【作者】 刘昕；

【导师】 杨官品；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 孔雀鱼(Guppy, Poecilia reticulata)又名百万鱼、彩虹鱼、库比鱼等,隶属于脊索动物门(SubphylumVertebrata)、辐鳍鱼纲(Class Actinopterygii)、鳉形目(Order Cyprinodontiformes)、花鳉科(Family Poeciliidae)、花鳉属(Genus Poecilia),原产于南美洲的委内瑞拉、圭亚那、巴西、巴巴多斯及特里尼达等国家和地区,是一种极具观赏价值的小型淡水鱼。孔雀鱼繁殖周期短、易饲养,且体表颜色和体型变化丰富,因此可作为遗传学研究的实验动物。孔雀鱼有23对染色体,是XY性别决定系统,细胞学和遗传学上X和Y染色体都有差别,因而为研究性别决定基因及性别相关标记提供了条件。本研究以孔雀鱼为主要研究对象,构建了SSR和AFLP标记的孔雀鱼雌、雄遗传连锁图谱,并筛选得到了一个性别特异的分子标记,为研究鱼类性别决定奠定了基础。1.孔雀鱼遗传连锁图谱的构建利用AFLP和微卫星标记,以孔雀鱼F1全同胞家系为作图群体,构建了孔雀鱼的雌性和雄性遗传连锁图谱。用120对AFLP引物组合对亲本和56个子代个体进行了分离分析。共得到667个在亲本间有多态性并在子代中1:1分离的AFLP标记,其中含385个父本分离标记和282个母本分离标记。卡方检验显示,父、母本分离标记中共有18个偏分离标记,其他649个标记可用于连锁分析。从304对微卫星引物中筛选出65对多态性引物(21.3%),得到43个父本标记和29个母本标记。另外,将性别作为表型标记一同进行连锁图谱构建。分别用父本和母本标记构建了孔雀鱼雄性和雌性遗传连锁图谱。雄性连锁图谱含25个连锁群(包括1个三联体及1个连锁对),共332个标记(31个微卫星DNA标记,301个AFLP标记),图谱总长1120.5cM,覆盖率为84.1%。标记间最大间隔21.2cM,平均间隔3.6cM,平均每个连锁群上13.3个标记。雌性连锁图谱含25个连锁群(包括1个三联体及2个连锁对),共233个标记(20个微卫星DNA标记,213个AFLP标记),图谱总长901.2cM,覆盖率为77.3%。标记间最大间隔22.9cM,平均间隔4.3cM,平均每个连锁群上9.3个标记。性别标记定位于雄性图谱第六连锁群上。2.性别特异标记的筛选利用AFLP技术对比分析孔雀鱼雌雄基因组间的差异,筛选到1个雄性特异的AFLP标记(PreF262)。对这个标记进行回收、克隆、测序,根据所得核苷酸序列设计了一对引物,仅能在雄性个体基因组DNA中扩增到到特异条带,成功的把AFLP标记转化为SCAR标记。建立了快速鉴定孔雀鱼遗传性别的PCR技术。更多还原

【Abstract】 Guppy fish (Poecilia reticulata) belongs to class Actinopterygii (ray-finned fish), order Cyprinodontiformes, family Poeciliidae and genus Poecilia. It mainly inhabits the coastal streams and rivers of Venezuela, Guyana, Surinam and several of the Lesser Antilles including Trinidad and Tobago. Guppy has become popular among aquarists and hobbyists and played an important role in the freshwater ornamental fish industry worldwide. Guppy is also a model fish for studying the genetic bases of heterosis, aging and inbreed depletion crucial for aquaculture because of its relatively short life cycle, easiness of breeding and reproduction, availability of commercial strains homologous for the traits such as body color and tail shape. In the present study, we constructed linkage maps for guppy using microsatellite DNA and AFLP markers, and isolated a sex-specific AFLP marker by screening the genomes of guppy using AFLP technique. This study provides strong support for the study of sex determination and sex differentiation in fish.1. Construction of linkage maps for guppyGenetic linkage maps were constructed for using AFLP and microsatellite markers in an F1 family. A total of 667 polymorphic loci were produced with the 120 AFLP primer pairs. Among all the polymorphic markers,282 dominantly inherited bands were from the female parent,385 were from the male, and 62 markers showed a significant distortion from the expected 1:1 ratio(P<0.05),then 649 markers were used for linkage analysis. Among the 65 informative microsatellites ,29 and 43 markers valued for the female and the male map, respectively.Separate linkage maps were constructed for male and female parents. The male map contained 25 linkage groups (including 1 triplets and 1 doublets), to which 332 markers (31microsatellites and 301AFLPs) were assigned. The map spanned a length of 1120.5cM, covering 84.1% of the estimated genome size (1333.1cM). The size of linkage groups ranged from 9.2cM to 108.7cM with an average of 44.8cM. The number of markers per group varied from 2 to 35 with an average of 13.3. The female parent map contained 25 linkage groups (including 1 triplets and 2 doublets), to which 233 markers (20microsatellites and 213AFLPs) were assigned. The map spanned a length of 901.2cM, covering 77.3% of the estimated genome size (1059.8cM). The maximum and average spaces between 420 loci were 22.9cM and 4.3cM respectively. The size of linkage groups ranged from 14.1cM to 97.8cM with an average of 36.0cM. The number of markers per group varied from 2 to 26 with an average of 9.3.2. Isolation of sex-specific AFLP markersThe differences of genomic DNA between females and males of guppy were studied by using AFLP technique. As a result ,a male-specific maker were found to be only present in all males but absent in all females. The marker was excised, cloned and sequenced. A pair of PCR primers was designed, which could amplify sex-specific bands in male genome DNA. So we converted the AFLP marker into a PCR marker, SCAR. Selection in identifying the sex of guppy with this SCAR marker will be more efficient and less expensive than with AFLP markers.更多还原