【作者】 姜黎明；

【导师】 张全启；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2013， 博士

【摘要】 半滑舌觸<iCynoglossus semilaevis、是我国黄激海区珍稀、名贵的大型养殖鱼类，其野生资源日益减少，渔业资源形势严峻。目前该物种工厂化养殖主要采取捕获野生亲鱼后驯化的方式获得苗种，十分不利于其养殖业的长期发展。育种工作中急需解决如何将人工繁殖子代培育成可繁殖亲本的问题，解决这一问题需要将传统的选择育种与分子辅助育种的工作相结合。本研宄针对这一现状开展了三个方面的工作：半滑舌鳎微卫星分子标记的开发；遗传连锁图谱的构建；染色体定位的初步研宄。为半滑舌鳎分子标记辅助育种工作的开展奠定了基础。1.半滑舌鳎微卫星分子标记的开发本部分利用三种方法针对半滑舌鳎开发微卫星分子标记：富集文库+菌落原位杂交法、cDNA文库序列筛选法、Fosmid文库序列筛选法。通过富集文库+菌落原位杂交法，筛选了1718个阳性克隆，经过进一步的筛选送测了1192个克隆，在得到的序列中共设计了784对引物，在6尾野生半滑舌鳎个体中检测标记多态性，共得到474对多态性引物；通过cDNA文库序列筛选法，在908条本实验室构建的cDNA文库序列中设计了71对引物，得到12对多态性引物；通过Fosmid文库序列筛选法，在768条Fosmid文库双末端测序序列中设计了117对引物，得到74对多态性引物。对这三种方法筛选微卫星标记的效率的比较中发现：富集文库+菌落原位杂交法的标记获得率（65.8%)远高于其他两种方法（8.5%和15.2%)； Fosmid文库序列筛选法的引物多态率（63.2%)稍高于富集文库+菌落原位杂交法（60.5%)，远高于cDNA文库序列筛选法（15.6%)。2.半滑舌鳎遗传连锁图谱的构建本部分利用本研宄开发的半滑舌鳎多态性微卫星标记及少量本实验室发表的微卫星标记，以一个全同胞n代家系为作图群体，构建了半滑舌鳎首个完全由微卫星标记构成的性别特异遗传连锁图谱。雌性图谱由21个连锁群组成，总长1041cM，共有193个标记，定位了174个位点，21个连锁群中长度最短的2cM，最长的88cM，包含四个二联体，平均间隔最小的2.0cM，最大的17.0cM，图谱平均间隔6.8cM，平均每个连锁群含有8.3个标记，预期长度1356.9cM，推算其图谱的覆盖率为76.7%。雄性图谱也由21个连锁群组成，总长1154cM，共有195个标记，定位了181个位点，21个连锁群中长度最短的6cM，最长的102cM，包含三个三联体和两个二联体，平均间隔最小的3.5cM，最大的16.5cM，图谱平均间隔7.2cM，平均每个连锁群含有8.6个标记，预期长度1477.3cM，推算其图谱的覆盖率为78.1%。雌雄重组率的比例大约为1:1.02。3.半滑舌觸染色体定位的初步研宄本部分利用FISH技术定位了3个含有微卫星标记的Fosmid克隆；对克隆B106-62通过Fosmid文库步移筛选得到2个克隆，并对这两个克隆进行了FISH定位，验证了利用Fosmid-FISH技术整合微卫星遗传连锁图谱与染色体定位的可能性，同时找到了3个能够同时定位于W染色体和一对常染色体的Fosmid克隆。设计PCR探针定位了半滑舌鳎的18S rDNA和5S rDNA，结果发现18S rDNA的定位结果在半滑舌鳎雌鱼和雄鱼中略有不同：雌鱼中存在7个杂交信号，其中6个信号定位于三对常染色体着丝粒区域，另外1个信号定位于W染色体臂末端；雄鱼中存在6个杂交信号，定位于三对常染色体着丝粒区域，与雌鱼中常染色体上定位形式类似。5S rDNA的定位结果在半滑舌鳎雌鱼和雄鱼中相同：共有12个信号定位于6对常染色体的着丝粒区域，W染色体上没有发现信号。对18SrDNA和5S rDNA进行双色杂交时发现：两对常染色体上相近的位置存在共线性的18S rDNA和5S rDNA信号，其余的杂交信号都位于不同的常染色体对上，W染色体上有一处18S rDNA信号。通过对克隆B106-62及其步移得到的另外两个Fosmid克隆进行FISH定位发现：W染色体上存在至少两个较大的重复片段富集区域。利用上述杂交结果推测半滑舌鳎原始性染色体在进化过程中可能发生了染色体融合、重复片段积累及非同源染色体易位，经过一个渐进的过渡过程形成了现在W染色体的形态。更多还原

【Abstract】 Half smooth tongue sole {Cynoglossus semilaevis) is a kind of large farmed ifsh inYellow Sea and Bohai Sea. The wild resources of this species are dwindling and thesituation of ifshery resources is grim. The present factory farming fries are mainlygenerated from captured and domesticated wild broodstock，which is very detrimentalto the long-term development of the aquaculture industry. Three parts of work aroundthis situation were carried out in this study: development of microsatellite molecularmarkers; construction of genetic linkage map; preliminary study of chromosome map.Our results laid the foundation for molecular marker-assisted breeding work in halfsmooth tongue sole.1. Development of microsatellite molecular markersWe developed microsatellite molecular markers of half smooth tongue sole in threeways: enriched library+colony in situ hybridization; screening in cDNA librarysequences; screening in Fosmid library sequences. Using the way of enriched library+colony in situ hybridization, we screened1718positive clones and sequenced1192clones. A total of784pairs of primers were designed，474of which were polymorphicdetected in six wild individuals. Using the way of screening in cDNA librarysequences,71pairs of primers were designed in908cDNA library sequences,12ofwhich were polymorphic. Using the way of screeing in Fosmid library sequences,117pairs of primers were designed in768Fosmid library sequences,74of which werepolymorphic. By comparing the eiffciency of the three methods, we found that markerobtaining rate by way of enriched library+colony in situ hybridization (65.8%) washigher than that of the other ways (8.5%and15.2); polymorphic primers rate by screening in Fosmid library sequences (63.2%) was slightly higher than that by theway of enriched library+colony in situ hybridization (60.5%) and was much higherthan that of screening in cDNA library sequences (15.6%).2.Construction of genetic linkage mapWe constructed the ifrst microsatellite genetic linkage map of half smooth tonguesole with polymorphic markers developed in this research and published by ourlaboratory. Mapping population employed a full-sib F1family. The female map wascomposed of21linkage groups with the total length of1041cM. There were193markers in this map and174loci were located. Among the21linkage groups, theshortest was2cM and the longest was88cM. The minimum average interval was2.0cM and the maximum was17.0cM，with an average resolution of6.8cM. There were8.3markers per linkage group contained, and the expected length was1356.9cM. Thecoverage rate was reckoned to be approximately76.7%. The male map was alsocomposed by21linkage groups with the total length of1154cM. There were195markers in this map and181loci were located. Among the21linkage groups, theshortest was6cM and the longest was102cM. The minimum average interval was3.5cM and the maximum was16.5cM，with an average resolution of7.2cM. Therewere8.6markers per linkage group contained. The expected length was1477.3cMand the coverage rate was approximately78.1%. The recombination ratio offemale/male was about1:1.02estimated in the sex-speciifc frame map.3.Preliminary study of chromosome mapWe located3Fosmid clones containing microsatellite markers and2Fosmid clonesfrom library walking by Fluorescence in situ hybridization (FISH). We veriifed thepossibility of integrating the microsatellite genetic linkage map and the chromosomemap using Fosmid-FISH technology. Three Fosmid clones were found, which couldlocated in W chromosome and a pair of autosomes simultaneously. The chromosomallocalization of both18S rDNA and5S rDNA were studied through designing PCR probes. The results showed the positioning pattern of18S rDNA was slightly differentin females and males. Seven signals of the major rDNA (18S rDNA) were mapped tothe telomeric region of the long arm on W chromosome and the centromeric regionsin three pairs of autosomes in females. While there were only six signals mapping tothe centromeric regions of three pairs of autosomes in males. As to the minor rDNA(5S rDNA), twelve signals were found mapped to the centromeric regions of six pairsof autosomes in females and males. Double FISH further certiifed that two pairs ofthe18S rDNA signals and two pairs of the5S rDNA signals were correspondinglylocated at the same positions in the same autosomes. Only18S DNA signal could beidentiifed on the W chromosome in female genome. Signals of18S rDNA were alsolocated on another pair of autosomes, while those of5S rDNA were observed on other4pairs of autosomes. It was found that there were at least two repeat fragment richregions on W chromosome through locating clone B106-62and the other two Fosmidlibrary walking clones. We speculated that original sex chromosomes of half smoothtongue sole might undergo chromosome fusion, repeat fragment accumulation andnon-homologous chromosome translocation during evolutionary process, and theformation of W chromosome was a gradual transition process.更多还原