【作者】 王磊；

【导师】 陈松林；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2013， 博士

【摘要】 牙鲆是中国、日本和韩国等国家的重要海水经济养殖鱼类之一，随着第四次海水养殖浪潮的到来，牙鲆养殖业蓬勃发展起来。近些年来，病害频发严重阻碍了牙鲆养殖业的发展，培育具有抗病力强和养殖存活率高等优良性状的牙鲆新品种是解决这一问题的重要途径之一。从培育抗病新品种的实际需求出发，主要对以下4个方面进行了探讨：1、牙鲆抗淋巴囊肿病和高养殖成活率家系的筛选与分析；2、连续三代牙鲆抗鳗弧菌病家系的筛选与分析；3、牙鲆抗鳗弧菌病和淋巴囊肿病相关分子标记的筛选；4、牙鲆连续两代雌核发育及F1近交家系的遗传分析。主要研究结果如下：1、牙鲆抗淋巴囊肿病和高养殖存活率家系的筛选与分析2009年的牙鲆家系在2010年爆发了淋巴囊肿病，对16个家系及对照组的发病率进行了评估，得到4个发病率低于40%的家系，其中有一个家系F0939完全没有发病；2011年，在2009年牙鲆家系长到580日龄时，统计了各个家系的养殖存活率和生长情况，发现养殖存活率超过55%的7个家系中有6个家系的至少一个亲本家系F0750，说明以F0750做亲本可以有效的提高家系的养殖存活率；相关分析表明，牙鲆淋巴囊肿病的发病率与感染时的生长和580日龄的生长呈极显著负相关(r=-0.790)；另外，牙鲆鳗弧菌感染存活率与养殖存活率呈低度正相关（r=0.371）。2、牙鲆连续三代抗鳗弧菌病家系的筛选与分析2012年，为了继续稳定抗病家系的抗病性状，同时筛选新的抗鳗弧菌病家系，以实验室多年建立的牙鲆育种群体和家系为基础，共建立牙鲆家系65个，选取其中43个家系进行鳗弧菌感染实验，共筛选出8个抗病力强的家系，其中1个家系极显著高于对照组（P<0.01），其他7个家系显著高于对照组（P<0.05），这8个家系包括4个F1代家系、2个F2代价系、1个F3家系和1个雌核发育二代家系。同时对连续三代16个抗病力强的家系进行分析，发现其中14个家系与抗病基础群体有关，说明牙鲆抗鳗弧菌病性能能够遗传给后代。3、牙鲆抗鳗弧菌病和抗淋巴囊肿病相关分子标记的筛选(1)牙鲆抗鳗弧菌病QTL的定位为了加快抗鳗弧菌病的选育进程，以抗病家系F0750和易感家系F0743的杂交F1代家系F0975为资源家系，采用混合分群分析(BSA)和QTL定位相结合的方法从高密度遗传连锁图谱上每10cM左右筛选1个标记共178个标记对感染材料进行分析，利用2种模型对表型性状进行定义，通过这两种模型分别定位了1个（qVT-0）和3个(qVT-1, qVT-2, qVT-3)QTL位点，这些QTL区域的可解释变异率超过60%。同时，qVT-0和qVT-2定位区域重合，这个重合的QTL区域有可能是牙鲆抗鳗弧菌病的最主要的区域。（2）牙鲆抗鳗弧菌病家系相关分子标记的筛选由于QTL定位是基于家系的，因此很少具有广泛的适用性，为了筛选具有较广泛适用性的抗病相关标记，利用本实验室从牙鲆的表达序列标签中筛选到46对多态的微卫星标记（EST-SSR）对2009年感染的30个家系进行了抗病标记筛选，最终得到3个与抗病显著相关（P<0.01）的分子标记。（3）牙鲆抗淋巴囊肿病相关分子标记的初步筛选在牙鲆抗淋巴囊肿病相关分子标记筛选方面，本文以一个发病率接近50%的家系为材料，首先从高密度连锁图谱上筛选178个微卫星标记，通过BSA法进行初步筛选，然后用来自3个家系的52个个体进行验证，最后筛选到1个与抗淋巴囊肿病相关的分子标记。4、牙鲆连续两代雌核发育家系和全同胞近交家系的遗传分析全同胞近交和雌核发育是鱼类育种中经常用来纯合基因和稳定性状的方法，为了准确评估这两种方法的遗传纯合效果，本研究利用来自于高密度遗传连锁图谱上的30个微卫星对牙鲆全同胞近交F1代家系、雌核发育一代、二代家系及其亲本家系进行了分析，发现牙鲆减数分裂雌核发育可以快速降低后代的遗传多态性，提高后代个体间的遗传相似度，但由于一些位点的重组率很高，很难通过简单的多次减数分裂雌核发育来得到牙鲆纯系，而全同胞近交方式不受重组率高低的影响，可以将两者结合起来培育纯系；同时发现雌核发育及近交后代的纯合性与其亲本的纯合度密切相关，因此在牙鲆育种中，对符合目标性状的个体进行纯合性分析，选出纯合性较高的个体再进行近交或雌核发育可以大大加快纯系培育的进程。这些研究为最终培育出具有多种优良性状于一体的牙鲆新品种奠定了基础。更多还原

【Abstract】 Japanese flounder （Paralichthys olivaceus） is an important economical fish inChina, Japan, Korea and so on. In recent years, the development of flounderaquaculture has been seriously interfered with bacterial and viral diseases. Toprevent or at least diminish the devastating effects of these diseases, one usefulmethod is to cultivate families or strains with good characters such as fast growth,strong disease resistance, high breeding survival rate（BSR） and so on. To achievethis purpose, four studies have been carried out as follows: firstly, lymphocystisdisease（LD） resistance families and high BSR families were analyzed; secondly,the families of three successive generations with strong resistant to Vibrioanguillarum were screened and analyzed; thirdly, the molecular markers associatedwith resistant to the V. anguillarum and LD were screened; fourthly, two successivegenerations in meiotic gynogenesis families and inbreeding（brother sister mating）strain of the first finial generation（F1） family have been genetically analyzed. Themain results are as followings:1. Comparative analysis of resistant to LD and BSR among flounder familiesIn2010, LD outbreak in some families established in2009. After evaluating thehealth rate of16families and a control group，four families with incidence below40%were selected and one of the four families with0%incidence was F0939.Around580days after hatching （DAHS） in2011, BSR and body mass （BM） weremeasured and seven families were discovered with BSR above55%. At least one parent of the above six families derived from F0750. It indicates that a family with aparent from F0750may effectively improve the BSR. Correlation analysis showedthat it was significantly negative correlation between the incidence of LD and bodymass at the time of infection （r=-0.790）. In the mean time, the survival rate afterinfection with V. anguillarum （VSR） was relatively positive correlation with BSR（r=0.371）.2. Comparative analysis of disease resistance among flounder families of threesuccessive generationsIn order to enhance V. anguillarum disease resistance and screen new familieswith high resistance to V. anguillarum disease, sixty-five families of flounder wereestablished in2012. Forty-three families were selected as candidate materialschallenged with V. anguillarum using intraperitoneal injection. After infection,different families exhibited variable abilities of disease resistance and8families withstrong disease resistance were discovered. In contrast with the control family, thesurvival of one family was very significantly higher （P<0.01） and seven weresignificantly higher（P<0.05）which contained an F3family and a second generationof gynogenesis family. In the mean time, sixteen families with strong resistance to V.anguillarum from the last few years were analyzed. Fourteen of these families wererelated with Chinese resistance stock （selection by spontaneous challenge andintraperitoneal injection of Vibrio anguillarum）. This indicated that the ability ofresistance to Vibrio anguillarum in flounder could inherit to their offsprings.3. Screening of molecular markers associated with resistant to the V. anguillarumand LD（1） A genome scan for quantitative trait loci （QTLs） associated with V. anguillaruminfection resistance in flounder by bulked segregant analysisIn order to speed up the process of disease resistance breeding, it is necessary toscreen some molecular markers associated with V. anguillarum disease resistance. Inorder to map QTLs, a high-density genetic linkage map was employed to detectedQTLs. F1population was established by crossing of F0750and F0743andchallenged with V. anguillarum in2009. A total of178simple sequence repeat （SSRs） markers were selected around every10cM from genetic map. To screeneffectively, bulked segregant analysis （BSA） and QTL mapping were combined todetect association with this disease. One （qVT-0） and three （qVT-1, qVT-2, qVT-3）QTLs which explained more than60%phenotypic variance in the two models werelocated in a new map, respectively. At the same time, qVT-0and qVT-2were at thesame region. The common QTL maybe a major candidate region for disease resistantto V. anguillarum infection.（2） Screening of EST-SSR markers associated with high VSR families in flounderAs most of QTLs were detected from special families, only a few QTLs wereuniversal. In order to filter out some molecular markers associated with V.anguillarum disease resistance witch suited the entire breeding populations, a total offorty-six simple sequence repeats （SSRs） derived from expressed sequence tags（EST）were used to genotype individuals in thirty infected families. In the end, three SSRswere verified to significantly association （P<0.01） with disease resistance.（3） Initial screening of SSR markers associated with LD resistance in flounderTo screen SSR markers associated with resistant to LD, a family of which theincidence of LD was nearly50%was employed as a resource family. In initialscreening, a total of178simple sequence repeat （SSR） markers were selected aroundevery10cM from a high-density genetic linkage map to be used in BSA. Fifty-twoindividuals derived from three families were selected to confirm the associationbetween markers and LD and one SSR（scaffold185₅97）was verified in the end.4. Genetic analysis of two successive generations in meiogynogenetic family and F1familyFull-sib inbred and gynogenesis are two common methods to make genehomozygous and traits stable. To accurately assess the impact of the two methods,thirty SSRs were employed to analyze the families of F1, the first-generation diploidgynogenesis, the second-generation diploid gynogenesis and the family from whichthe parents of F1came. The results indicated that meiotic gynogenesis of floundercould rapidly reduce the offspring’s genetic polymorphism and increase the genetic similarity between offsprings. As high recombination rate in some loci, it’s difficultto get pure line following simple repeatedly meiotic gynogenesis. But full-sibinbreeding is free from the influence of recombination, it’s necessary to combine thetwo methods to cultivate pure line. In the mean time, parental homozygosity isclosely related with their offspring in meiotic gynogenesis. As a result, selectingindividuals with high homozygosity could rapidly improve breeding program offlounder combining inbreeding with gynogenesis. All these studies laid thefoundation of cultivating a new breed with several good traits.更多还原