厦门大学学位论文原创性声明本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中以适当方式明确标明, 并符合法律规范和厦门大学研究生学术活动规范 ( 试行 ) 另外, 该学位论文为 ( ) 课题 ( 组 ) 的研究成果,

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1 学校编码 :10384 分类号密级学号 :B UDC 博士学位论文大黄鱼群体遗传的微卫星分析及微卫星标记在石首鱼类系统进化研究中的应用 Study on the Population Genetics of Pseudosciaena crocea and Phylogenetic Perspective in Sciaenidae with Microsatellites 林能锋指导教师姓名 : 苏永全教授专业名称 : 海洋生物学论文提交日期 :2008 年 12 月论文答辩时间 :2008 年 12 月学位授予日期 : 2008 年 12 月

2 厦门大学学位论文原创性声明本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中以适当方式明确标明, 并符合法律规范和厦门大学研究生学术活动规范 ( 试行 ) 另外, 该学位论文为 ( ) 课题 ( 组 ) 的研究成果, 获得 ( ) 课题 ( 组 ) 经费或实验室的资助, 在 ( ) 实验室完成 ( 请在以上括号内填写课题或课题组负责人或实验室名称, 未有此项声明内容的, 可以不作特别声明 ) 声明人 ( 签名 ): 年月日

3 厦门大学学位论文著作权使用声明本人同意厦门大学根据中华人民共和国学位条例暂行实施办法等规定保留和使用此学位论文, 并向主管部门或其指定机构送交学位论文 ( 包括纸质版和电子版 ), 允许学位论文进入厦门大学图书馆及其数据库被查阅借阅本人同意厦门大学将学位论文加入全国博士硕士学位论文共建单位数据库进行检索, 将学位论文的标题和摘要汇编出版, 采用影印缩印或者其它方式合理复制学位论文本学位论文属于 : ( )1. 经厦门大学保密委员会审查核定的保密学位论文, 于年月日解密, 解密后适用上述授权 ( )2. 不保密, 适用上述授权 ( 请在以上相应括号内打或填上相应内容保密学位论文应是已经厦门大学保密委员会审定过的学位论文, 未经厦门大学保密委员会审定的学位论文均为公开学位论文此声明栏不填写的, 默认为公开学位论文, 均适用上述授权 ) 声明人 ( 签名 ): 年月日

4 目录中文摘要...i ABSTRACT...iv 第一章文献综述...1 第一节分子遗传标记在鱼类群体遗传学研究中的应用鱼类群体遗传学研究内容群体遗传学研究中常用的分子遗传标记微卫星 DNA 标记及其在鱼类遗传学研究中的应用...6 第二节我国沿海几种石首鱼类的群体遗传学研究大黄鱼的资源状况及群体遗传学研究几种石首鱼类的群体遗传学研究现状...24 第三节石首鱼类的系统发育研究系统发育石首鱼类的系统发育研究...30 第四节本研究的目的和意义...34 第二章大黄鱼部分基因组文库的构建及微卫星 DNA 的筛选引言材料与方法实验材料实验方法结果与分析大黄鱼基因组 DNA 的提取基因组 DNA 及 puc18 质粒的双酶切及目的片段的回收小片段基因组文库的构建与保存从基因组文库中筛选微卫星 DNA 微卫星序列的识别与分类讨论微卫星探针的选择 PCR 法筛选文库方法微卫星序列在大黄鱼基因组中分布微卫星长度的变异...44 第三章大黄鱼群体遗传多样性的微卫星分析 I

5 0 引言材料与方法实验材料实验方法结果与分析微卫星引物的多态性分析大黄鱼群体的遗传变异讨论微卫星座位的多态性分析大黄鱼群体的遗传结构...56 第四章大黄鱼微卫星标记在石首鱼科近缘种中的应用...57 第一节大黄鱼微卫星标记在石首鱼科近缘种中的适用性研究引言材料与方法实验材料实验方法结果与分析大黄鱼微卫星引物在几种石首鱼类中的扩增微卫星扩增图谱的分析讨论大黄鱼微卫星标记引物在石首鱼科鱼类中的适用性大黄鱼微卫星标记在不同种中扩增的差异...62 第二节应用大黄鱼微卫星引物分析小黄鱼的遗传多样性引言材料与方法实验材料实验方法结果与分析小黄鱼群体在 7 个微卫星位点上的遗传多样性各微卫星位点的杂合度及 Hardy-Weinberg 平衡分析讨论大黄鱼微卫星引物在小黄鱼群体中的多态性舟山小黄鱼群体的微卫星遗传多样性...66 第三节应用大黄鱼微卫星引物分析棘头梅童鱼的遗传多样性引言...68 II

6 1 材料与方法实验材料实验方法结果与分析舟山棘头梅童鱼在 6 个微卫星位点上的遗传多样性棘头梅童鱼的微卫星位点的杂合度及 Hardy-Weinberg 平衡分析讨论大黄鱼微卫星引物在棘头梅童鱼群体中的多态性舟山棘头梅童鱼群体的微卫星遗传多样性...71 第五章微卫星标记在石首鱼类系统进化中的应用研究引言材料与方法实验鱼的分类及来源 DNA 的提取微卫星引物的选择微卫星位点的扩增 PCR 产物的纯化及测序各微卫星位点的序列及系统进化分析结果与分析几个微卫星位点在 10 种石首鱼中的扩增种石首鱼类微卫星位点扩增序列的比对利用 3 个可通用扩增序列所构建的系统树微卫星位点的变异讨论石首鱼科鱼类的系统发生研究方法微卫星侧翼序列在石首鱼科系统发生研究中的应用...85 图版...87 参考文献在读期间发表及已完成的论文致谢 III

7 Contents Abstract in Chinese... i Abstract in English... iv Chapter 1 Review...1 Section 1 Molecular markers and their applications in fish population genetics Population genetics of fishes Molecular markers in population genetics researches Microsatellite and its applications to fish genetics researches...6 Section 2 Population genetics research of sciaenidae in China Nature resource of P. crocea and its population genetics researches Population genetics researches of the other sciaenidae...24 Section 3 Review of phylogenetics research of sciaenidae Phylogeny Phylogenetics research of sciaenidae...30 Section 4 The contents and intentence of this paper...34 Chapter 2 Construction of partial genome library of P. crocea and isolation of microsatellites Introduction Material and Methods Material Methods Results Extraction of genomic DNA of P. crocea Double enzyme digestion of genomic DNA and puc18 and recovering of IV

8 object fragment Construction and storage of partial genomic library Isolation of microsatellites Identification and label of microsatellites Discussion Slection of microsatellite s probes Methodology of Screen library with PCR Distrubution of microsatellites in P. crocea genome Variation of repeat number of microsatellites...44 Chapter 3 Genetic analysis of P. crocea stocks using polymorphic microsatellite Introduction Material and Methods Material Methods Results Polymorphism of P. crocea microsatellites Genetic structure analysis of different P. crocea stocks using microsatellites52 3 Discussion Polymorphism analysis of microsatellites from P. crocea Population genetic structure of different P. crocea stocks...56 Chapter 4 Usage of microsatellites from P. crocea in close-related species...57 Section 1 Cross-species amplification of the microsatellite markers from P. crocea in sciaenidae Introduction Material and Methods Material...57 V

9 1.2 Methods Results Versatility of PCR primers of P. crocea Genetics variation of microsatellites at different species Discussion Utility of microsatellite primers from P. crocea in sciaenidae Difference of amplification of sciaenidae with PCR primers of P. crocea...62 Section 2 Genetics diversity analysis of P. polyactis using polymorphic microsatellite from P. crocea Introduction Material and Methods Material Methods Results Analysis of genetics variation of P. polyactis at 7 microsatellite loci Analysis of heterozygosity and Hardy-Weinberg equilibrium of microsatellite loci in P. polyactis Discussion Genetic variability of microsatellite primers of P. crocea in P. polyactis The genetics diversity of zhoushan P. polyactis stock in microsatellite loci..66 Section 3 Genetics diversity analysis of C. lucidus using polymorphic microsatellites from P. crocea Introduction Material and Methods Material Methods Results Analysis of genetics variation of C. lucidus at 6 microsatellite loci Analysis of heterozygosity and Hardy-Weinberg equilibrium of microsatellite loci in C. lucidus...70 VI

10 3 Discussion Genetic variability of microsatellite primers of P. crocea in C. lucidus The genetics diversity of zhoushan C. lucidus stock in microsatellite loci...71 Chapter 5 Study on phylogenetic perspective in sciaenidae with microsatellites Introduction Material and Methods Taxonomic status and collection locations of the species used in this study Extraction of DNA Choose microsatellite primer pairs Amplified microsatellite loci of 10 sciaenidae Purification of PCR products and sequencing Analysis of phylogeny of scieanidae Results Results of amplification of microsatellite primer pairs in ten sciaenidae Aligned DNA sequences of PC10F10 PC10G6 and PC10G6-500 from 10 species of scieanidae fishes Molecular phylogenetics relationship among 10 species of scieanidae fishes Variation of the microsatellite loci Discussion Study strategies for phylogeny of sciaenidae Use of microsatellite flanking regions to investigate phylogeny of sciaenidae...85 Plates...87 References...99 Publications Acknowledgements VII

11 摘要大黄鱼 (Pseudosciaena crocea) 是我国重要的海水养殖鱼类, 研究其不同群体的遗传结构对促进大黄鱼资源的保护和开发具有重要意义本文通过建立大黄鱼小片段基因组文库, 应用 PCR 技术快速筛选含 (CA)n 及 (GA)n 微卫星序列的克隆, 共得到 32 个含微卫星序列的阳性克隆, 选择核心序列重复次数较多的 14 个序列登录 GenBank 应用所得微卫星序列的侧翼区设计引物, 对大黄鱼样品进行扩增, 得到 8 对 (PC1C4, PC4H12, PC5E11, PC7A2, PC7H4, PC8F5, PC10F10, PC10G6) 可在大黄鱼 DNA 样品中清晰扩增的微卫星引物在大黄鱼群体中, 各位点平均得到 6.52 个有效等位基因 ; 观测杂合度 (Ho) 为 ~0.6404( 平均 ), 期望杂合度 (He) 为 ~0.9321( 平均 ); 各微卫星座位的多态性信息含量 (PIC) 为 ~0.9281( 平均 ), 除 PC8F5 表现为中度多态外, 其它 7 个位点均表现出高度的多态性在 8 个微卫星位点中, 除 PC7A2 位点外, 其余位点的等位基因频率均偏离 Hardy-Weinberg 平衡 (P<0.05) 微卫星标记分析表明, 我国海区大黄鱼群体仍保持一定的遗传变异水平, 但也表现出较明显的近亲繁殖现象应用上述 8 个微卫星位点分析 4 个大黄鱼不同地理群体 ( 舟山群体, 宁德群体, 漳浦群体, 海口群体 ) 的遗传结构数据分析显示, 大黄鱼不同地理群体的平均遗传分化系数为 , 表明群体中大部分遗传变异来自群体内部 ; 群体中存在明显的近亲交配 ( 平均近交系数为 ); 各地理群体间平均基因流为 , 反映出不同地理群体间存在较频繁的基因交流 UPGMA 聚类分析结果显示, 宁德群体与漳浦群体首先聚类 ( 闽 - 粤东族 ), 而后与舟山群体 ( 岱衢族 ) 聚类, 海口群体 ( 硇洲族 ) 则与上述 3 个群体最后聚类其结果基本反映了大黄鱼群体的地理分布特点, 为首次应用分子遗传标记的方法证实徐恭昭等人依据形态及生态特征将我国沿海的大黄鱼种群分为 3 个不同的地理种群的研究结果舟山群体与宁德漳浦群体先聚类, 与目前养殖现状中浙江福建大黄鱼养殖区中苗种的较广泛流通有关分析上述 8 对大黄鱼微卫星标记引物在石首鱼科 5 个亚科 8 个属 10 个种中 i

12 的适用性结果表明, 大黄鱼微卫星标记引物在黄鱼亚科鱼类中有较高的适用性, 其中位点 PC1C4,PC4H12,PC5E11,PC8F5,PC10F10,PC10G6 的引物可在小黄鱼 (P. polyactis) 和棘头梅童鱼 (C. lucidus) 中获得清晰扩增条带, 位点 PC7H4 引物还可在小黄鱼中扩增, 上述可扩增位点在两个种中均得到多个等位基因, 表现出较高的多态性大黄鱼微卫星标记引物在白姑鱼亚科叫姑鱼亚科牙亚科及石首鱼亚科鱼类中也有较高比例的通用性, 其中银牙 (O.argenteus) 得到 6 个可扩增位点 (PC1C4,PC4H12,PC7A2,PC7H4,PC10F10,PC10G6), 鮸状黄姑鱼 (N.miichthioides) 丁氏 (W.tingi) 和眼斑拟石首鱼 (S.ocellatus) 各得到 5 个可扩增位点, 浅色黄姑鱼 (N. chui) 和尖头黄鳍牙 (C. aureus) 各得到 4 个可扩增位点, 皮氏叫姑鱼 (J.belengerii) 得到 3 个可扩增位点 (PC7H4,PC10F10, PC10G6) 引物 PC5E11 和 PC8F5 只在黄鱼亚科中通用, 对其是否可作为鉴定黄鱼亚科的特异性标记, 还应分析其在更多石首鱼类中的扩增情况位点 PC10F10 和 PC10G6 引物在 10 个种中都得到清晰的扩增产物, 是首次筛选到的可在石首鱼类中通用的微卫星标记, 对其进行深入的研究有可能为石首鱼类的遗传多样性及系统进化提供重要的遗传信息应用可在小黄鱼中扩增的 7 对大黄鱼微卫星引物分析舟山小黄鱼群体的遗传多样性扩增结果显示, 各位点的等位基因数为 6-14 个, 平均每个位点扩增出等位基因 8.29 个, 各位点的 PIC 均大于 0.5, 表明这些位点在小黄鱼群体显示出高度的多态性 ; 各位点的平均期望杂合度为 , 显示舟山小黄鱼群体仍然保持了较为丰富的遗传多样性微卫星位点的等位基因频率偏离 Hardy-Weinberg 平衡, 且多个位点表现为杂合度缺失, 结合历史上小黄鱼曾因遭受过度捕捞造成比较严重的资源衰退, 推测舟山小黄鱼群体在历史上经历过瓶颈效应, 遗传漂变是造成微卫星位点等位基因频率偏离 Hardy-Weinberg 平衡的主要原因本文首次应用分子遗传标记对棘头梅童鱼的遗传多样性状况进行了分析 6 对大黄鱼微卫星引物在舟山棘头梅童鱼群体中总共扩增出 53 个等位基因, 各位点平均等位基因数 8.83 个, 平均有效等位基因数 5.31 个 ; 各位点的 PIC 均大于 0.5, 为高度多态性位点, 因此这些微卫星位点可做为棘头梅童鱼群体遗传学研究的分子遗传标记 ; 各微卫星位点的平均观测杂合度为 , 平均期望杂合度值 , 表明现有棘头梅童鱼群体仍具有较丰富的遗传多样性 Fis 值表明, 除 PC1C4 位点在群体中出现了杂合过剩现象外, 其它位点均表现为杂合缺失 ; ii

13 据此推测舟山棘头梅童鱼群体在历史上也经历过瓶颈效应应用可在 10 种石首鱼中通用的大黄鱼微卫星引物 PC10F10 和 PC10G6-500 本文首次应用核基因序列对石首鱼类的分子系统发生进行了探讨对上述位点的微卫星扩增产物进行测序,Clustal X 软件比对分析微卫星侧翼序列特征, 利用 MEGA4.0 软件绘制系统进化树从各微卫星位点侧翼序列所构建的系统发生树来看, 同一位点侧翼序列所构建的系统树间,NJ 树与 ME 树各分支的拓扑结构相似, 而与 MP 树有较明显的区别 ; 在不同位点间, 往往体现出序列越长, 黄鱼亚科鱼类的进化关系越接近传统树 ; 所有各位点构建的系统树中, 叫姑鱼亚科的 2 个种总是表现出最近的亲缘关系但在黄姑鱼亚科和牙亚科几个种的系统发育关系分析中, 在不同位点所构建的系统树不同构树法所生成的系统发生树间差别较大, 因此, 用目前的几个微卫星位点的侧翼序列还不足以清晰的阐明石首鱼类的系统发生关系要应用微卫星的侧翼序列研究石首鱼类的系统发育, 还应开发出更多的可通用的微卫星引物, 得到更长的侧翼序列以更好的反映系统进化的信息关键词 : 微卫星标记 ; 大黄鱼 ; 群体遗传 ; 石首鱼 ; 通用性 ; 系统发育 iii

14 Study on the Population Genetics of Pseudosciaena crocea and Phylogenetic Perspective in Sciaenidae with Microsatellites Abstract Sciaenidae are the most commercially important fishes in China. Among all seawater cultured fishes, large yellow croaker (Pseudosciaena crocea) has the largest yield. However, wild caught sciaenidae fisheries have been in severe decline. And some species are in the danger of extinction without efficient fishery management. But the genetic background of these species remains still unknown. Therefore, it is necessary to understand the current situation of the natural resources of these species. Microsatellite markers, which are characterized even distribution, high polymorphism, co-dominance and selective neutrality are widely employed in the studies concerning with variety evaluation, linkage-map construction and physical-map construction, genetic diversity of population and evolution. Population genetics has recently gained popularity in the protection and management of sciaenidae. In this paper, the genetic diversity of P. crocea and some other sciaenidae were evaluated with microsatellite markers. In the first part of this paper, a partial genomic library of P. crocea was constructed, and then microsatellites were isolated. The PCR-based screening library method was used to isolate microsatellite loci containing the repeat motif of (CA)n and (GA)n. A total of 55 positive clones were isolated. Among them, 32 positive clones were sequenced, of which 14 sequences were submitted to GenBank, with the accession number being EF to EF pairs of primers were designed on the basis of the unique sequences flanking each repeat motif by using Primer3. All these primers could successfully be amplified in large yellow croakers. In the second part of this paper, 92 individual of large yellow croakers from four stocks were applied to evaluate polymorphism of these markers and the prelimilary iv

15 genetic structure of these stocks were analyzed. GENPOP (v.1.32)was used to analyse the allele data. The number of the alleles per locus varied from 6 to 22. Mean observed heterozygosities (Ho) and expected heterozygosities (He), were and , respectively. These markers showed that the polymorphism information content (PIC) were between and The observed allele frequency of most of microsatellites loci deviated Hardy-Weinberg equilibrium, the main reason for which may be that the population mating system of P. crocea may not follow the random mating principle. Genetic differentiation were not significant between stocks (F ST =0.0848). F IS values within stocks were high (average ). The gene flow between all four stocks was The data showed that there were significant gene exchanges between stocks. All sampled stocks were tested and it was found that every stock contained unique alleles. The findings indicated that there were variations among the different stocks of the P. crocea populations, which might be caused by the large geographical differences of tested sample stocks. It implied that the germplasm of large yellow croaker could renew in some measure if suitable conservation and management strategies were adopted. The UPGMA tree of the four stocks of P. crocea showed that the stocks of Ningde and Zhangpu matched firstly, we could infer that they might belong to the same geographical population of Minyuedong population. Then, they matched with Zhoushan stock which belongs to Dai-ju population. Finally, they matched with Haikou stock which might represent Naozhou population. These findings reflected the distrubtion of P. crocea populations and the actual conditions of culture. They were the first molecular proof that similar to the conclusion drawn by Xu gongzhao et al., which showed that there were three geographical populations of P. crocea along the coast of China sea. Studies have indicated that there was conservation of flanking regions of microsatellite sequences among related species. If the 8 microsatellites isolated from large yellow croaker had conservative characteristics, costly and time-consuming operations would be avoided to isolate microsatellites from other sciaenidae and it v

16 would serve as a prerequisite for inter-specific comparisons. In the third part of this paper, the primers of 8 microsatellites loci developed from P. crocea were amplified in ten species of Sciaenidae fishes which were collected from the coast of Zhejiang Province and Fujian Province. We found that the ratio of successful amplifications in species belonging to the same genus or the same subfamily as the target species was high. In the subfamily of Pseudosciaeninae, 7 loci (PC1C4, PC4H12, PC5E11, PC7H4, PC8F5, PC10F10, PC10G6) could be amplified in Pseudosciaena polyactis. In Collichthys lucidus, there were 6 of the 8 loci, could acquire legible amplified products, except for PC4H12 and PC7A2. Primers of these loci were also useful in species of Argyrosominae, Johniinae, Otolithinae and Sciaeninae. 6 loci (PC1C4, PC4H12, PC7A2, PC7H4, PC10F10, PC10G6) were amplified successfully in Otolithes argenteus. Loci PC1C4, PC4H12, PC7H4, PC10F10, PC10G6 could obtain plainly discernible amplified bands in Nibea miichthioides and Sciaenops ocellatus, while PC1C4, PC7A2, PC7H4, PC10F10, PC10G6 in Wak tingi. Loci PC1C4, PC7A2, PC10F10, PC10G6 and loci PC1C4, PC7H4, PC10F10, PC10G6 had been amplified in Nibea chui and Chrysochir aureus, respectively. Meanwhile, only 3 loci (PC7H4, PC10F10, PC10G6) were amplified successfully in Johnius belengerii. Cross-species amplification data also indicated that allelic diversity was very low in nontarget species when compared with target species in some cases. For example, locus PC10F10 had high allelic diversity in P. crocea but it had only one allele in S. ocellatus. Loci PC5E11 and PC8F5 were amplified in subfamily Pseudosciaeninae only. Maybe they were specific loci in Pseudosciaeninae. This suggested that we could use these loci to identify species of Pseudosciaeninae from other sciaenid fishes. In addition, differences were found between closely related sciaenid fishes in average allelic length and the number of alleles which had been scored. Thus the microsatellite s footprints of each species might be used to distinguish closely related species, while more data were still needed to confirm the inference. Furthermore, the design of highly versatile MFR-primers might be useful for population-level work in the species belonging to this highly species-rich suborder of fishes. vi

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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