: 稀有鮈鲫近交系的遗传质量检测
作者: 邵燕
答辩日期: 2007-07-10
导师: 曹文宣
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 稀有鮈鲫 ; 近交系 ; 遗传纯度 ; 外部形态 ; 骨骼 ; 同工酶 ; 鳞片移植 ; 微卫星
其他题名: Genetic quality control of an inbred strain of Gobiocypris rarus
摘要: 稀有鮈鲫(Gobiocypris rarus)是我国特有的一种小型鲤科鱼类,隶属鲤形目(Cypriniformes)鲤科(Cyprinidae),仅分布于四川省汉源县、石棉县、都江堰市、双流县、彭州市等地。从1990年开始,中国科学院水生生物研究所以培育鱼类实验动物为目的对其进行了大量的生物学研究,证实了它是培育鱼类实验动物的理想对象,并从饲养管理、品系培育等方面开展实验动物化研究。目前,通过全同胞兄妹交配已近交至第22代,按照实验动物近交系的定义,稀有鮈鲫的近交系已经建立,但需要对其进行遗传质量检测,鉴定近交系。本研究参照哺乳类实验动物的遗传质量监测的方法,从外部形态、骨骼、免疫、生化、分子五个方面对近交系的遗传纯度进行检测,以期获得近交系的遗传背景,建立品系鉴定的方法。 1. 对稀有鮈鲫的可数性状、传统可量性状及框架可量性状进行了检测,采用聚类分析、判别分析、主成分分析等多元统计分析方法,对非近交(野生和F1)和近交(F10和F20)稀有鮈鲫的外部形态变异进行了研究。结果发现,不同群体可数性状的变幅重叠,众数接近或相等,据此难以对群体进行鉴别。对可量性状多元分析发现稀有鮈鲫不同群体外部形态差异明显。判别分析的综合判别率为80%以上,F10和F20的判别准确率均高于野生种群;聚类分析表明野生种群和F1在形态上较为接近,与F10、F20在形态上却存在较大的差异;主成分分析和判别分析的结果表明,非近交和近交稀有鮈鲫在外部形态上存在的明显差异,主要是由于鱼体头部和躯干部垂直轴向的形态特征差异引起的。稀有鮈鲫群体间外部形态的差异主要是受遗传因素和环境因子共同影响。外部形态多元分析方法可以作为近交系鉴别的手段。 2. 对采自汉源县的稀有鮈鲫和HAN系F20头部骨骼变异进行研究,发现主鳃盖骨的外部形态在不同群体间具有明显差异。在判别分析中,野生种群和HAN系的判别准确率分别为100%、93.8%,表明结合判别分析利用主鳃盖骨进行稀有鮈鲫群体间的鉴别是可行的。 3. 采用鳞片移植方法评价稀有鮈鲫HAN系的遗传纯度,结果表明HAN系F22鳞片异体移植的成功率略低于同体移植的,但显著高于非近交个体鳞片异体移植的成功率,证明HAN系F22个体间具有较强的遗传一致性。采用鳞片移植的方法进行稀有鮈鲫近交系的遗传质量检测是可行的。 4. 运用聚丙烯酰胺凝胶电泳技术,对稀有鮈鲫4种组织6种同工酶和1种肌蛋白进行了电泳研究,并以同工酶最具代表性的组织对稀有鮈鲫的近交和野生个体进行分析和比较。结果表明,稀有鮈鲫的同工酶系统具有明显的组织特异性。肝脏可作为群体间乙醇脱氢酶检测的实验材料,其它同工酶检测采用肌肉组织。在所研究的6种同工酶系统中,共记录出13个基因位点,其中在野生种群中单态位点有11个,有2个位点为多态,它们是EST-2和EST-3,多态位点的比例为15.56%;在HAN系F22中则无多态性位点,不同个体的同工酶酶谱均是一致的。在肌蛋白3个区段表达中,HAN系F22表现一致,个体间无差异,而野生种群的区段1、2则明显表现出多态性。以上结果表明经过多代近亲交配,稀有鮈鲫的遗传纯合度已有了明显的提高,近交系具有较高的遗传均一性。 5. 利用17对微卫星引物对稀有鮈鲫野生种群和近交系F20和F22进行了遗传分析。结果表明在野生种群中17个微卫星位点均为多态位点,但在F20中仅有6个多态位点,F22中则仅有4个多态位点。在野生种群中共检测到64个等位基因,F20、F22分别为26、21个。HAN系的平均基因纯合率均较高,其中F20为86.18%,F22达91.96%,而野生种群平均基因纯合率为46.84%。HAN系平均杂合度和平均多态信息含量均较野生种群低。在HAN系F20和F22中,群体间遗传相似性指数最大,其遗传距离最小,说明二者之间的亲缘关系最近。因此,HAN系遗传多样性明显降低,已具有较高的遗传纯度。
英文摘要: Gobiocypris rarus is an endemic cyprinid fish in China, distributed only in paddy fields, ditches or gullies in some counties of Sichuan Province, such as Hanyuan county, Pengzhou county and so on. It belongs to Cypriniformes, Cyprinidae. This species has many attactive attributes, which make it possible to become a potential candidate of experimental model fish in future in China, or even all over the world. In order to use G. rarus as an laboratory animal, an inbred strain has been established by brother-sister mating to the 20th generation in the laboratory since 1990. According to the definition of inbred strain, it is time to examine genetic quality for the inbred strain. In the present study, genetic quality of G. rarus was studied by means of the methods, which are usually used in genetic monitoring of mammal experimental animal mouse, from the following five levels: 1. Morphological variability between non-inbred populations (3 wild populations and 1 F1) and inbred stocks (F20 and F22) of G. rarus was studied by multivariate analysis on morphometric and meristic characters determined by traditional method and truss network, including stepwise discriminant analysis, principal component analysis and cluster analysis included. The results showed tiny intraspecies variations on meristic characters were found, but they were not effective for population distinctions. On the contrary, analysis on morphometric characters revealed abundant variations among stocks, especially between wild populations and inbred stocks . The average discriminant accuracy for all populations was over 80%, whereas the discriminant accuracy for inbred strains was much higher. In the cluster analysis, fully and excellent classification could be obtained between the two inbred stocks F10, F20 and the other populations. In the principal component analysis and stepwise discriminant analysis, the characters related to body depth of fish played important role in the discrimination of populations. It was argued that morphological differences among stocks were resulted from both genetic differences and environmental factors. Multivariate analysis on morphometric characters could be used to distinguish inbred strains of G. rarus. 2. Based on the observation of head skeleton of wild population and inbred stock F20 of G. rarus, there was obvious shape difference on operculum between different populations. In the discriminant analysis, the discrimintant accuracy of wild population and F20 was 100% and 93.8%, respectively. All the results indicated that it is feasible to differentiate populations of G. rarus by means of performing discriminate analysis on fish operculum. 3. Genetic purity of inbred stock of G. rarus was evaluted by scale transplantation. The results showed that the survival rate of transplanted scales in inbred stock was lower than that of in scale autotransplantation of either inbred stock or wild population, but it was much higher than that of allotransplanted scales in wild population, indicating that genetic uniformity existed among inbred stock. So it is feasible to perform genetic quality control by means of scale transplantation. 4. By the polyacrylamide gel electrophoresis, six isozymes and one muscle protein from four different tissues of G. rarus were studied, and also were analyzed in both wild population and inbred strain F22. The results showed that all isozymes presented tissue specificity. Liver can be used as the suitable material to do ADH isozyme analysis among populations, and muscle used for the others. Six isozymes presumably were encoded by 13 gene loci, 11 of which were found monomorphic in wild population. And only 2 loci with two alleles are polymorphic, being EST-2, EST-3. Mean proportion of polymorphic loci was 15.56%. However, no polymorphic loci was found in F22. In addition, no difference between individuals was found in inbred stock with regard to three belts of muscle protein, whereas belt I and II presented polymorphic in wild population. That is to say, after successive mating cross of brother and sisiter, inbred stock of G. rarus has reached a high degree of genetic purity. 5. The polymorphism of 17 microsatellites in wild population and inbred strain F20 and F22 of G. rarus was analyzed. All primers showed polymorphic in wild populations, yet only six of these primers were polymorphic in F20, and four pairs of primers showed polymorphic in F22. There were totally 64 alleles of these microsatellite loci in wild population, while 26 and 21 alleles were found in F20 and F22, repectively. The mean rates of homozygote in inbred stocks was 91.96%,86.18% and 46.84% in inbred stock F22, F20 and wild population, respectively. In contrast to wild population, high homozygosity and low heterzygosity were found in inbred stocks. Among all populations, the genetic distance was the closest and genetic similarity indices was the biggest between inbred stocks F20 and F22, showing they the nearest in relationship. So it was argued that genetic diversity of inbred stock was much lower than wild populations, and high degree of inbreeding existed in inbred stocks.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12088
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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稀有鮈鲫近交系的遗传质量检测.邵燕[d].中国科学院水生生物研究所,2007.20-25
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