coi条形码在鳗鲡种质鉴定和系统发育研究中的应用.docx

摘要本研究通过比较鳗鲫Col基因序列的差异对玻璃鳗(欧洲鳗鲫、日本鳗鲫、 花鳗鲫和莫桑比克鳗鲫)进行种质鉴定，建立相应的Col条形码同时对鳗鲫属 中9种鳗鲫(欧洲鳗鲫、美洲鳗鲫、双色鳗鲫、吕宋鳗鲫、花鳗鲫、日本鳗鲫、 莫桑比克鳗鲫、云纹鳗鲫和太平洋双色鳗鲫)进行遗传多样性和系统发育的研究， 以期为鳗蛆养殖过程中存在的种质混乱，养殖种类过于单一等问题提供帮助1. 玻璃鳗种质鉴定：采集玻璃鳗样品共46尾并提取DNA,通过PCR扩增 Col基因片段后进行序列测序，用相关软件进行数据处理和分析后得到613bp的 序列46条序列共定义了 16个单倍型以GenBank中已有的鳗蛆线粒体Col 基因序列为参照，经分析碱基组成、单倍型网络进化关系、遗传距离和NJ分类 树等，鉴定结果为欧洲鳗鲫、日本鳗鲫、花鳗鲫、莫桑比克鳗鲫欧洲鳗鲫和莫 桑比克鳗蛆亲缘关系较近，日本鳗鲫和花鳗鲫亲缘关系较近，但欧洲鳗鲫、莫桑 比克鳗鲫与日本鳗鲫、花鳗鲫聚为两大类46条Col序列中有69处发生碱基转 换，14处发生碱基颠换，转换与颠换的发生数量比值约为15:1,其中T与C之 间的转换发生最多4种鳗鲫种间遗传距离为0.0549〜0.0926,种内遗传距离为 0.16〜0.0062。

本试验中用mtDNACoI序列进行的鳗鲫分类和形态学鉴定结果 是一致的，表明用线粒体Col序列的DNA条形码技术鉴定鳗鲫种类是可行的 单倍型hapl-4为莫桑比克鳗鲫独有；hap5-6为日本鳗蛆独有；hap7-ll为日本鳗 鲫独有；hapl2-16为花鳗鲫独有，可作为DNA条形码鉴别不同种鳗鲫2. 鳗蛆属遗传多样性和系统发育研究：采集9种鳗鲫(共83个样品)提取 DNA,通过PCR扩增Col基因片段后进行序列测序，用相关软件进行数据处理 和分析83条序列定义了 36种单倍型鳗鲫的核昔酸多样性(0.00047〜0.00470) 远远低于单倍型多样性(0.286〜0.944),遗传多样性相差较少欧洲鳗鲫、吕宋 鳗鲫单倍型多样性较高，分别为0.909、0.944,日本鳗鲫单倍型多样性仅为0.286, 该种鳗蛆资源丰富度偏低欧洲鳗鲫的平均核昔酸差异(K)、核昔酸多样性(Pi) 最大，分别为2.897、0.00470,多态位点9个；日本鳗鲫平均核昔酸差异(K)和 核昔酸多样度(Pi)最小，分别为0.286、0.00047,多态位点仅有1个，各单倍型 序列间的差异少，需采取相关措施保护日本鳗苗资源。

序列中碱基转换与颠换比 值为4.24,碱基转换值明显高于颠换碱基替换大部分发生在密码子的第三位点， 第二位点未有碱基替换，最为保守种间平均遗传距离在0.0212-0.0933之间， 种内遗传距离为0.0016〜0.0060物种的系统发育与地理分布位置密切相关发 育系统树显示分为两大支，其中一支中花鳗鲫与云纹鳗鲫亲缘关系最近，其次是 吕宋鳗鲫，日本鳗蛆则稍远，太平洋双色鳗鲫和双色鳗鲫亲缘关系较近，但是与 花鳗鲫和云纹鳗鲫则是亲缘关系最远的另一支上欧洲鳗鲫首先与美洲鳗鲫亲缘 关系最近，莫桑比克鳗鲫相对稍远莫桑比克鳗鲫分布地虽与花鳗鲫、双色鳗鲫 等相近，都分布于印度洋，但反而是与分布于大西洋的欧洲鳗鲫和美洲鳗鲫聚为 一大支，这可能是于地球洋流运动有一定的相关性关键词：鳗鲫；种质鉴定；遗传多样性；系统发育AbstractThis study completes identification of glass eels(A.anguilla,A.japionca, A.marm- oratafA.mossambic)by analyzing difference of Col gene sequences,establish the corresponded Col barcode.Research genetic diversity and phylogenetic development among 9 different species of eel in Anguill (A.anguilla,A.rostrata, A.bicolorbicolor,A.luz- onensis,A.marmorata,A.japionca,A.mossambic\Help to solve problems of eel seedings confusion and single.1 .Identify glass eels on the molecular level.Collected 46 samples of glass eels and extracted DNA of 46 samples.Through Polymerase Chain Reaction amplification to adopte 613bp sequences.After sequencing,the PCR products are analyzed by relevant software. Number of haplotypes is 16.As the reference of mitochondrial Col gene sequences of eel in GenBank,analysis base composition, variation,genetic distance, network of haplotypes and NJ system tree,we know that samples taken are A.anguilla.A.japionca^.marmorata.A.mossambic.A.anguilla and A.mossambic are w- ith closer phylogenetic relationship.A.japionca and A.marmorata are with closer phylogenetic relationship,but they are clustered into two categories.There are 69 transitions of nucleotide and 14 transversion of nucleotide in 46 sequences of Col gene. The number of transitions: transversion is 15:1.Transitions are the most between T and C. Genetic distances between species of Anguilla are 0.0549〜0.0926. Within species of Anguilla, genetic distances are 0.0016〜0.0062. In the experiment the result of using mtDNA Col to identify different species of Anguilla is consistent with the result of morphological identification, showed that Col DNA barcode is feasible to identify 庆〃q.Haplotypes 1-4 is unique in A.mossambic, haplotypes5-6 is unique inAhaplotypes?-11 is unique in A.庆〃《haplotypes 12-16 is unique in A.marmorata. They can be as DNA bar code to identification different species of Anguilla.2.Study about genetic diversity and phylogenetic development of Anguilla. Collected 9 different species of 讥〃o(total 83 samples)and extracted DNA.Throu- gh polymerase chain reaction amplification to adopte respectively .After sequencing,the PCR products are analyzed by relevant software.Number of haplotype is 36. Nucleotide diversity(0.00047〜0.00470) is considerably reduced from haplotype diver- sity(0.286〜0.944),with less genetic diversity. Haplotype diversity of A.anguilla and A.luzonensis are higher, respectively are 0.909, 0.944. Haplotype diversity of A.japio- nca is only 0.286, the richness of this resources is low.Average number of nucleotide differences and nucleotide diversity of A.anguilla are the most, respectively are 2.897, 3., and polymorphic sites of A.anguilla is 9.The least is averagenumber of nucleotide differences and nucleotide diversity are 0.286,0.00047,polymorphic sites is only 1. The differences between haplotypes of A.japionca is less,need to take relevant measures to protect resources of A.japionca.Ratio of nucleotide transition and transversion is 4.24,transition are much higher than transbersion.Bases substitution mostly occurs in the third codon site,not occurs in the second site.The second site is comparatively conservative.Genetic distances between species of A.An- guilla are 0.0212〜0.0933. Within species of A.Anguilla, genetic distances are 0.0016〜 0.17Phylogenetic development and geographical distribution are strongly coupled.NJ tree indicates that they are clustered into two categories.In one of them, A.marmoratcCs phylogenetic relationship is closer to A.nebulosa,then is A.luzonensis. but far to A.japio。