【作者】 郭明兰；

【导师】 苏永全；

【作者基本信息】 厦门大学 ， 海洋生物学， 2009， 博士

【摘要】 本文从形态、细胞和分子水平对分类上存在混淆的云纹石斑鱼(Epinephelusmoara)和褐石斑鱼(E.bruneus)进行了比较研究，以澄清它们在分类上的疑问、弄清其遗传背景并筛选可以鉴别两者的特异性分子标记，为云纹石斑鱼和褐石斑鱼的理论研究、生产实践及种质资源的保护提供重要资料。主要结果如下：1．2种石斑鱼的体形和体表横带等外部形态特征非常相似，但体表斑纹不同，Fisher逐步判别法表明两者在背鳍鳍条、鳃耙和幽门盲囊等方面差异达到极显著水平(p＜0．001)。两者骨骼系统均由脑颅、咽颅、脊柱、肋骨和附肢骨骼组成，主轴和附肢骨骼的形态结构基本相同，但在脑颅的眶前骨和眶后骨、咽颅的前鳃盖骨和尾舌骨、附肢骨骼的髓棘间骨及尾下骨与尾鳍鳍棘连接方式等方面存在差异，骨骼差异可作为属间或种间的分类依据。结果确认云纹石斑鱼和褐石斑鱼为为形态特征可以鉴别的2种石斑鱼。2．利用常规核型、Ag-NORs、C-带、重复序列(5S rDNA、18S rDNA和(TTAGGG)n)的荧光原位杂交技术分析了2种石斑鱼的细胞遗传学特性：2种石斑鱼二倍体染色体数目均为48，但单臂染色体和双臂染色体个数不同，因此核型不同：云纹石斑鱼为2n=48，4SM+44T(NF=52)；褐石斑鱼为2n=48，2M+4SM+42T(NF=54)。银染和FISH均显示，云纹石斑鱼和褐石斑鱼中期分裂相中NORs的数目不同。Ag-NORs主要为4个和5个，分别位于2对(第9和24)和3对(第2、9和24)双臂染色体短臂或亚着丝粒区，种内Ag-NORs数目和分布模式均具多态性；2种石斑鱼18S rDNA的FISH杂交信号分别为4个和6个，位于双臂染色体亚着丝粒区，不同染色体对杂交信号强弱不同。2种石斑鱼均有1对5S rDNA杂交信号位于1对中等大小端部着丝粒染色体亚着丝粒位置。2种石斑鱼大多数染色体均具有着丝粒C-带，有些同时还具有端粒C-带；云纹石斑鱼第9对染色体和褐石斑鱼第2对染色体短臂相应的Ag-NORs区均具有恒定异染色质C-带。2种石斑鱼大多数染色体着丝粒位置及部分染色体近端粒区呈DAPI阳性带，与C-带结果一致。2种石斑鱼均仅在染色体的端粒位置而没有在中间部位检测到端粒序列(TTAGGG)n探针杂交信号，云纹石斑鱼信号强度及大小比较均匀，而褐石斑鱼染色体组有10对端部着丝粒染色体末端的端粒序列杂交信号强度和大小均明显强于其它染色体上的杂交信号(约4倍以上)。结果显示2种石斑鱼具有较近的亲缘关系，为石斑鱼属中的特化类群，在进化上云纹石斑鱼比褐石斑鱼更原始。3．采用AFLP、RAPD及ISSR分子标记分析了2种石斑鱼遗传多样性。10对AFLP引物、30个RAPD引物和21个ISSR引物分别产生997、226和148条扩增片段，多态位点百分比分别为36．31％、34．51％和27．03%；种间分化明显，平均遗传距离D分别为0．2987、0．3178和0．1792，Shannon’s信息指数Ⅰ分别为0．2247、0．2251和0．1753，遗传分化系数Gst分别为0．7997、0．8298和0．6460；种内遗传多样性水平均不高，遗传相似度S分别为0．9632和0．9630、0．9775和0．9691、0．9414和0．9769。mtDNA的16S rRNA、Cytb和ND2基因及核基因组中核糖体基因簇的ITS1区进化分析均表明：石斑鱼属是石斑鱼亚科中最新演化的类群，云纹石斑鱼和褐石斑鱼为石斑鱼属内的近缘种。将物种特异性的ISSR及RAPD标记转化并获得4个能鉴别2种石斑鱼的稳定SCAR标记。选取ND2基因和ITS1区为目的片段，首次成功地通过改良建立的Nest-Tetra-primer specific PCR方法对2种石斑鱼进行鉴别，分别获得3个和5个可以鉴别2种石斑鱼特异性分子标记，为2种石斑鱼的鉴别提供了稳定、可靠、快捷的特异性分子标记技术，也为鱼类近缘种的DNA鉴别提供了新的途径。此外，验证实验进一步说明福建南部水域的这2种石斑鱼是具有不同的生态位，没发现杂交现象。更多还原

【Abstract】 Whether Epinephelus moara and E.bruneus,with quite similarity in externalcharacters and distribution,belong to one species or not,has being existed theargument and confusion for a long time.To clarify the questions in classification,study and ratify the genetic status and provide specific molecular markers identifyingthe two grouper species,comparative studies were investigated based on themorphological,cytogenetic and molecular analysis.It provided solid foundation forfurther research,practice and protection of germplasm of E.moara and E.bruneus.Main results revealed as followings:1.Outer shape,color and bands were very similar in E.moara and E.bruneus,but their bars on the body were different.By means of the Fisher Discriminant FoundAnalysis,statistically significant difference would be found to exist between E.moara and E.bruneus （p＜0.001） in meristic and morphometric characters,such as thenumbers of dorsal fin rays,gill rakers and pyloric caeca.Although both species werebasically homologous on the configuration and composition of neurocranium,splanchnocranium,vertebrate,rib and appendicular skeleton,there were manyremarkably differences between them,such as preorbital,post-orbital,rreoperculare,urohyal,predorsal interneural spine and the connection of hypurals and caudal spineand so on.These skeletons could be used as the important characters identifyinggenera or species in fishes.Accordingly,we considered that E.moara and E.bruneuswere different species that could be discriminated by morphological characteristics.2.Cytogenetic characteristic analysis of Epinephelus moara and E.bruneus wasperformed by using karyotype,Ag-NORs,C-banding,FISH with 18S rDNA,5SrDNA and telomere sequences.Although both species shared 2n=48 chromosomes,their karyotypes were different in terms of the number of uni- and bi-armedchromosomes and the localization of nucleolar organizer regions （NORs） revealed byAg-staining and FISH.Karyotypic formulaes were 4SM+44T （NF=52） in E.moaraand 2M+4SM+42T （NF=54） in E.bruneus.Different Ag-NORs sites could beobserved on the short arms or sub-centromere regions of those bi-armed chromosomes in E.moara （chromosome pairs No.9 and 24） and E.bruneus（chromosome pairs No.2,9 and 24）.Result of 18S rDNA by FISH was consistent.The signal strength of Ag-NORs was varied within species.Meanwhile,two 5S rDNAsignals were localized on the sub-centromere regions of one pair telocentricchromosomes differed from those chromosomes of 18S rDNA.Most chromosomes exhibited C-banding hetreochromatins and DAPI positivebandings on the centromere regions,some even on the sub-telemere regions in bothspecies.These NORs on chromosome pair No.9 of E.moara and No.2 of E.bruneuswere C-positive heterochromatic sites.Telomere sequence （TTAGGG）n hybridizingsignals were found on the telomere region but not intermediate position of allchromosomes in these two species.E.moara was characterized by uniform signalstrength and size.E.bruneus was different,of which 10 chromosome pairs weresignificantly stronger and larger （about 4 times） than others.All results indicated thatE.moara and E.bruneus,with close relationships,were different species specializedin genus Epinephelus,and E.moara was more primitive than E.bruneus.3.Genetic diversity of E.moara and E.brunues was investigated by AFLP,RAPD and ISSR.997,226 and 148 amplified fragments were recorded,of which thepercentage of polymorphic bands were 36.31%,34.51% and 27.03%,from ten pairsAFLP primers,thirty RAPD primers and twenty-one ISSR primers respectively.Differentiation of inter-species was obvious in both speices.Average genetic distance（D） was 0.2987,0.3178 and 0.1792,Shannon’s information index （Ⅰ） was 0.2247,0.2251 and 0.1753,and coefficient of population differentiation (G_st) was 0.7997,0.8298 and 0.6460,respectively.Intra-specific genetic diversity was not at a highlevel in the Fujian south coastal population of E.moara and E.bruneus.Geneticsimilarities （S） were 0.9632 and 0.9630,0.9775 and 0.9691,0.9414 and 0.9769separately.Molecular phylogenetic evolution was estimated based on mitochondrial16S rRNA,Cytb and ND2 gene,as well as nuclear ribosomal ITS1 region.All resultsaffirmed that the genus Epinephelus located at the top of the phylogenetic tree andwas the mostly recently diverged group,and E.moara and E.bruneus were closelyrelated species in genus Epinephelus. Then,four stable SCAR molecular markers,translated from the species-specificISSR and RAPD amplicons,were obtained to discriminate E.moara and E.bruneus.To identify these two species,an improved Nest-Tetra-primer specific PCR assay wasdeveloped and established based on ND2 gene and ITS1 region sequences firstly.Itamplified three and five species-specific molecular markers,respectively.Thismethod provided a highly reliable,precise,and rapid molecular marker technique todiscriminate the two grouper species,as well as a new method of DNA differentiationof closely related species in fishes.Furthermore,the results of verification testsshowed that,in the south sea of Fujian,China,E.moara and E.bruneus had differentniches and no hybridization occurred between them.更多还原