【作者】 肖俊；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2010， 博士

【摘要】 在物种进化过程中,多倍化在进化中的作用得到人们越来越多的重视。在脊椎动物中,鱼类的多倍化现象最为普遍。鲫鱼更是被认为是古老的四倍体鱼。鲫鱼(Carassius auratus L.)由于其适应能力、繁殖能力和抗病能力极强,分布很广,且在洞庭湖水域具有丰富的资源。我国的野生鲫鱼在很长时间内一直被认为是二倍体(2n=100)。但自二十世纪80年代以来,中国各省的野生鲫鱼中陆续报道有三倍体的存在。本研究以洞庭湖水域的野生鲫鱼为研究对象,对其倍性进行了大规模的检测,发现其中有二倍体、三倍体和四倍体三种不同倍性的野生鲫鱼存在,并对这三种倍性野生鲫鱼的多种生物学特性进行了比较研究,探讨它们可能的多倍化起源和进化方式。主要内容如下：1.通过DNA含量检测及染色体制片方法,从2005年起系统地检测了洞庭湖水域的野生鲫鱼的倍性及其倍性组成。检测结果表明：在洞庭湖水域的野生鲫鱼群体中,除了存在染色体数目为2n=100的二倍体野生鲫鱼外,还存在染色体数目为3n=150+的三倍体野生鲫鱼及少量染色体数目为4n=200的四倍体野生鲫鱼。三倍体野生鲫鱼的数量在群体中占大多数,并且比例有逐年上升的趋势；而四倍体野生鲫鱼在群体中的数量极少,并且只发现了雌性个体。其中,在中国野生鲫鱼群体中存在四倍体尚属首次报道。三种不同倍性的野生鲫鱼在洞庭湖水域中同时发现,对探讨鲫鱼多倍化和在染色体水平的遗传多样性具有重要意义。2.对洞庭湖水域三种不同倍性野生鲫鱼的外型及血细胞进行了比较研究,结果表明：洞庭湖水域野生鲫鱼在外型上与已报道各地三倍体鲫鱼有较大区别,但三种不同倍性野生鲫鱼彼此间在外型上并没有明显区别；三种不同倍性野生鲫鱼血细胞大小与倍性成正比,且能在三倍体和四倍体野生鲫鱼血细胞中观察到一定数量的无丝分裂的现象。3.使用了组织学切片、扫描电子显微镜和透射电子显微镜,对洞庭湖水域三种不同倍性野生鲫鱼性腺进行了系统的比较研究。卵巢组织学切片结果表明：三种不同倍性野生鲫鱼的卵巢都有正常的结构,能产生正常的卵子。用扫描电子显微镜观察二倍体野生鲫鱼和三倍体野生鲫鱼成熟精子结果表明：二倍体和三倍体野生鲫鱼精子都具有正常的头部和尾部,但头部大小随倍性增加而增大。透射电子显微镜观察二倍体和三倍体野生鲫鱼精巢结果表明：二倍体和三倍体野生鲫鱼精巢结构基本相似,但三倍体野生鲫鱼精子头部,观察到一定数量的空泡存在。对三种不同倍性野生鲫鱼的性腺研究表明：洞庭湖水域野生鲫鱼性腺发育均正常,能够形成正常的精子和卵子。以上结果为证明三倍体野生鲫鱼可育提供了直接的细胞学证据。4.使用一系列与鲫鱼亲缘关系远近不同的雄鱼(二倍体野生鲫鱼、三倍体野生鲫鱼、鲤鱼、团头鲂及紫外灭活的团头鲂精子)与三种不同倍性野生鲫鱼雌鱼进行繁殖实验,比较研究三种不同倍性野生鲫鱼的生殖方式,结果表明：三种不同倍性的野生鲫鱼有其各自不同的生殖方式。其中,二倍体野生鲫鱼行正常的两性生殖；四倍体野生鲫鱼只有雌性,但通过行天然雌核发育的生殖方式产生全雌后代来繁衍；而三倍体野生鲫鱼为中间类型,如遇同源的三倍体野生鲫鱼精子受精则行两性生殖的生殖方式,产生雌雄两种性别的后代。如遇亲缘关系较近的异源精子激活则行天然雌核发育生殖,产生全雌的后代。这个结果极大的丰富了我们对鲫鱼生殖方式的认识,该结果对今后野生鲫鱼的繁殖育种工作具有重要的指导意义。5.线粒体基因组具有完全母性遗传的特点,对三种野生鲫鱼的线粒体全序列测序并比较,结果表明：三种野生鲫鱼具有很近的亲缘关系,彼此间的遗传距离小于与红鲫的遗传距离,分化的时间很短。而通过Sox基因HMG框分子标记检测不同倍性野生鲫鱼,比较了它们的基因组在分子水平的差别,发现在三倍体野生鲫鱼与四倍体野生鲫鱼中存在异源片段。同时,使用只有在鲫属鱼类中才能扩增出的1900bp的Sox4基因建立系统树,发现二倍体野生鲫鱼与红鲫聚为一支,而三倍体和四倍体野生鲫鱼聚为一支。这两个分子生物学的证据表明：不同倍性的野生鲫鱼起源于同一母系,但其中三倍体和四倍体野生鲫鱼很可能不是二倍体野生鲫鱼直接加倍得到,而是通过二倍体野生鲫鱼与近缘种通过杂交得到。这从分子生物学水平说明高倍性野生鲫鱼的起源与进化,并探讨了使野生鲫鱼出现染色体加倍的可能原因。上述结论为探讨杂交在物种进化中起到的作用提供了非常有价值的信息。更多还原

【Abstract】 The importance of polyploidization is found enlarging in the evolution process and has gained more and more public attention. In fish species, polyploidization occurred more commonly than in other kinds of vertebrates. The crucian carps are regarded as ancient tetraploids. It has been one of the most important freshwater species for Chinese aquaculture and is especially abundant in the Dong-ting water system of Hunan province. Crucian carp used to be considered as all diploid forms (2n=100). However, triploid forms (3n=150+) have been reported in China since 1980s.In this study, the wild population of crucian carp living in the Dong-ting water system in Hunan province were sampled. After a large scale of detection was set up to monitor the ploidy diversity of the population, totally three different ploidy of crucian carps were found co-existent in the Dong-ting water system. The biological characteristics of the population were comparatively studied, leading to further consideration of the possible origin and probable evolutionary pathway of polyploid. The primary coverage includes:1. Since 2005, the ploidy diversity of the wild population of crucian carps in Dong-ting water system was systematically monitored by DNA content measurement and further by chromosome count on kidney tissue. Accordingly, besides the diploid forms (2n=100), there were a great amount of triploid forms (3n=150+) and a few of tetraploid forms (4n=200) found in the population. The triploids made up the majority of the population with an increasing proportion every year. Only a few tetraploids were detected, all of which were identified as all female. Most importantly, this is the first reports on the discovery of tetraploid forms in the wild crucian carp population. The discovery of coexistence of three different ploidy in the wild crucian carp population of Dong-ting water system was of great significance in discussing the genetic diversity of crucian carps at chromosome level, providing excellent material for study on the origin of polyploidization and fish evolution.2. Morphological analyses on the three different ploids of wild crucian carp population in Dong-ting water system and comparative investigation of their erythrocytes were employed. Accordingly, there was no obvious variety in their countable and measurable traits. The three different ploids with similar appearance could not be directly distinguished by their configuration. The erythrocyte nuclear volumes increased among diploids, triploids and tetraploids with the increasing ploidy. Erythrocytes with two nuclei resulting from amitosis were observed in triploids and tetraploids but not found in diploids.3. The gonads of the three ploids of common crucian carps in Dong-ting water system were systemically compared by tissue section, scanning electron microscope (SEM) and transmission electron microscope (TEM). The tissue section of the ovaries showed that the ovary structures of the three forms were normal with isochronous development of common crucian carps and could produce functional eggs. Under SEM observation, the mature spermatozoa of diploids and triploids had the normal external form, comprising by a head, a connecting piece, and a tail. With the increase of ploidy level, the mean head size of spermatozoa increased. Under TEM observation, all the mature spermatozoa in the testis of diploids and triploids had normal ultra-structures. However, in the testis of 3nCC there were certain a few of vacuoles in the head of the spermatozoa. According to these results, the three different forms in the wild crucian carp population possessed normally developed gonads which could produce functional spermatozoa and eggs, forcefully supporting the fertility of triploids at cytology level.4. Series of crossings between the females of three different ploidy of wild crucian carps and the males of some differently related species were set up to compare the reproductive patterns of the wild crucian carps. The results came back that the three different forms possessed specific reproductive patterns. The diploids were indentified as normal bisexual fertile. The all-female tetraploids could produce progenies through natural gynogenesis. The triploids possessed a complex pattern that they could be bisexual fertile when their eggs met homogenous spermatozoa of the male triploids or they reproduced all-female progenies through natural gynogenesis when their eggs were activated by related spermatozoa of other kinds.5. The complete mitochondrial genomes of the three different forms of common crucian carp were sequenced. Comparative analyses on the sequences showed that the relationship among the three different ploidy were very close, and the genetic distance within the population of common crucian carps was smaller than that between the common crucian carps and red crucian carps. Novel Sox-HMG markers were employed to compare the nuclear genomes of the three different ploidy forms at molecular level. Allogenetic DNA fragments were found in triploids and tetraploids. Moreover, the 1900-bp fragments of Sox4 gene which could be amplified only in species of the genus of C. were also obtained in common crucian carps. The phylogenetic tree that was structured by the sequences of these 1900-bp fragments were divided into two clades:one including the diploid common crucian carp and red crucian carp; and the other including the triploid and tetraploid forms of common crucian carp. The molecular evidences that were mentioned above directly proved that the different ploidy forms in the wild population of common crucian carp shared the original maternal parent. The triploid and tetraploid genomes probably not resulted from direct duplication of the diploid genome but from hybridization between diploid individuals and some other related species. Consequently, the possible origin and evolutionary pathway of polyploid common crucian carps were investigated at the molecular level. Considering the appearance time of the polyploid forms, human economic activity was further presumed as the main reason of genome duplication of the common crucian carps. The conclusion provides valuable information to study how hybridization contributes to animal evolution.更多还原