【作者】 朱树人；

【导师】 王群； 李家乐；

【作者基本信息】 华东师范大学 ， 水生生物学， 2014， 博士

【摘要】 乌鳢(Channa argus)是我国重要的淡水经济鱼类之一,但在养殖过程中需要投喂冰鲜鱼类,会导致水质恶化。杂交鳢(斑鳢♀×乌鳢♂)可以全程投喂人工饲料养殖,极大减少了对养殖环境的危害,并且在其生长、抗病等方面优于亲本,已在生产上得到广泛推广。本研究利用部分COI、16S及全部ITS序列进行了研究,为两者杂交提供了分子依据,并且测序获得了斑鳢、乌鳢及杂交鳢(斑鳢♀×乌鳢♂)的线粒体DNA全序列,用SRAP分子标记技术研究了斑鳢、乌鳢及杂交鳢(斑鳢♀×乌鳢♂)遗传关系,用SSR技术对杂交鳢F2及其亲本杂交鳢Fl的遗传信息传递进行初探,并分析了5个乌鳢养殖群体的遗传多样性。具体结果如下：1.斑鳢和乌鳢杂交的分子依据本研究对4种鳢属鱼类的COX1基因和16S基因部分序列进行了分析,发现与其它鱼类研究一样,COX1和16S基因在4种鳢属鱼类中高度保守,种内变异低,种间有明显的不同。种间差异明显大于种内差异。4种鱼类COX1共获得19个单倍型,每个单倍型所获得的COX1序列为652bp,没有发现插入或缺失现象。在K2P模型下,最远的种间遗传距离发生在乌鳢和线鳢之间(0.219),最近的种间距离发生在乌鳢和斑鳢之间(0.091)。4种鱼类16S基因发现有10个单倍型,乌鳢、斑鳢和月鳢单倍型长度为573bp,线鳢单倍型长度为572bp.在K2P模型下,最近的遗传距离发生在乌鳢和斑鳢之间(0.021),最远的遗传距离发生在乌鳢与线鳢之间(0.077)、斑鳢和线鳢之间(0.077)。种内和种间没有重叠,表示COX1基因和16S基因都可以作为条形码鉴定4种鱼类。基于COX1和16S基因ML和NJ进化树显示乌鳢、斑鳢、月鳢和线鳢的单倍型分别聚在一起,然后乌鳢和斑鳢聚在一起,再与其它鱼类聚在一起。这表示乌鳢和斑鳢之间遗传距离最小,亲缘关系最近。将所得到的ITS1和ITS2碱基序列进行拼接,得到斑鳢和乌鳢ITS序列长度分别为927bp和902bp。两者ITS的G+C含量高于A+T,占72%左右。两者在ITS有明显的差异性片段(特别是在ITS-2中斑鳢明显多出30bp大小的片段)。乌鳢和斑鳢种内差异较小,两者之间核苷酸差异明显大于种内。采用NJ和ML两种方法建立进化树,两者同时表明乌鳢和斑鳢的遗传距离较近(约0.020),亲缘关系最近,而与同属的月鳢遗传距离相对较远(约为0.073)。COX1、16S以及ITS序列都显示乌鳢和斑鳢之间序列差异最小,乌鳢和斑鳢的遗传距离非常近,两者是最晚从鳢属鱼类中分化出来的,这为乌鳢与斑鳢比较容易杂交提供了分子依据。2.斑鳢、乌鳢及杂交鳢(斑鳢早×乌鳢♂)线粒体DNA全长经过测序拼接,乌鳢、斑鳢及杂交鳢的线粒体全长分别为16558bp,16559bp和16558bp。这三种鱼符合经典脊椎动物线粒体的特征,包含13个蛋白编码蛋白,2个rRNA,22个转运RNA,和一个假定的控制区域。在编码氨基酸的第三个位置没有明显的偏好性。在乌鳢PCG中,CUC (193)是最常见的密码子,而在斑鳢及杂交鳢中CUA(204)是使用最多的密码子。在这三种鳢科鱼类中,亮氨酸是使用最频繁的氨基酸,乌鳢(17.9%)、斑鳢(17.5%)及杂交鳢(17.5%)。COX1起始密码子为GTG,其它12种PCG基因用ATG作为起始密码子。ATP6和ATP8基因有10bp相互重叠,ND4L-ND4, ND5-ND6和ATP6-COX3之间分别有7bp,4bp和1bp的重叠。ND5在乌鳢中的终止密码子是TAG,在斑鳢及杂交鳢线粒体基因组中终止密码子是TAA。ND1、ND2、COX1、ATP6、ATP8、COX3和ND4L终止密码子是TAA, ND6终止密码子为TAG,其余基因(COX2、 ND4和CYTB)有不完整的终止密码子T。ML和NJ聚类结果同样表明,在鲈形目中同一个科的鱼类先聚在一起,然后再与其它邻近科聚在一起。同样,杂交鳢因为与斑鳢母性遗传的原因先聚在一起,再与乌鳢聚在一起,最后与其它鲈形目鱼类聚在一起。3种鳢属鱼类为单源性分支,而鲈形目其它科鱼类聚在一起,本研究可以给鳢科鱼类在硬骨鱼类中的分类地位提供一定的基础资料。3.斑鳢、乌鳢及杂交鳢的SRAP分析选取14对SRAP引物对斑鳢、乌鳢及杂交种F1扩增,在3个群体中共扩增出105个位点,每个组合引物扩增条带从5到11不等。其中有88个位点具有多态性,多态性比率为83.81%,在乌鳢中多态性位点有48个,多态位点比例为45.71%,在斑鳢中多态性位点有27个,多态位点比例为25.71%,在杂交F1中多态性位点有34个,多态位点比例为32.38%。在105个扩增位点中检测到有17个为乌鳢、斑鳢及杂交F1共有位点。斑鳢、乌鳢及杂种F1的群体内Nei’s遗传多样性指数(H)为0.0964、0.1489和0.1076。遗传距离结果表明杂交鳢与父本(0.2195)或母本(0.4009)的遗传距离要比其父母本(0.6367)之间的遗传距离近,这表明杂交鳢F1是父母本杂交所产生的后代,杂交鳢F1遗传距离更偏向乌鳢,与乌鳢的亲缘关系更近。4.杂交F2与亲本F1的遗传信息传递初探采用10个SSR位点对3组Fl亲本及F2子代30个体进行了研究,发现有7个位点(WL-28、CHA13、CHA25、CHA31、CHA41、CarC7和CarD139)的遗传符合孟德尔定律,子代分别遗传了父母本的信息,而在其它3个位点(WL-8、CarD108和CarD121)中,发现有相当数量的子代个体没有基因型,没有遗传父母本相关的遗传信息。3个位点在3个重复组合90个个体中有近一半的个体没有发现基因型,这说明这3个位点所在的1条或几条染色体在近一半个体中不存在。这表明Fl亲本在进行有性繁殖时有1条或几条染色体虽然不能配对,但在其它同源染色体相互分离移向两极时可能随机进入其中一个细胞,进行第二次减数分裂。本研究丰富了鱼类有性繁殖理论的内容。5.5个养殖乌鳢群体的遗传多样性采用10个SSR位点对5个养殖乌鳢群体(LQ、YJ、YH、XS、YG)的遗传多样性进行了分析。10个位点共发现有160个等位基因,平均期望杂合度(He)和表观杂合度(Ho)分别为0.65-0.91和0.67-0.85,说明5个养殖群体具有较高的遗传多样性,YJ群体遗传多样性最高。经Hardy-Weinberg平衡分析,在YJ、YG和LQ群体中都发现有个别位点偏离平衡。YJ群体偏离了突变-漂移平衡,近期可能经历了遗传瓶颈。为更好地育种策略,建议补充YJ群体有效个体数目。除XS与YH群体间没有发现分化,LQ群体与其它群体之间出现中等程度分化(0.05<FST<0.15),其它群体间均由轻微程度分化。XS群体与YH群体之间没有发现分化,两者之间遗传结构相似,为同一个群体分离出来。LQ群体与YH群体遗传距离最远,可以作为种群间杂交的一个良好的育种策略。更多还原

【Abstract】 Channa argus is an important economic freshwater fish. During the breeding process, it needs to be fed chilled fish, resulting in deterioration of water quality. The hybrids (C. maculata(?) and C. argus(?)) can be fed with artificial feed, which greatly reduces the harm to the environment farming. The hybrids also obtained excellent traits from its parents, which has been promoted in the production. We studied the taxonomic status of C. maculata and C. argus by part sequence of COX1gene and16S gene and full length sequence of ITS, which could be provided for the molecule basis for hybrid. And we also sequenced the whole mtDNA length for C. maculata, C. argus and the hybrid (C. maculata (?) and C. argus (?)).-By the technology of SRAP to study the genetic raltionships of C. maculata, C. argus and the hybrid (C. maculata (?) and C. argus (?)), and by the technology of SSR to research the transfer of genetic information from F1parents to F2generations. Finally, we analysised the gentic diversity for five cultured population of C. argus. The results are summarized as follows:1. The molecule basis for hybrid between C. maculata (?) and C. argus (?)Four kinds of snakehead fish were analyzed using part gene of COX1and16S. High conservative in four kinds of snakehead fish, low intraspecific variation and large differences between species were detected. The interspecific K2P distances were higher than intraspecific distances. A total of19haplotypes were identified in COX1gene in this study. And each haplotype is652bp. No insertions or deletions phenomenon were founded in this study. Under the model of K2P, the lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. A total of10haplotypes were obtained in16S gene in this study. The length sequence of haplotypes of C. argus, C. maculata, C. asiatica and C. striata is573bp,573bp,573bp and572bp. Under the model of K2P, the highest interspecific distance (0.077) was between C. argus and C. striata, between C. maculata and C. striata, while the lowest interspecific distance (0.021) was between C. argus and C. maculata. No overlap was found between intraspecific and interspecific distances, suggesting the existence of a distinct barcoding gap. The ML and NJ tree show all the snakehead haplotypes belong to one of the four Channa species. First, C. argus was clustered with C. maculata, then were clustered with other fish. It indicates that C. argus and C. maculata had the lowest genetic distance and the closest relationship.The length of the rDNA sequences of C. maculata and C. argus were927bp and902bp. The content of G+C (about72%) is higher than A+T. Distinct differences fragments were found between C. argus and C. maculata(particularly30bp more in ITS2of C. maculata). The interspecific K2P distances were higher than intraspecific distances. Phylogenetic analysis showed that C. argus and C. maculata (0.020) had the lowest genetic distance, while C. argus and C. asiatica (0.073) had the highest genetic distance.Minimal sequence differences and lowest distant were observed in C. argus and C. maculata. And these two fishes were latest differentiation from other snakehead fishes. These provides the molecular basis between C. argus and C. maculata.2. The complete mitochondrial genome of C. maculata, C. argus and hybridsnakehead fish [C. maculata ((?))XC. argus ((?))]The total length of the C. maculata mitochondrial DNA was16559bp, which was slightly longer than C. argus (16558bp) and hybrid snakehead fish (16558bp). The structural organization and location of different feature in the snakehead mt genomes conformed to the common vertebrate mt genome model and consisted of13protein-coding genes,2rRNAs,22tRNAs and1putative control region. There was no obviously favor base pair in third position of encoding amino acids. The CUC (193) codon was used most frequently in C. argus while the CUA (204) codon was used most frequently in C. maculata and hybrid snakehead fish. The amino acids Leu is the most frequently used encodon in C. argus (17.9%), C. maculata (17.5%) and hybrid snakehead fish (17.5%). COXl started with GTG while other PCGs genes used ATG as the initiation codon. The ATP6and ATP8shared10bp overlap, whereas ATP6-COX3and ND4L-ND4shared1and7bp overlap, respectively. TAG was the termination codon in C. argus while TAA was the termination codon in C. maculata and hybrid snakehead fish for ND5. Seven PCGs ended with TAA (ND1, ND2, COXl, ATP6, ATP8, COX3, and ND4L), one ended with TAG (ND6) and the rest had incomplete stop codon T (COX2, ND3, ND4and CYTB). This feature is common among vertebrate mitochondrial PCGs, of which incomplete stop codons are presumably completed as TAA via posttranscriptional polyadenylation.C. argus and C. maculata, as the representative of the suborder Channoidei, formed the most basal branch having a sister relationship with a monophyletic clade including the other suborders of Perciformes. These could provide some basic information for fish taxonomy.3. The SRAP analysis of C. maculata, C. argus and their hybrid [C. maculata ((?))×C. argus ((?))]The genetic structure of C. argus, C. maculata and their hybrid [C. maculata ((?))×C. argus ((?))], the heterosis of the hybrid F1, were analyzed by the SRAP technique with14primers.105loci were amplified in three population, and each combination of primers amplified bands ranging from5to10. Out of105loci,88loci occurred polymorphism band in three population, accouted for83.81%,48loci (45.71%) occurred polymorphism band in C. argus,27loci (25.71%) occurred polymorphism band in C.maculata,34loci (32.38%) occurred polymorphism band in hybrids. The H for C. argus, C. maculata and hybrids were0.1489,0.0964,0.1076, respectively. The results showed that the genetic distance of hybrids and male parent (0.2195) or female parent (0.4009) was closer than the genetic distance of male parent and female parent (0.6367), which indicated hybrids F1was the hybrid offspring produced by parents. It also indicated that the relative genetic distances were not equal (closer to male parent) between the hybrids and their parents.4. Transfer of genetic information from F1parents to F2generationsTransfer of genetic information from F1parents to F2generations were analyzed by10SSR loci in3group (90individuals). The inheritance of7loci (WL-28; CHA13; CHA25; CHA31; CHA41; CarC7; CarD139) was shown to be in agreement with Mendel’s law. However, nearly half F2offsprings had no genotypes by3loci (WL-8, CarD108and CarD121), which indicated one or several chromosomes do not exist in nearly half individuals. Combined with the number of F2offspring chromosomes44and45, indicating that there is one or several chromosomes can not pair during meiosis. During the first meiotic division, the one or several chromosomes may randomly into one cell. It then conducted the second meiotic division. In this study, the research enriched the contents of the theory of sexual reproduction of fish.5. Genetic diversity of five cultured C. argus populationA total of160alleles were detected at the ten microsatellite loci. The average heterozygosity (HE) and observed heterozygosity (Ho) varied from0.65-0.91and0.67-0.85in five cultured populations, indicating high genetic diversity in these five populations. Among them, the highest genetic diversity was observed in YJ population. Some loci in YJ, YG, LQ population deviated from H-W. Population YJ deviated from mutation-drift equilibrium and may have experienced a recent bottleneck. It is necessary to increase effective population sizes in YJ population for better breeding. No differentiation and some gentic structure between XS population and YH population, which indicated the two population were the some population. There is moderate differentiation between LQ population and other poplation. The farest genetic distance was observed between LQ population and YH population, which could be served as a good breeding strategy.更多还原