【作者】 毕潇潇；

【导师】 高天翔；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2013， 博士

【摘要】 松江鲈（TrachidermusfasciatusHeckel）隶属于鲉形目（Scorpaeniformes）、杜父鱼科（Cottidae）、松江鲈属（Trachidermus），为小型降河洄游性鱼类，是我国野生动物重点保护二级水生动物、中国优先保护鱼类一级物种名录，于2004年被列入中国物种红色名录。本文利用形态学和线粒体DNA序列及ISSR（Inter-simple sequencerepeat）技术和微卫星DNA（Microsatellite DNA）方法分别对采自丹东、秦皇岛、文登、杭州湾、荣成、东营、大连、日本有明海等松江鲈群体进行了研究。（1）应用单因子方差分析、判别分析、主成分分析和聚类分析4种多元统计分析方法，对松江鲈20组形态学参数及5个群体分节特征进行分析。结果显示不同群体间分节特征差异不显著；多元统计分析结果表明地理距离较近的大连群体和秦皇岛群体存在较少的形态差异，同时，丹东和文登群体间形态差异不显著，这些差异仍属于种内不同地理群体间的差异。（2）利用基于电子探针（EPMA）的耳石Sr:Ca比初步开展了采自大连（野生群体）和东营（1个野生群体和1个养殖群体）等3个群体的松江鲈的生活履历及生活史类型研究。结果发现，松江鲈存在两种生活史类型，包括：海淡水洄游性个体生活史型以及出生并生活于河口或近海的非洄游性个体生活史型。该结果与传统的松江鲈为降河性洄游鱼类结论不同，为松江鲈种质资源保护提供了重要依据。（3）利用PCR技术扩增了丹东、秦皇岛、文登、东营、荣成、杭州湾、日本有明海等8个群体线粒体DNA控制区，在获得的854bp的D-loop全序列中，共发现51个多态核苷酸变异位点，研究结果表明松江鲈的中国与日本群体之间遗传分化显著，中国世系的各群体没有明显的地理谱系结构。（4）在8个群体松江鲈144个个体的控制区高变区486bp序列上，共检测到50个变异位点，72种单倍型，丹东群体的单倍型多样性指数最高（0.996），荣成群体最低（0.533）；利用Kimura模型构建的NJ树显示：松江鲈8个群体内存在着两个明显的单倍型类群，比较两两群体之间遗传分化指数FST值发现，松江鲈中国群体和日本群体间存在显著遗传分化，最大遗传距离出现在丹东与日本群体之间，中性检验和核苷酸不配对分布揭示松江鲈经历了近期群体扩张事件。（5）扩增并测定了松江鲈细胞色素b基因全序列，在1141bp的Cyt b基因序列中，47个个体出现了31种单倍型，8个群体有2个共享单倍型，共发现38个变异位点，研究结果表明松江鲈中国沿海群体和日本有明海群体间的遗传差异显著，两个世系分歧时间约为41万年前。（6）对松江鲈的线粒体16SrRNA基因片段进行了扩增和序列测定，得到长度为572bp的序列片段，碱基含量的平均值为（%）：A=28.8，T=23.9，C=23.8，G=23.5，11条序列片段共发现3个变异位点，4种单倍型h=0.4909，π=0.12%。其进化速率最慢，最为保守。（7）利用6个ISSR引物在3个群体85个松江鲈个体中扩增出的条带数目22～97不等，片段大小在0.2～2.5kb之间，其中多态片段为324条。丹东野生群体的Shannon多样性指数最高（H=0.3507），秦皇岛人工繁育群体最低（H=0.2769），即松江鲈野生群体的遗传多样性水平高于人工繁育群体。秦皇岛人工繁育群体与丹东和文登两野生群体间遗传分化系数（Gst=0.34900；Gst=0.39646）明显高于丹东野生群体和文登野生群体间的遗传分化系数（Gst=0.07240），而丹东和文登两野生群体间的基因流（6.40608）明显高于这两个群体与秦皇岛人工繁育群体的基因流（0.93266；0.76116）。（8）利用8个SSR引物对黄河口松江鲈进行了初步分析，研究结果表明50个个体在8个微卫星位点上具有8-15个等位基因，多态性适中，并具有较高的PIC值，表明东营松江鲈具有较高的遗传多样性；雌、雄个体间没有产生显著的遗传差异，显示松江鲈的遗传变异并非仅存在于雌、雄个体间。更多还原

【Abstract】 Roughskin sculpin, Trachidermus fasciatus, which belongs to Scorpaeniformes,Cottidae, is a ClassⅡ of the National Key Protected Wildlife List and in China Red DataBook of Endangered Animals-Pisces. It feeds on zooplankton, fish and shrimp, and has acatadromous life style. Eight geographical groups of T. fasciatus, collected from Dandong,Qinhuangdao, Wendeng, Dalian, Hangzhouwan, Rongcheng, Dongying and Ariake Sea, aredescribed and compared on morphological characteristics and sequence comparison analysisof mtDNA, ISSR, SSR.（1）Fivemeristiccharactersandtwentymorphometriccharactersaremeasuredandthedata are analyzed by one-way ANOVA, discriminant analysis, principle component analysismethod and multivariate analysis method to study the morphological variations among5populations of T. fasciatus. There are no significant differences among the five groups onmorphological characters. As can be seen from the results, there are no significant differencesbetween Dandong and Wendeng geographical groups of T. fasciatus. Although there aresome differences between Daliang and Qinghuangdao geographical groups of T. fasciatus,which are still within the population level.（2）In the present study, the migratory history of Trachidermusfasciatus from Dalian(wild population) and Dongying (one wild population and one cultured population) wasinvestigated using the otolith Sr:Ca ratios by X-ray electron probe microanalyzer (EPMA).Results showed that both estuarine anadromous individuals and estuarine originnon-anadromous ones occurred in the T. fasciatus populations. The result didn’t agree withthe point that this species is only estuarine anadromous. The present is helpful for the fisheriesmanagement. （3）By amplyfing with PCR technique, mitochondrial control region sequences aredetermined from8groups, including Dandong, Qinhuangdao (aquacultured), Wendeng,Dalian, Hangzhouwan, Rongcheng, Dongying and Ariake Sea. Sequence length of thecomplete control region in individuals of T. fasciatus is854bp. There are51polymorphicnucleotide mutation loci in the sequences. The genetic distance between groups of china andgroup of Japan is significant.（4）SequencelengthofthehypervariablecontrolregioninindividualsofT.fasciatusis486bp, including50variable sites, and72haplotypes are found in144individuals. Thehaplotype diversity index of Dandong group is the highest (0.996) while Rongcheng group isthe lowest(0.533). A molecular phylogenetic tree constructed using the neighbor-joining (NJ)method suggested that there are2significant haplotypes in8groups. From the comparison ofthe genetic differentiation index FSTbetween two groups, we can found there is a significantgenetic differentiation between Chinese groups and Japanese groups. The maximum geneticdistance showed between Dandong group and Japanese group (Ariake Sea).（5）Bothmismatchdistributionanalysesandneutralitytestssuggestedalatepopulationexpansion for T. fasciatus population.1141bp sequence which encode the cytochrome bgene of mitochondrial DNA are amplified.31haplotypes are found in47individuals,including38variable sites. Two haplotypes are shared among8groups. The divergence timebetween two lineage (Chinese lineage and Japanese lineage) is410thousand years agoapproximately.（6）Mitochondrial16S rRNA gene is amplified and sequenced,4haplotypes aredetected in11individuals of T. fasciatus. Of the572bp in the16S rRNA gene,3sites arepolymorphic. The average base content are: A=28.8, T=23.9, C=23.8, G=23.5. The resultsshowed that16S rRNA is the most conservative part among the three parts in mtDNA.（7）Inter-Simple Sequence Repeat (ISSR) systems are used to detect the geneticdiversity in T. fasciatus. Eighty-five individuals from three populations are used6ISSR primers screened,22~27clear and reproducible bands are generated. A total of324reproducible loci ranging from0.2to2.5kp were amplified from all the85individuals. TheShannon’s diversity indices are0.3507in wild population (Dandong population) and0.2769in cultured population (Qinhuangdao population). Shannon’s index and the Gst showed thatthe wild stock in Dandong and Wendeng have the highest genetic variation. Apparently theinter-stock’s Gst (0.34900;0.39646) are higher than the wild population (0.07240). And thegene flow between wild populations (Dandong and Wendeng)(6.40608) are higher than thereared groups of Qinhuangdao (0.93266;0.76116), indicating that the genetic differencebetween wild and reared stocks hasn’t reached the population level yet.（8）SimpleSequenceRepeats(SSR)systemsareusedtodetectthegeneticdiversityinT. fasciatus. Fifty individuals from T. fasciatus populations are used8SSR primers screened.As the results showed,8-15alleles on8microsatellite site, which has high PIC and geneticdiversity. There is no significant genetic difference between male and female individuals,which indicate the genetic variation is not only exist in males and females.更多还原