牙鲆群体遗传多样性及鲽形目鱼类分子系统学初步研究

【作者】 尤锋；

【导师】 相建海； 王可玲；

【作者基本信息】 中国科学院海洋研究所 ， 海洋生物学， 2001， 博士

【摘要】 本文以山东近海野生和养殖牙鲆Paralichthys olivaceus（T. ＆ S.）为研究对象，采用同工酶电泳和随机扩增多态性DNA（RAPD）两种方法，进行了群体遗传学研究；另外，用PCR扩增了牙鲆、桂皮斑鲆Pseudorhombus cinnamomeus（T.＆ S.）、石鲽Kareius bicoloratus，Basilewsky和大菱鲆Psetta maxima 4种鲽形目鱼类mtDNA 16S rRNA基因区的部分片段，采用生物信息学方法构建了鲽形目分子系统树。主要结果如下： 1．首先建立了适于牙鲆同工酶分析的水平淀粉凝胶和垂直聚丙烯酰胺凝胶电泳系统；对获得的牙鲆15种同工酶基本酶谱进行了生化遗传分析，进而对自然和养殖群体的生化遗传结构进行了分析，共记录了29个基因座位，发现了9个多态座位。 2．野生群体的生化遗传参数多态基因座位比例（31.0％）、等位基因平均数（1.38）和群体平均杂合度（0.0802）都明显高于养殖群体（24.1％，1.28，0.0788）；在野生群体中有9个多态基因座位，而养殖群体仅7个多态基因座位；其中，除了Cat和Idhp-1（仅养殖群体）（P＜0.05）有显著差异、Ldh-C（P＜0.01）完全偏离Hardy-Weinberg定律外，其余多态座位基因频率均符合Hardy-Weinberg遗传平衡定律。野生和养殖群体的遗传相似性系数（Ⅰ）为0.9877，它们的遗传距离（D）是0.0124；两群体间的遗传分化系数G_st为0.0681，D_m为0.01，表明总变异中的6.8％的遗传变异产生于群体间的基因差异。 3．采用11个随机引物对20个野生个体和24个养殖个体进行了RAPD群体遗传多样性分析，分别扩增出88条和86条DNA带，片段大小在200-2500bp之间，平均每个引物扩增的带数是7.8-8.0。两个群体的多态座位比例分别是43.2％和34.9％，平均杂合度是0.2739和0.2255，而Shannon遗传多样性指数表明两群体的遗传变异中有88.12％的遗传变异来自种群内，只有11.88％的变异来自群体间。遗传分化指数G_st的结果也验证了Shannon遗传多样性指数的结果：总群体的遗传变异中约有12％是由两群体间的基因差异产生的。 4．本文对牙鲆两个群体的同一批样品分别采用经典的同工酶方法和RAPD 我N卧 牙鲜群体遗传多样性及煤形图鱼类分子系统学初步研究 治e学位论文 方法进行了较系统的比较分析。发现，KAPD所显示的多态性要比同工酶的高 得多，因为大部分RAPD的变异是源于非编码区和重复DNA，可以遍布整个基 因组，而同工酶仅是功能基因的产物，只表现编码区的变异。因此，自然选择 在同工酶编码区的作用要多于 RAPD标记。在遗传相似性系数目)和遗传距离 （D）上，KAPD的分析结果与同工酶的分析结果也是有差异的，用同工酶分 析两个群体遗传距离只有0乃124，而用RAPD研究可达0刀508。遗传分化指数 的差异也很大，同工酶为0．068，RAPD为0．1237。 5．RAPD和同工酶的分析结果是类似的，即自然群体的多态座位比例和平 均杂合度要比养殖群体高，降低幅度在同工酶中界于1．7～22．3％之间，在KAPD 中则界于 15．9～19．2％之间。这充分证明了养殖群体的遗传多样性水平己有明显 的丧失，值得我们注意。 6．构建了毁形目鱼类 mtDNA 6S rRNA基因的分子系统树。通过分子克 隆法将牙均、桂皮斑稣、大菱鲜和石煤 mtDNA 6S rANA目的基因片段连接到 质粒载体上，经MegaBACE测序仪坝序，分别获得了 590、595、582和 590hp 序列，通过生物信息学方法对其进行了序列分析和核酸变异比较，结合NCBI 上6种群形目鱼类的同源序列探讨了这4种鱼类在群形目中的遗传分化和分子 系统进化，构建了系统树，其中，桂皮斑稣的 16s rRNA基因在系统树中的位 置与物种形态资料的系统演化不相符，而其它三种很好地呈现了它们在线形目 中的系统位置。同时，可以看出 mtDNA 6S rRNA基因片段可以构建一个相对 准确的树，特别是NJ树和ML树比较接近，更为客观一些。由比对序列获得 的物种之间的遗传距离也基本可以反映种、属、科间的不同变异水平。更多还原

【Abstract】 Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks off Shandong coastal waters of China was analyzed using isozyme and RAPD techniques. And partial DNA sequences of mitochondrial DNA 1 6S rRNA gene from P. olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus were amplified by PCR and sequenced in order to address the phylogenetic relationships of 4 flatfishes. The main results shown as follows: 1. Pm and 25 isozymes, such as LDH, MDH, MEP, IDHP, G3PDH, AK, CK, ACP, PGM, SDH, ADH, CAT, SOD, GDH, GPI, GAPDH, ALP, HK, G6PDH, PGDI-1, MPI, EST, POD, AMY and GOT in 4 buffer systems (TC, pH=6.9; TCI,pH8.O; EBT, pH?.9; TG,pH=8.3) were studied by horizontal starch gel and vertical po]yacrylamide gel electrophoresis. Among them, 15 isozymes (LDH. MDH. MEP. IDHP.. G3PDH. AK.. CK. ACP, PGM. SDH. ADHS CAT.. SOD.. GDH.. GPI) in 3 buffer systems (TC, pH=6.9; EBT, pH=8.9; TG,pH=8.3) were analyzed, and their expression in 6 tissues and organs-tissues specify were also compared. The basic electrophoretic patterns of these 15 isozymes were gained and their biochemical genetic parameters such as locus number, structure, alleles et al., were also analyzed. The electrophoregrams of the rest isozymes were too bad to be recorded. 2. The natural sample size was 79 live left-eyed flounders, which were collected from the coastal waters of Qingdao. The cultured samples (52 live left-eyed flounders) were collected from a fish farm in Rongcheng of Shandong Province, the parental generation of which was also captured in the coastal waters of Shandong. The biochemical genetic structures of both stocks have been studied and compared. Altogether 29 loci in each stock were recorded, among them 9 gene loci (Ldh-A, Ldh- C, Idhp-1, Acp-1, Pgm, Sdh, Adh, Cat and Gdh) belong to the polymorphic loci in the natural stock and its mean proportion of polymorphic loci (P) was 31.0%. While in the cultured stock, 7 gene Joci (Ldh-A, Ldh-C, Idhp-1, Sdh, Adh, Cat and Gdh) belong to the polymorphic loci and its P was 24.1%. The allelic frequencies of both stocks were also gained, with the average heterozygosities (H) of stocks being 0.0802 in the natural stock and 0.0788 in the cultured stock. Both values ofF and H in the cultured stock are lower than those in the natural stock. The chi-square test showed that all polymorphic loci but Cat (P<0.05), Idhp-1 (P<0.05, only in the cultured population) and Ldh-C (P<0.0 1) in both of the natural and cultured stocks were in Hardy-Weinberg equilibrium. The observational values of heterozygosity (Ho), Hardy-Weinberg expected heterozygosity (He) and the genetic departure index (d) also expressed the difference in genetic variance of these two stocks. The genetic similarity and genetic distance between the two stocks were 0.9877 and 0.0 124, respectively. 3. 20 natural individuals and 24 cultured individuals of]? ollvaceus were investigated using 11 random primers, respectively. 88 and 86 RAPD bands ranging from 200 to 2,500 bp were recorded separately, an average of 7.8-8.0 bands gained by per primer. The mean percentages of polymorphic loci of two stocks were 43.2% and 34.9%. The average heterozygosity (H) of them were 0.2739 and 0.2255. And the average Shannon indices of phenotypic diversity of them (H0) were 0.1120 and 0.0942. The genetic similarity ano genetic distance between these two stocks were 0.9505 and 0.0508 respectively.更多还原