【作者】 徐汗福；

【导师】 黄鹤忠；

【作者基本信息】 苏州大学 ， 水生生物学， 2011， 硕士

【摘要】 乌苏里拟鲿(Pseudobagrus ussuriensis)隶属于硬骨鱼纲(Dsteichthyes)、辅鳍亚纲(Actinopteri)、鲇形目(Silurformes)、鲿科(Bagridae)、拟鲿属(Pseudobagrus),因其肉质细嫩、味道鲜美、营养价值高而备受青睐。然而随着滥捕现象和环境污染日趋严重,其野生资源数量锐减;另一方面,由于野生亲本数量有限,普遍存在近亲繁殖现象,从而可能导致种质退化。本文从基因组DNA、mtDNA基因片段等多个层次研究了乌苏里拟鲿不同群体间的遗传多样性,初步筛选了乌苏里拟鲿的性别相关分子标记并对其线粒体控制区做了初步的研究。具体研究结果包括如下3个方面:(1)采用SRAP分子标记技术对乌苏里拟鲿东北黑龙江水系野生群体(DB)、洪泽湖野生群体(HL)、淮安养殖群体(HA)、苏州养殖群体(SZ)进行了遗传多样性分析。从不同的引物组合中筛选出12对条带清晰、重复性强、多态性好的引物组合,每组引物对乌苏里拟鲿4群体扩增均产生11~21个扩增位点,片段大小在572~111bp,从4个群体中共扩增出212个位点,其中多态位点有130个,多态位点比例为64.36%,显示了较高的多态性。野生DB群体、HL群体、与养殖HA群体、SZ群体的多态位点比例分别为:65.67%,63.13%,56.54%,54.88%;Nei’s基因多样性指数(H)为0.2597、0.2641、0.2546、0.2469;Shannon’s信息指数(I)为0.4097、0.4118、0.4050、0.3861。两个野生群体间的遗传距离最大(0.1924),遗传相似度最小(0.6718),两个养殖群体间的遗传距离最小(0.1087),遗传相似度最大(0.8970)。UPGMA聚类图显示:HA群体和SZ群体首先聚类为一个分支,再与HL群体聚为一支,最后与DB群体聚类在一起。结果表明:2个野生群体的遗传距离最大,亲缘关系最远,存在明显的遗传分化;养殖群体遗传多样性水平较低,且存在较为明显的遗传分化趋势;但从遗传距离及遗传分化的角度分析,人工养殖群体还没有形成独立的遗传结构。(2)利用SRAP分子标记技术对乌苏里拟鲿雌雄基因组DNA的多态性进行了检测,并筛选与其性别相关的分子标记。8对引物组合在乌苏里拟鲿雌雄群体中共检测到164个位点,其中雌性个体142个位点,多态位点(AP)85个,多态位点比例(P)为55.19%,平均等位基因数(Na)为1.5519、平均有效等位基因数(Ne)为1.3001、Nei’s基因多样性指数(H)为0.1797、Shannon’s信息指数(I)为0.2732;8对引物组合检测到雄性个体154个位点,多态位点102个,多态位点比例(P)达到66.23%,平均等位基因数(Na)为0.6623、平均有效等位基因数(Ne)为1.3113、Nei’s基因多样性指数(H)为0.1887、Shannon’s信息指数(I)为0.2917,表明雄性乌苏里拟鲿群体的遗传多样性要比雌性群体丰富,遗传变异比雌性群体大。通过分析8对引物组合的SRAP扩增图谱,得到一条与雄性相关的SRAP分子标记F10R6—215bp以及雌性个体特异性SRAP分子标记F8R7—450bp。(3)对乌苏里拟鲿线粒体控制区进行扩增并与圆尾拟鲿、长吻鮠、光泽黄颡鱼和大鳍鳠mtDNA控制区序列进行比较。结果表明:乌苏里拟鲿的ETAS序列为TACATTAATTTACTATGGTATAGT ;中央保守序列区CSB-F的序列为ATGTAGTAAGAAACCATCAACCCTGTAT,它是终止序列区和中央保守序列区的分界点,CSB-E的序列为:AGGGGCAAAACTTGTGGGGG,在这个序列中存在一个GTGGGG-box的稳定结构,CSB-D的序列为:TATTACTGGCATCTGGTTCC TA;保守序列区CSB-1的序列为:ATAATATATGTATGTCTTAATGACATA,其识别的关键序列为ACATA,CSB-2序列为AAACCCCCCTACCCCC,CSB-3序列为TGTTAAACCCCTAAACCAG,其中,CSB-3比CSB-2的变异稍大些。乌苏里拟鲿线粒体控制区的A、T含量高,G含量低,这与其他四种鲿科鱼类碱基的组成具有相同的趋势。更多还原

【Abstract】 Pseudobagrus ussuriensis which belongs to Dsteichthyes,Actinopteri,Silurfor- mes,Bagridae. Pseudobagrus ussuriensis is a kind of important economic fish,which taste delicious and has high nutritional value. The success of its artificial propagation improved the status of its fry in short supply and accelerated the foundation of many small breeding populations. In limited of conditions and scales, many seed farm kept few parents and apart of them were offspring of their inbreeding. Its germ plasm, however, such as prematurity, the decline of hatching ratio,and the ability of disease resisitance and spawning has been seriously degenerating every year in recent years.In this study, the genetic structure and genetic diversity of Pseudobagrus ussuriensis were analyzed by mtDNA and SRAP.The main results were shown as follows:(1) The sequence-related amplified polymorphism(SRAP)molecular marker technique was used to compare genetic structures among three populations of Pseudobagrus ussuriensis-one wild and two cultured populations.Samples of two wild population were collected from HeiLongjiang and Jiangsu Hongze Lake,the two cultured populations from Fisheries Research Institute of Huaian (F2 generation) and Suzhou Dongshan Aquatic Breeding Plants (F3 generation). 12 pairs of SRAP primer, which produced good amplified patterns were selected from 100 primer combinations. 212 amplified loci were obtained from the three populations, among which 130 were polymorphic. The percentage of polyrnorphic loci in the HeiLongjiang (DB) population,Hongze Lake(HL) population, Huaian populations(HA), Suzhou Dongshan populations(SZ)were65.67%,63.13%,56.54%,54.88%,respectively. The results indicated that genetic polyrnorphism decreased in two cultured populations. The Nei’s gene diversity of four populations were0.2597, 0.2641, 0.2546 and 0.2469, and the Shannon’s Information index of four populations were0.4097,0.4118, 0.4050 and 0.3861, respectively. The genetic distance between two wild population was 0.1924, while the genetic simility was 0.6718; The genetic distance between two cultured population was 0.1087, while the genetic simility was 0.8970. The noticeable decrease in the number of rare loci and the increase in the number of homozygous recessive loci in the cultured population suggested a considerable loss of low frequency alleles in the cultured populations, which may have resulted from small effective population sizes during artificial seed production. With the method of UPGMA on the basis of genetic distanee,the results showed the populations of HA and SZ assemble done branch first, then with HL assemble done branch second, the last did DB populations because of these populations’s the different sites. The results showed that: two wild populations has the largest genetic distance and the farthest genetic relationship, there is significant genetic differentiation; low level of genetic diversity of culture, and there was obvious genetic differentiation trend; but the genetic distance and genetic differentiation perspective Analysis, artificial breeding groups have not formed an independent genetic structure.(2) SRAP analysis was applied to study sex and genetic diversity in Pseudobagrus ussuriensis.A total of 60 samples,which are 30 males and 30 females,were used in the test.Of 100 random oligonueleotide primers for the amplification of Pseudobagrus ussuriensis genomic DNA,8 could produce reproducible, distinctive and characteristic bands from 572~111bp.164sites were deteeted,112 of which(68.29%) were polymorphic. Genetic diversity quantifled by Shannon index was 0.2917(male) and 0.2732(female) respectively. Only F10R6 primer was successful to amplify a specific band of 215 bp from male populations, named F10R6—215bp. This marker was not associated with male populations and can be used as SRAP marker for sex identification of Pseudobagrus ussuriensis.(3) Complete sequence of Pseudobagrus ussuriensis mtDNA control region is amplified and compared with sequence of Mystus macropterus,Leiocassis longrostris, Pseudobagrus tenuis and Pseudobagrus nitidus downloaded from GenBank.In analogy,the conserved region of these four species are identified as extended terminal associated sequenee,Certrol conserved sequence bloke,Conserved sequence block; These sequences of ETAS is TACATTAATTTACTATGGTATAGT; These sequences of CSB-F is ATGTAGTAAGAAACCATCAACCCTGTAT;These sequences of CSB-E is AGGGGCAAAACTTGTGGGGG;These sequences of CSB-D is TATTACTGGCATCT GGTTCCTA;These sequences of CSB-1 is ATAATATATGTATGTCTTAATGACATA; These sequences of CSB-2 is AAACCCCCCTACCCCC; These sequences of CSB-3 is TGTTAAACCCCTAAACCAG.The sequences of CSB-D and GTGGG-box are stringently conserved while a few site variations are found in other regions among species.更多还原