【作者】 马洪雨；

【导师】 陈松林；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 圆斑星鲽、条斑星鲽及半滑舌鳎是我国北方非常重要的三种海水养殖比目鱼类。其中，圆斑星鲽主要分布于我国黄渤海海域及朝鲜半岛周围，条斑星鲽主要分布于日本北海道太平洋沿岸，我国黄渤海海域有少量分布，而半滑舌鳎为我国的本地种，主要分布于我国北方沿海。目前，这三种鱼类的野生资源量非常稀少，渔业资源已处于非常严峻的形势。在养殖业中，它们与大菱鲆、牙鲆一起成为我国北方海水鱼类养殖业的主导。本研究从分子育种的实际需要出发，主要探讨以下三个关键问题：一是筛选和克隆性别特异分子标记，建立遗传性别鉴定方法；二是开发多态的微卫星标记；三是构建遗传连锁图谱。主要研究结果如下：1．性别特异标记筛选与克隆：（1）利用AFLP技术对比分析圆斑星鲽雌雄基因组间的差异，首次筛选到3个雌性特异的AFLP标记（VevaF160、VevaF218和VevaF533），它们只在雌性圆斑星鲽中出现，而在雄性圆斑星鲽中不出现。对这3个标记进行克隆、测序，结果表明，它们之间无同源性，是圆斑星鲽的新序列。进一步建立了鉴定圆斑星鲽遗传性别的分子技术，可帮助缩短育种周期，提高育种进程。此外，探讨了标记VevaF218在一个杂交家系（圆斑星鲽（?）×条斑星鲽♀）中的遗传分离规律，结果表明该标记的遗传方式是由母亲遗传给女儿，该结果对深入理解圆斑星鲽的性别决定机制有一定的启发。（2）根据前人的研究结果，克隆了半滑舌鳎4个雌性特异的AFLP标记（CseF783、CseF464、CseF305和CseF136），并成功将其转化为SCAR（sequence characterizedamplified regions）标记，建立了快速鉴定半滑舌鳎遗传性别的PCR技术。此外，将该技术应用于候选伪雄鱼（遗传性别为雌性而生理性别为雄性）、伪雄鱼与正常雌鱼产生的后代及正常亲鱼的后代的性别鉴定，得到了很好的效果。（3）利用AFLP技术和SSR技术对比分析条斑星鲽雌雄基因组DNA差异，发现了若干个候选的性别相关标记，但未筛选到性别特异的标记。2．多态微卫星标记开发：（1）采用FIASCO（fast isolation by AFLP of sequences containing repeats）方法构建了圆斑星鲽3个基因组富集文库（分别为GT、GA和GATA重复），通过克隆、测序获得了229条序列，其中191条序列含有微卫星重复序列，成功设计了141对微卫星引物，最终获得40个多态的微卫星位点。为圆斑星鲽的种质资源评估、遗传连锁图谱构建等研究提供了候选标记。（2）采用构建基因组富集文库和查找公共数据库中基因序列相结合的方法开发条斑星鲽的多态微卫星标记。构建了2个基因组富集文库（分别为GT和GATA重复），通过克隆、测序获得了200条序列，成功设计了82对微卫星引物，最终获得28个多态的微卫星位点。在GenBank数据库中查找到条斑星鲽5个基因含有微卫星重复序列，共设计7对微卫星引物，最终首次获得3个多态的Ⅰ型微卫星位点。为条斑星鲽的种质资源评估、遗传连锁图谱构建等研究提供了候选标记。（3）通过查找公共数据库中基因序列的方法开发半滑舌鳎的Ⅰ型多态微卫星标记。发现有19个基因含有微卫星重复序列，大多位于内含子和非翻译区。根据基因序列设计了19对引物。群体检测表明，有7个微卫星位点具有多态性。首次报道了半滑舌鳎与已知功能基因连锁的Ⅰ型微卫星标记。为条斑星鲽的种质资源评估、遗传连锁图谱构建及分子标记辅助育种等研究提供了候选标记。3．遗传连锁图谱构建：（1）首次利用AFLP标记构建了半滑舌鳎遗传连锁图谱。整个连锁图谱共定位205个AFLP标记，分布于27个连锁群中。LOD值≥3．5。每个连锁群的标记数目从2～28个不等，平均为7．6个。连锁群长度在4．4cM～87．2cM之间，相邻标记间最大间隔为27．2cM，平均间隔（s）为5．05cM。图谱总长度(G_oa)为899．4 cM，预期长度（G_e）（基因组的预期大小）为1229．3cM，而整个图谱的基因组覆盖率(C_oa)为73．2％。该图谱为第一张基于AFLP标记的半滑舌鳎遗传连锁图谱，为QTL定位、分子标记辅助育种提供了平台且为进一步构建中、高密度的遗传图谱奠定了基础。（2）利用SSR标记和AFLP标记初步构建了条斑星鲽（♀）和圆斑星鲽（（?））的遗传连锁图谱。条斑星鲽（♀）遗传图谱包含98个标记（40个SSR标记和58个AFLP标记），分布于27个连锁群中。各连锁群的标记数在2～7个之间，平均3．6个，长度在2．6cM～48．2cM之间，相邻标记间最大间隔为26．2cM，平均间隔（s）为8．9cM。圆斑星鲽（（?））遗传图谱包含86个标记（38个SSR标记和48个AFLP标记），分布于24个连锁群中。各连锁群的标记数在2～7个之间，平均3．6个；长度在1．3cM～52．5 cM之间，相邻标记间最大间隔为30．8cM，平均间隔（s）为10cM。两张图谱中共有的微卫星标记为5个。该图谱的密度还比较低，不能满足QTL定位的要求，今后必须继续定位更多的微卫星标记，以提高图谱的密度，为这两种鱼类的遗传育种工作提供平台。本文首次构建了条斑星鲽和圆斑星鲽的遗传连锁图谱。更多还原

【Abstract】 Spotted halibut (Verasper variegatus), barfin flounder (Verasper moseri) and half-smoothtongue sole (Cynoglossus semilaevis) are the three important cultured marine fish species inthe northern China. Spotted halibut mainly distributed in the Bohai Sea, the Yellow Sea, aswell as along Korean Peninsula, barfin flounder mainly distributed in cold sea basins aroundEast Hokkaido, Japan, as well as the Bohai Sea and the Yellow Sea, China, while half-smoothtongue sole which is the native fish species of China, mainly distributed in the north Chinacoast. At present the number of wild individuals of the three fish species is little, and thefisheries resource is rather critical. In aquaculture, they have been the most popular fishspecies together with turbot (Scophthalmus maximus) and olive flounder (Paralichthysolivaceus) in the northern China. In order to benefit the molecular breeding of fish species,the three items were studied in this paper including (1) Isolation, cloning sex-specific DNAmarkers and development genetic sex identification technique; (2) Isolation of polymorphicmicrosatellite markers (ⅠandⅡtypes); (3) Construction of genetic linkage maps. The mainlyresearch results are as follows:1. Isolation and cloning of sex-specific markers:(1) The differences of genomic DNA between females and males of spotted halibut (Veraspervariegatus) were studied by using AFLP technique. As a result, three female-specific AFLPmarkers (VevaF160, VevaF218 and VevaF533) were found to be only present in all femalesbut absent in any males. The three markers were all cloned and sequenced, and analysisindicates that they are different and novel markers for spotted halibut. Furthermore, thegenetic sex identification technique was developed which could be useful to shorten the breeding cycle, and improve the breeding process. The inheritance patterns of thefemale-specific marker VevaF218 in an interspecific hybrid family was examined, and theresult showed a female-specific inheritance patterns from mother to daughter. It may behelpful for better understanding the sex determination system in spotted halibut (Veraspervariegatus).(2) Based on the previous study, four female-specific AFLP markers (CseF783, CseF464,CseF305 and CseF136) were cloned in half-smooth tongue sole (Cynoglossus semilaevis), andsuccessfully converted into SCAR (sequence characterized amplified regions) markers.Subsequently, the simple and fast technique for genetic sex identification was developed. Atlast, this technique was applied in the genetic sex identification of three different derivedindividuals including the candidate neo-males (the genetic sex is female but the phenotypicsex is male), the progeny from the cross of the neo-male with the common female, theprogeny from the cross of common male with female.(3) The differences of genomic DNA between females and males of barfin flounder (Veraspermoseri) were studied by using AFLP and SSR markers. As a result, several candidatesex-linked AFLP markers were found but none of them were sex-specific.2. Isolation and characterization of polymorphic microsatellite markers(1) Three repeat-echriched genomic libraries (the repeat units were GT, GA and GATA,respectively) were constructed by FIASCO (fast isolation by AFLP of sequences containingrepeats) in spotted halibut (Verasper variegatus). 229 sequences were obtained, of which191 contained microsatellite repeats. 141 pairs of primers were designed successfully, and 40microsatellite loci were proved to be polymorphic. These markers will be benefit for theevaluation of genetic resource and the construction of genetic linkage map in spotted halibut.(2) A total of 31 polymorphic microsatellite loci were reported, of which, 28 loci wereisolated from two repeat-echriched genomic libraries (the repeat units were GT and GATA,respectively), and threeⅠtype loci were isolated from the function known genes. This studycould provide the candidate markers for the evaluation of genetic resource and theconstruction of genetic linkage map in barfin flounder.(3) Functional genes of half-smooth tongue sole (Cynoglossus semilaevis) were screened fromGenBank database. 19 microsatellites were identified and successfully designed 19 pairs of primers. After tested on 30 individuals, seven microsatellite loci showed polymorphic. It is thefirst time to reportⅠtype micrsatellites linked with functional genes in half-smooth tonguesole (Cynoglossus semilaevis), and will be benefit for evaluation of genetic resource,construction of genetic linkage map and molecular marker-assisted breeding (MAS) inhalf-smooth tongue sole (Cynoglossus semilaevis).3. The construction of genetic linkage maps(1) A first consensus AFLP-based genetic linkage map of half-smooth tongue sole(Cynoglossus semilaevis) was constructed. A total of 205 AFLP markders were mapped in 27linkage groups. The linkage between the loci was identified by an LOD score of≥3.5. Thenumber of markers per group ranged from 2 to 28, with an average of 7.6. The length ofgroups ranged from 4.4 cM to 87.2 cM (Kosambi), with an average distance between markersof 5.05cM. The observed consensus map length and the estimated consensus map length were899.4 cM and 1299.3 cM, respectively, with the coverage of 73.2%. This linkage map may behelpful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding)and construction of middle or high density linkage map of half-smooth tongue sole(Cynoglossus semilaevis).(2) Preliminary genetic linkage maps of barfin flounder (Verasper moseri) (♀) and spottedhalibut (Verasper variegatus) ((?)) were constructed using microsatellite and AFLP markers.The barfin flounder map consisted of 98 markers (40 SSR markers and 58 AFLP markers)grouped in 27 linkage groups, with an average resolution of 8.9 cM. The spotted halibu mapconsisted of 86 markers (38 SSR markers and 48 AFLP markers) grouped in 24 linkagegroups, with an average resolution of 10 cM. Five microsatellite markers were common toboth maps. More markers especially microsatellites should be mapped on the two maps, as theresolution of the two maps is too low. It is the first time to construct genetic linkage maps forbarfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus), and could beuseful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding)and construction of middle or high density linkage map of this two fish species.更多还原