【作者】 王解香；

【导师】 白俊杰；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 草鱼(Ctenopharyngodon idella),隶属于鲤形目(Cypriniformes),鲤科(Cyprinidae),雅罗鱼亚科(Leuciscus),草鱼属(Ctenopharyngodon),具有生长快、养殖成本低等优点,为池塘、湖泊和水库的主要养殖对象,是我国淡水渔业中最重要的养殖品种之一。草鱼分布在亚洲的各大江河水系,在中国除新疆和青藏高原无自然分布外,在其它主要的河流、湖泊都有分布,形成了草鱼丰富的种质资源。近年来,由于酷捕和水环境的污染使得自然界草鱼种群数量明显下降。因此,为有效利用这一淡水鱼类资源,需对不同水系的草鱼群体的遗传背景进行分析,为草鱼选育奠定基础。另一方面,由于草鱼性成熟周期长,亲鱼个体大等原因,给良种选育带来困难,目前草鱼尚未获得遗传改良的品系。利用DNA遗传标记进行辅助选育已经成为动物育种过程中的重要方法。筛选和利用与草鱼经济性状相关的分子标记,可有效提高选择效率,缩短育种年限,对加快培育具有优良经济性状的草鱼养殖品系,具有重要的意义。本研究拟从草鱼的EST数据库中筛选微卫星分子标记,利用微卫星标记对长江水系群体(石首、监利、长沙)和珠江水系草鱼群体(清远、肇庆)的遗传结构进行分析。同时,筛选与草鱼生长性状相关联的微卫星分子标记,具体研究内容如下:1、草鱼EST-SSR标记的开发。以草鱼脑、肌肉、肝等组织构建cDNA文库,经测序获得ESTs序列760000个,再用trf(tandem repeats finder)软件从中筛选微卫星序列共5556个,据此设计EST-SSR引物118对,其中19对EST-SSR引物能够扩增出带型清晰且多态性较高的谱带。2、草鱼5个不同地域群体的遗传结构分析。利用已开发的19个EST-SSR标记研究三个长江水系群体(石首CS、监利JL、长沙SS)和两个珠江水系群体(清远QY、肇庆ZQ)草鱼的遗传结构,共获得93个等位基因,每对引物检测到的等位基因数为2~8个,平均为4.89个;群体遗传多样性分析表明:5个群体的平均多态信息含量(PIC)范围为0.4154~0.4604,显示草鱼群体的遗传多样性偏低;平均观测杂合度(Ho)范围为0.4158~0.5013,平均期望杂合度(He)范围为0.4506~0.5028,其中,长沙群体平均期望杂合度最高0.5028,监利群体的平均观察杂合度和平均期望杂合度均最低,分别为0.4158和0.4506,石首群体、肇庆和清远群体居中,即长沙群体的遗传多样性最高,监利群体的遗传多样性最低;对数据进行F-检验,结果表明群体间的遗传分化程度低。Hardy-Weinberg平衡的卡方检验结果表明5个群体均一定程度上偏离了平衡;聚类分析显示长沙群体(CS)、石首群体(SS)与监利群体(JL)聚成一支;肇庆群体(ZQ)与清远群体(QY)聚成一支,这与草鱼群体的流域分布相一致。3、草鱼生长性状相关联的EST-SSR标记筛选。利用草鱼EST(expressed sequence tags)数据库开发的18条EST-SSR标记对草鱼养殖群体进行基因型与生长性状关联分析和群体的遗传多样性分析,结果表明:关联分析得到6个微卫星位点(13118、13305、24017、35939和40698)与体重,体长和体高显著或极显著相关(P<0.05或P<0.01)。对差异显著的位点进行不同基因型间与生长性状的多重比较,获得了与体重,体长和体高等生长性状相关的有利基因型,他们是13118位点的BB、13305位点的AD、24017位点的AC、25085位点的BE、35939位点的BB和40698位点的BB。将上述6个微卫星位点上的EST序列与GenBank数据库进行BLAST比对,其中有2条EST序列与已知功能的基因序列高度同源,24017序列与鲤鱼自然杀伤细胞增强因子(NCEF)同源性水平高达86%,25085序列与草鱼反应元件结合蛋白(CREB)的基因同源性水平达到80%。应用这18个微卫星位点对草鱼随机群体进行遗传多样性分析,共检测到82个等位基因,平均等位基因4.556个,每个位点检测到的等位基因数为2～9个,群体的平均观测杂合度为0.4529,平均期望杂合度和平均多态信息含量分别为0.4571和0.4017,表明该群体遗传多样性处于低水平。更多还原

【Abstract】 Grass carp(Ctenopharyngodon idella) is one of most important cultured freshwater fishes which belongs to Cypriniformes, Cyprinida, Leuciscus, Grass carp. Because of fast growth and low cost farming, it has been the main breeding species in the ponds, lakes and reservoirs. It distributes in major river systems and lakes in Asia except for Xinjiang and Qinghai Tibet Plateau without the natural distribution, forming rich germplasm resources. In recent years, the unclear genetic background of broodstock and inbreeding led to germplasm degradation; executive arrest and pollution of the natural environment makes natural grass carp populations decreased significantly. Therefore, in order to protect and use the freshwater fish resources, the genome of different groups in grass carp should be researched. We need to develop and use a variety of molecular genetic markers to find out their genetic polymorphism and relationship and study their genetic background for the basis of grass carp breeding population. On the other hand, due to long period of sexual maturity of grass carp, big broodstock individuals and other reasons, so it is difficult to breed superior strains. The current has not yet acquired the genetic improvement of grass carp strains. Marker assistant selection(MAS) has become an important method to improve breed. Selection and use of grass carp economic traits associated molecular markers can effectively improve the selection efficiency and shorten the breeding period. It is important significantly to accelerate the development of superior economic traits of grass carp breeding strains. In this paper, microsatellite markers were selected from the EST database of grass carp. They were employed to detect the genetic diversity of three groups of grass carp populations from the Yangtze River System(SS, JL, CS) and two groups from the Pearl River System(QY, ZQ). Meanwhile, we identified microsatellite loci associated with growth traits in grass carp. The detailed content was as follows:1. Development of EST derived Microsatellites in grass carp. 5556 microsatellite sequences were found from ESTs database (760000 items) which were constructed from brain, muscle,liver tissues of grass carp. According to these microsatellite sequences, 118 pairs of primers were designed with Primer Premier 5.0. Nineteen pairs of primers can amplified clear and highly polymorphic products by method of PCR.2. Development of EST derived Microsatellites and Analysis of Genetic Diversity in Five Populations of Grass Carp. Nineteen pairs of EST SSRs primers were employed to detect the genetic diversity of three groups of grass carp populations from the Yangtze River System(SS, JL, CS) and two groups from the Pearl River System(QY, ZQ) . The result displayed that 93 polymorphic loci were amplified and each primer were detected 2 to 8 alleles or 4.89 alleles by average. Genetic diversity data showed that: the average polymorphism information content(PIC) ranged from 0.4154 to 0.4604 displayed that grass carp population had lower genetic diversity; the average observed heterozygosity was from 0.4158 to 0.5013, the average expected heterozygosity was 0.4506 and 0.5028 , CS had the highest mean expected heterozygosity(0.5028). JL had the lowest mean expected heterozygosity(0.4506) and mean observed heterozygosity(0.4158). SS, QY and ZQ had the middle mean observed heterozygosity and mean expected heterozygosity. These observation indicated that CS has the highest genetic diversity, whereas JL the lowest; Fst value indicated that the populations were lowly differentiated. Chi square test was used to analyze the genotypes based on Hardy Weinberg equilibrium, the P value denoted that the five populations deviated equilibrium partially. The dendrogram based on genetic distance showed two major clusters according with basin distribution of grass carp populations: the stock from CS , SS and JL were in one cluster, which were sampled from the Yangtze River. The other cluster consists of ZQ and QY from sampling localities distributed in the Pearl River.3. Screening of EST SSRs markers related to growth traits in grass carp. Eighteen pairs of EST SSRs primers selected from EST base of grass carp were employed to examine the associations between their genotypes and growth traints and detect the genetic diversity of random Grass carp populations. Microsatellite loci of 13118, 13305, 24017, 25085, 35939 and 40698 were significantly associated with body weight, body length., and body height (P<0.05 or P <0.01). The most favorable genotypes for growth traits were AA at 13118, AD at 13305, AC at 24017, BE at 25085, BB at 35939 and BB at 40698. In addition, a total of alleles for the loci were detected (2~9 alleles for each locus). The average effective number of alleles(Ne), observed heterozygosity(Ho), expected heterozygosity(He) and mean polymorphic information content(PIC) was 4.556, 0.4529, 0.4571 and 0.4017, respectively, both of which indicated that the population genetic diversity was low. The homology identity of the 6 correlative ESTs was determined by GenBank of NCBI blast programmer on the amino acid levels. It was found, through BLAST analysis, that 2 ESTs with significant similarity to the known functional sequences in GenBank. 24017 was highly homologous to the known natural killer enhancing factor(NKEF) with an identity of 86%. 25085 was highly homologous to the known cAMP response element binding protein with an identity of 80%. The other 4 ESTs were not significantly homologous to any of the known functional genes from GenBank.更多还原