【作者】 赵兰；

【导师】 孙效文；

【作者基本信息】 大连海洋大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 鲤(Cyprinus carpio)在世界水产养殖上是一种重要的养殖品种，年产量已达340万吨，占所有淡水水产品的比例高达14%；鲤在我国有着悠久的养殖历史，并且还具有一定的文化意义；除此之外，鲤还可以作为观赏鱼。近年来，国内外研究者在鲤的遗传学和基因组学方面开展了广泛的研究，比如开发大量分子标记、构建低、中等密度的遗传连锁图谱、重要经济数量性状位点的定位、构建大片段基因组文库、末端测序以及构建物理图谱等。但是鲤较高密度的遗传连锁图谱、遗传—物理整合图谱构建工作还没有完成。本论文的目的就是要建立鲤较高密度的遗传连锁图谱以及遗传—物理整合图谱；另外，还通过比较基因组学手段对鲤与斑马鱼进行了比较作图，最终将鲤较高密度的遗传连锁图谱、鲤物理图谱和斑马鱼的全基因组图，三者有机的结合在了一起。通过本研究，以期丰富鲤的基因组学和比较基因组学方面的研究并且用于验证鲤全基因组测序和组装结果。具体研究结果如下：1、从鲤物理图谱中选择较大的230个重叠克隆群(contig)，从每个contig的BAC末端序列里分别查找微卫星并设计微卫星引物2-3对。一共设计了629对BAC锚定(BAC-anchored)微卫星引物，其中550对引物有扩增产物；226对有效扩增的引物是多态的，并且在作图群体中进行了基因分型。2、通过将黑龙江水产研究所随机开发的664个微卫星和5000个SNP侧翼序列与鲤物理图谱上BAC末端序列数据库的比对，发现有40个微卫星以及137个SNP是属于BAC关联(BAC-associated)标记。3、一共使用了1359个标记分型结果进行遗传连锁图的连锁分析(包括新开发的226个BAC-anchored微卫星分型结果、原先开发的332个BAC-anchored微卫星分型结果、黑龙江水产研究所开发的664个微卫星分型结果和137个BAC-associated SNP分型结果)，JoinMap4.0软件连锁分析后，得到的鲤遗传连锁图谱上一共有1209个标记分布于50个连锁群上。遗传连锁图谱长度为3565.9cM，每个连锁群长度范围是17.3-126.7cM，标记间平均间隔为3.077cM。4、所有的BAC-anchored及BAC-associated的标记共计有620个作为锚定点，将遗传连锁图谱与物理图谱联系在一起，达到了整合的目的。遗传—物理整合图谱上一共有463个物理图谱的contig (其中新开发微卫星标记整合了166个最大的contig)和88个物理图谱的单个克隆，共计498.75Mb的物理长度(其中新开发的微卫星标记共整合了258.28Mb)，约占鲤全基因组大小的30%。5、通过Blastn比对发现，鲤遗传连锁图谱上的1209个标记中有597个标记可以定位到斑马鱼基因组(ZV9)上，并且鲤相应的两个连锁群上几乎所有的同源标记都位于斑马鱼相对应的一条染色体上，这很好的证明了鲤染色体与斑马鱼染色体2:1的比例，即鲤比斑马鱼多经历了一轮全基因组复制现象。6、通过Blastn比对发现，整合的463个物理图谱contig中有442个contig可以定位到斑马鱼相应的染色体上，这442个contig一共包含有13449个BAC末端序列，即鲤中一共有13449个BAC末端序列可以定位到斑马鱼染色体上。更多还原

【Abstract】 Common carp (Cyprinus carpio) is one of the most important aquaculture species with an annualglobal production of3.4million metric tons that accounts for nearly14%of all freshwateraquaculture production in the world. In China, common carp has quite a long history ofcultivation and certain cultural meanings. Besides, common carp becomes an importantornamental species. Nowadays, researchers from home and abroad have carried out extensivestudies on common carp, such as the development of new molecular markers, construction ofseveral low or medium density genetic linkage maps, location of important economicquantitative trait locis, construction of a bacterial artificial chromosome library, BAC endsequences and a physical map. However, higher density genetic linkage map andgenetic-physical integrated map are not available for common carp. The objective of this studywas to construct a higher density genetic linkage map and genetic-physical integrated map forcommon carp. In addition, through comparative mapping between common carp and zebrafishgenomes, the three maps (common carp genetic linkage map, common carp physical map andzebrafish whole genome map) are finally successfully connected to each other. The study willhelp us enrich the field in genomics and comparative analysis of common carp and verify thecommon carp whole genome sequencing and assembly. The results as follows:1. A total of629BAC-anchored microsatellite loci from the common carp230largest physicalmap contigs were developed. Of these contig-anchored microsatellites,550markers had PCRproduct and a total of226showed the presence of polymorphism and genotyped them in themapping family.2. The flank sequences of664microsatellite and5000SNP markers randomly developed byHeilongjiang Fisheries Research Institute were aligned to the BES database and a total of40microsatellite and137SNP markers were mapped to physical map contigs or single BAC clones.3. A total of1359polymorphic genetic markers, including newly developed226microsatellitemarkers, previously developed332microsatellite markers,664microsatellite markers and137SNP markers developed by Heilongjiang Fisheries Research Institute were used for linkageanalysis. After using software JoinMap4.0, a total of1209genetic markers were finally mappedto50linkage groups. Spanning3565.9cM of the common carp genome and the genetic length ofeach linkage group ranged from17.3cM to126.7cM, and the average interval between markersis3.077cM.4. A total of620BAC-anchored or BAC-associated markers serve as anchor points that connect genetic linkage map and physical map together. The genetic-physical integrated map composedof463physical map contigs and88single BACs, of which166contigs are newly developed.Combined lengths of the contigs and single BACs covered a physical length of498.75Mb(among them258.28Mb are from newly developed markers), or around30%of the commoncarp genome.5. The flank sequences of1209markers from common carp genetic linkage map were aligned tothe zebrafish reference genome (zv9) using BLASTn and successfully mapped597markers on25chromosomes of zebrafish. The results showed that two linkage groups of common carp werehomologs with one particular chromosome of zebrafish, which confirmed the two-to-onehomologous relationship of common carp and zebrafish chromosomes, which means commoncarp have experienced an additional round of whole genome duplication that doubled thechromosome number.6. A total of442physical map contigs (from the463integrated contigs) that formed synteniesbetween the common carp and zebrafish genomes, of which13449BAC end sequences hadsignificant hits to zebrafish chromosomes.更多还原