【作者】 匡刚桥；

【导师】 鲁双庆； 肖调义；

【作者基本信息】 湖南农业大学 ， 水产养殖学， 2007， 硕士

【摘要】 本研究采用生物信息学技术和FIASCO(Fast Isolation bv AFLP Sequences Containing repeats)法开发鳜鱼微卫星标记，并根据自行分离的鳜鱼微卫星序列设计20对引物，以鳜属鱼类4个物种翘嘴鳜、大眼鳜、斑鳜和暗鳜共80个个体为试验材料进行遗传多样性分析。实验结果如下：1、采用生物信息学技术从鳜鱼GenBank数据库中的304条核酸序列筛选获得6个高度多态性微卫星标记，其多态信息含量(PIC)在0.567～0.806之间，平均多态信息含量为0.692。2、采用FIASCO法首次成功构建鳜鱼基因组微卫星富集文库。随机挑取其中100个阳性克隆进行测序，60个(60％)含有微卫星序列(GenBank登录号：DQ789247～DQ789306)。成功设计了47对鳜鱼微卫星引物，随机合成其中21对引物进行PCR扩增，筛选获得18个多态性微卫星标记，预期基因杂合度在0.3556～0.9111之间，表明FIASCO法能有效提高筛选微卫星标记的效率。3、根据自行分离的鳜鱼微卫星序列(GenBank登录号：DQ789247～DQ789306)设计并合成20对微卫星引物，对鳜属鱼类4个物种翘嘴鳜、大眼鳜、斑鳜和暗鳜共80个个体进行遗传多样性分析，结果显示：20对微卫星引物在4个物种中的多态性位点百分率分别为90％、75％、85％和85％，累积个体识别率和非父排除率均达到0.9999，属于高识别力的微卫星标记系统；UPGMA聚类分析表明，翘嘴鳜与暗鳜之间亲缘关系最近，可归属于第Ⅰ类，大眼鳜为第Ⅱ类，斑鳜独自为第Ⅲ类。综上所述，本研究开发的鳜鱼微卫星标记可以用于鳜属鱼类的分类及进化研究、遗传背景分析和遗传连锁图谱构建，并将为其基因组结构分析、标记辅助育种以及数量性状位点(QTL)基因的定位等研究提供候选微卫星标记。更多还原

【Abstract】 The bioinforrnatic and FIASCO(Fast isolation by AFLP sequences containingrepeats) technique were used to development microsatellite markers from Siniperca inthis project.Twenty of microsatellite primers were designed using Primer Premier 5.0software according to the microsatellite DNA sequence (GenBank No:DQ789247～DQ789306) of Siniperca chuatsi isolated by ourselves.Then we used these 20microsatellite loci to analyze genetic diversity and genetic differentiation in the fourwild populations of Siniperca,Siniperca chuatsi, Siniperca kneri, Siniperca obscuraand Siniperca scherzeri.1、A bioinformatic analysis of 304 nucleotide sequences in Siniperca chuatsiGenBank identified 22 sequences containing 30 microsatellites, account for 9.87% ofwholly GenBank database. Cluster analysis indicated that the 16 dinucleotide pairswere the most abundant microsatellites,account for 53.33% of the totalmicrosatellite-containing sequences; 11 trinucleotide repeats and 3 tetranucleotiderepeats were found in these microsatellite sequences,account for 36.66% and 10.01%accordingly. 17 primer pairs were designed from these microsatellite sequences.Among the 17 primer pairs, 13 pairs have amplified products, 6 pairs can achieveclear PCR products by Electrophoresis. Genotype analysis results indicated that: theaverage polymorphism information contents(PIC) were 0.692(ranged:0.567-0.806),were all polymorphic loci.2、This project used the method of FIASCO(Fast Isolation by AFLP SequencesContaining repeats) triumphantly constructed the first microsatellite-enriched libraryof Siniperca chuatsi. One hundred clones were isolated and sequenced from Sinipercachuatsi microsatellite libraries. Sixty clones (60%) contained microsatellites(GenBank Accession Number:DQ789247～DQ789306). We designed 47SSR-primers and synthesized 21 of them,18 polymorphic microsatellite markers werescreened out.3、The Microsatellite DNA technique was used to analyze genetic diversity and genetic differentiation in the four wild populations of Siniperca,Siniperca chuatsi、Siniperca kneri、Siniperca obscura and Siniperca scherzeri.Twenty of microsatelliteprimers were designed using Primer Premier 5.0 software according to themicrosatellite DNA sequence (GenBank No:DQ789247-DQ789306) of Sinipercachuatsi isolated by ourselves. The results showed that:the number of alleles(Na) perlocus ranged from 5-25, the effective alleles(Ne) of each loci were 2.2-22.8.The totalof 293 alleles were found with allele size ranged from 80bp to 301bp. The averageheterozygosity(H) among the four populations of Siniperca chuatsi、Siniperca kneri、Siniperca obscura and Siniperca scherzeri was 0.64、0.58、0.67 and 0.65,respectively.At the 20 microsatellite loci among the. four wild populations,the value ofpolymorphism information content(PIC) was 0.269-0.812,0.375-0.857, 0.258-0.857and 0.332-0.843 respectively, averaged at0.5355,0.5106, 0.551 and 0.5478.Power ofdiscrimination (DP) was 0.32-0.82, 0.50-0.86, 0.36-0.86 and 0.42-0.85, respectively.and probability of paternity exclusion (PPE) was 0.338～0.866, 0.263～0.813,0.263-0.506 and 0.384-0.834, in sequence.The results indicated that the 20microsatellite loci selected were very sensitive and could be used in parentage andkinship determination of further genetic breeding studies in Siniperca.Clusteringanalysis indicated that Siniperca chuatsi and Siniperca scherzeri were classified as thefirst grouped as their genetic distance was relatively lower and kinship was relativelylate. Siniperca kneri and Siniperca obscura could be considered as one branch.Insummary,the UPGMA tree was reflected the evolutionary and breeding historyof thefour wild populations, they belong to the different species in the same genus.These microsatellite markers are useful for genetic background analyzing andgenetic linkage mapping construction of Siniperca chuatsi. Therefore, they provided amass of candidates for comparative genome analysis,marker-assisted selection andanalysis of Quantitative Trait Loci location of Siniperca chuatsi.更多还原