【作者】 陈永祥；

【导师】 周定刚；

【作者基本信息】 四川农业大学 ， 特种经济动物饲养， 2013， 博士

【摘要】 四川裂腹鱼(Schizothorax kozlovi Nikolsky)隶属鲤形目、鲤科、裂腹鱼亚科、裂腹鱼属,主要分布于长江上游的乌江上游、赤水河上游、金沙江、雅砻江水系,是长江上游特有的地方经济鱼类。近年来由于遭受酷鱼滥捕和生境严重破坏的影响,其天然资源量已十分稀少。本文通过对乌江上游水系四川裂腹鱼形态特征、生长特点、肌肉营养成分、同工酶、核型和mtDNA标记等的研究,描述其种质特性,以期为制定该鱼种质标准提供基础资料,为保护、开发和利用野生四川裂腹鱼资源提供科学依据。主要研究结果如下：1.对采自乌江上游总稽河段、横江洛泽河上游支流羊街河段、金沙江干流攀枝花段、雅砻江上游雅江段等4个河段的78尾四川裂腹鱼标本进行性状分析,结果表明：各河段间,四川裂腹鱼种群的可数性状和可量比例性状存在不同程度的差异(P<0.05)；4个河段四川裂腹鱼与采自岷江上游青衣江雅安段的31尾重口裂腹鱼标本进行比较,发现二者可数性状、可量比例性状和框架结构比较,多数指标差异极显著(P<0.01),不支持将二者合并为一个种。2.分别选择臀鳞、鳃盖骨、脊椎骨和耳石作为年龄鉴定材料,对四川裂腹鱼进行年龄鉴定。结果表明,与其它材料相比,耳石上呈现的年轮最为明显、清晰,几乎没有副轮,是较好的年龄鉴定材料。根据退算法原理,通过数学统计方法对乌江上游总稽河段50尾四川裂腹鱼的生长性能进行初步研究。结果表明：四川裂腹鱼体长(L,mm)与鳞径(R,mm)的关系式为L=27.009676R+107.260658(r=0.9591, N=50);体长(L,cm)和体重(W,g)的关系式为W=0.00002L2.9657(r=0.9719,N=50)；3龄及以下的个体生长较快。3.测定了25尾四川裂腹鱼和26尾昆明裂腹鱼的肌肉成分,以了解和比较其种质特性。结果表明：四川裂腹鱼肌肉中蛋白质、脂肪、灰分含量分别为15.49%、1.03%和1.06%；肌肉中不饱和脂肪酸含量占脂肪酸总量的71.72%；18种氨基酸总含量75.20%(占肌肉干重),其中必须氨基酸含量为31.95%,占氨基酸总量的42.51%,鲜味氨基酸含量达30.03%,占氨基酸总量的39.97%。与昆明裂腹鱼的肌肉成分无显著差异。4.对四川裂腹鱼9种组织(眼球、肌肉、心脏、性腺、肝脏、鳃、脾脏、肾脏、大脑)进行了5种同工酶(乳酸脱氢酶-LDH、苹果酸脱氢酶-MDH、谷氨酸脱氢酶-GDH、乙醇脱氢酶-ADH、酯酶-EST)的研究,并对几种酶的同工酶位点及酶谱进行了分析,结果表明：四川裂腹鱼9种组织的5种同工酶系统具有明显的组织特异性。5.本试验采用植物血细胞凝集素(PHA)体内注射法,对四川裂腹鱼染色体核型进行初步研究,结果表明：四川裂腹鱼标本的染色体数2n=96,核型公式为：2n=36m+16sm+10st+34t,臂数NF=148。未发现异形性染色体、随体染色体及次级缢痕。6.本试验采用PCR扩增和直接测序法分析了乌江上游总稽河段、横江洛泽河上游支流羊街河段、金沙江干流攀枝花段、雅砻江上游雅江段的四川裂腹鱼4个种群,92尾个体mtDNA控制区的序列差异和遗传多样性。在这4个种群ntDNA控制区长度为834bp的同源序列中,共计有84个变异位点,占所测序列的10.07%。在其控制区序列共鉴定出32个单倍型。其中雅砻江种群内平均遗传距离(P)为0.030,攀枝花种群内平均遗传距离(P)为0.0240,总稽河种群内平均遗传距离(P)为0.007,羊街河种群内平均遗传距离(P)为0.0040。4个种群的平均核苷酸多样性(π)为0.02244,平均单倍型多样性(h)为0.951。四个种群中,羊街河种群具有最低的单倍型多样性和核苷酸多样性(h=0.704,π=0.00302)；攀枝花河段种群具有最高的单倍型多样性(h=0.945)；雅砻江河段种群具有最高的核苷酸多样性(π=0.02430)。本研究AMOVA分析结果显示,四川裂腹鱼群体遗传变异主要发生在种群间(50.16%),而种群内的遗传变异相对较小(49.84%)。7.不同种群间的遗传分化指数表明,从整体水平上看四川裂腹鱼的遗传分化比较显著；除雅砻江种群与攀枝花种群间的遗传分化水平不显著外,其余种群间的遗传分化均比较显著；表明该物种具有较明显的种群结构。基因流估计显示,各种群间(除雅砻江与攀枝花种群间)的基因交流受到了限制,表明四川裂腹鱼种群间具有非常高的遗传分化水平,或其种群间已发生隔离。雅砻江种群和攀枝花种群遗传距离较小(0.027),基因流值较大(28.72),遗传分化指数Fst也不显著(P>0.05),表明这两个种群间的遗传分化水平较低。不配对分布分析、Tajima’s D检验和Fu’s Fs检验表明,四川裂腹鱼在整体水平上没有经历过瓶颈效应,但羊街河种群受到由瓶颈效应或奠基者效应引起的遗传漂变的影响。更多还原

【Abstract】 Schizothorax kozlovi Nikolsky is one of Schizothoracinae (Cypriniformes, Cyprinidae) fish species, which is widely distributed in the upstream of the Yangtze River (upstream of Wujiang, Hengjiang and Chishui, master stream of Jinsha and Yalong). It is one of the endemic large-scale commercial fishes in the upper section of the Yangtze River. In recent decades, the population sinks rapidly because of heavy fishing and habitat destruction, attracted a lot of attentions of the community, conservation scientists and other conservative organizations in the world. In order to design effective conservation strategies, this paper have did comprehensively research its Characters, Age&Growth, Nutritional Components, Isoenzyme, Karyotype and D-Loop of mitochondrial DNA on S. kozlovi. Genetic polymorphism of this species is also analyzed in our work. The major results are as follows:1. The Characters and morphology of78S. kozlovi specimens collected from Zongji River (the upper reach of Wujiang River), Yangjie River (the upper reach of Hengjiang River), Yalong River and Jinsha River were been analyzed using conventional methods. The results indicate that the countable and measurable characters differ in four different populations to varying degrees (P<0.05). By comparing to31S. davidi colected from Qingyi River(the upper reach of Minjiang River). The results show that, intra-specific variations of S. davidi and S. kozlovi were striking divergence (P<0.01) in most parameters and thus merging of the two species is not supported. 2. By comparing with different age assessment materials (Buttocks scales, Opercular, Vertebra, Otolith), otolith is the most adequate for age assessment, because its annual rings was easy distinct and no accessory mark almost. This experiment usied computer technology and according to the principle of back-calculation. The growth performance of S. kozlov (50samples collected from upstream of Wujiang River) were been preliminary researched through mathematical statistical method.The relation equation between body length and scale radius is L=27.009676R+107.260658(r=0.959147, N=50) and the relation equation between body length and body weight is W=0.00002L29657(r=0.9719, N=50). Individuals below3years old grow quicker.3. In order to understand and compare the genetic characteristics, muscle nutritional components in5. graham (26samples) and S. kozlovi (25samples) were been measured through the gas chromatograph-mass spectrometer(GS-MS), automatic amino acid analyzer and conventional method. The content of protein, fat and ash is15.49%,1.03%and1.06%, respectively. Unsaturated fatty acid content takes up71.72%of total fatty acid. Total contents of18amino acids are75.20%(dry weight accounted for muscles) in which essential amino acids contents are31.95%, delicious amino acids contents are30.03%, each taking up42.51%and39.97%of total amino acids contents, respectively. No significant differences in muscle composition compare with S. grahami.4. Polyacrylamide gel electrophoresis is used to analyze five isozymes (lactic dehydrogenase-LDH, malic dehydrogenase-MDH, glutamic dehydrase-GDH, alcohol degydrase-ADH and esterase-EST) from nine tissues (eyeball, muscle, heart, gonad, liver, gill, kidney and brain). Isozymes loci and zymogram are also analyzed. The results indicate that five isozymes from nine tissues have apparent tissue specificity in S. kozlovi.5. Chromosome karyotype was preliminary researched on S. kozlovi through injected phytohemagglutinin (PHA) The chromosome number is2n=96and karyotype formula is2n=36m+16sm+10st+34t,NF=148. Heteromorphic sex chromosomes, satellite chromosome and secondary constriction were not been found. 7. This experiment adopted the PCR amplification and direct sequencing method. The genetic structure and phylogenetic relationship of four wild populations (population of the Wujiang River upstream, population of the Hengjiang River upstream, population of the Yalong River, population of the Jinsha River) were examined according to mitochondrial DNA (mtDNA) control region. After being aligned,834bp sequences of mtDNA control region were acquired and revealed84variable sites in92samples, accounting for10.07%of the total number of sites. Among these polymorphic sites,17were singleton variable sites and67were parismony-informative sites. On average, base composition of all samples was T=33.4%, C=19.7%, A=33.1%, and G=13.7%. The mtDNA control region was an A+T-rich region of the mitochondrial genome. A total of32haplotypes were identified based on nucleotide variability. The total nucleotide diversity (%) and haplotype diversity (h) were0.951and0.02244, respectively. Haplotype and nucleotide diversities varied across the four populations. The geographic populations showed different genetic structure due to the influence of geographical isolation. Based on the Kimura2-parameter model, the overall average genetic distance among individuals was0.023. The overall mean intra-population genetic distance of the Yalong River Population (0.030) was the largest in the four populations, and that of Yangjie River Population (0.004) was the smallest. The neighbour-joining tree and the median-joining network which were constructed based on all haplotypes showed that the haplotypes from the Yalong and Panzhihua populations were promiscuously distributed, the Yangjiehe and Zongjihe populations were distributed similar. The Yalong population had a relatively distant relationship with the Panzhihua populations. The AMOVA revealed that most molecular variance was observed to occur among populations (50.16%), whereas variance within populations (49.84%) was relatively small.7. Pairwise Fsts demonstrated significant divergence between any two populations except between the Yalong and Panzhihua populations. Significant divergence across all samples was also observed, indicating a high level of geographical population structure. Gene flow (Nm) estimates among populations showed there were very limited genetic exchanges between any two populations except for the Yalong and Panzhihua populations. It appears that the level of divergence among populations (excluding Yalong and Panzhihua) was very high, and isolation has occurred among them. For the Yalong and Panzhihua populations, they had a low genetic distance (0.027), high gene flow (28.72), and no significant Fst (P>0.05), indicating a low level of divergence between them. The mismatch distributions, Tajima’s D and Fu’s Fs statistics showed that as a whole, Schizothorax kozlovi had not passed through a bottleneck, however, the Yanjiehe population had suffered a strong genetic drift might be related to a bottleneck or founder effect.更多还原