摘 要

圆尾斗鱼（Macropodus ocellatus）广泛存在分布在中国长江流域北部的池塘，沟渠，稻田中。圆尾斗鱼虽适应力强但栖息于极易受人类活动影响的生境，致使种群规模迅速减小，很多原来盛产的地区现已绝迹。但人们对于圆尾斗鱼的保护意识不强使得野生的圆尾斗鱼种群方面的研究过少导致无法对圆尾斗鱼的保护和利用提供理论上的支持。在这项研究中，利用线粒体细胞色素b基因序列的比较分析，用非损伤采样法调查来自该物种的分布范围内的东营、新乡、淮安、镇江、无锡、黄石、铜陵7个地区的87个个体的群体遗传多样性。在1135个碱基对长序列中发现了30个可变位点，在遗传多样性指数中单倍型指数和核苷酸多样性指数在总群中分别为0.899和0.00506；分子方差的分层分析（AMOVA）表明，总变异的34.89％起源于种群内的变异，65.11％来自种群间的变异；成对遗传分化（Fst）从0.12566变化到0.95765和基因流（Nm）0.02211至3.47899，显示圆尾斗鱼种群之间的高水平遗传差异。通过进化分析表明来自不同群体的细胞色素b单倍型混合在一起，这些发现可能有助于圆尾斗鱼的保护和利用。

关键词：圆尾斗鱼；CytB基因；地理种群；遗传变异

Genetic variation and population differentiation of the Macropodus ocellatus fish based on CytB sequence

Abstract

Macropodus ocellatus is widely found in ponds, ditches, and rice fields in the northern part of the Yangtze River valley in China. Although Macropodus ocellatus is highly adaptable but inhabits habitats that are highly susceptible to human activities, the population size is rapidly reduced, and many of the previously abundant areas are now extinct. However, people's awareness of the protection of the round-tailed fish is not strong, and the research on the wild population of the round-tailed fish is too small, which makes it impossible to provide theoretical support for the protection and utilization of the fish. In this study, a comparative analysis of mitochondrial cytochrome b gene sequences was used to investigate 87 regions from 7 regions of Dongying, Xinxiang, Huai'an, Zhenjiang, Wuxi, Huangshi and Tongling within the distribution range of the species using non-invasive sampling method. Individual population genetic diversity. Thirty variable sites were found in the 1135 base pair long sequence. In the genetic diversity index, the haplotype index and nucleotide diversity index were 0.899 and 0.00506 in the total population, respectively; Analysis (AMOVA) showed that 34.89% of the total variation originated from intra-population variation, 65.11% from inter-population variation; pairwise genetic differentiation (Fst) changed from 0.12566 to 0.95765 and gene flow (Nm) 0.02211 to 3.47899, showing a circle High levels of genetic differences between tailed fish populations. Evolutionary analysis showed that cytochrome b haplotypes from different populations were mixed together and these findings may contribute to the conservation and utilization of the fish.

Key words: round-tailed fighting fish; CytB gene; geographical population; genetic variation

目 录

1.前言………………………………………………………………1

1.1圆尾斗鱼的介绍………………………………………………………………1

1.1.1圆尾斗鱼的生物学特征………………………………………………………………1

1.1.2圆尾斗鱼生态学及观赏价值………………………………………………………2

1.2遗传多样性保护…………………………………………………………3

1.2.1保护遗传学………………………………………………………3

1.2.2遗传多样性保护的意义和目的………………………………………………………3

1.2.3遗传多样性保护的调查和检测方法………………………………………………3

1.3线粒体DNA介绍………………………………………………………………4

1.3.1鱼类线粒体DNA生物学特性……………………………………………………4

1.3.2线粒体DNA在鱼类检测的应用……………………………………………………5

1.4研究目的和意义………………………………………………………………6

2. 实验材料和方法………………………………………………………………8

2.1实验材料……………………………………………………………… 8

2.1.1圆尾斗鱼的样本采集………………………………………………………………8

2.1.2主要试剂………………………………………………………………8

2.1.3主要仪器………………………………………………………………8

2.2实验方法………………………………………………………………9

2.2.1DNA提取………………………………………………………………9

2.2.2PCR的扩增………………………………………………………………9

2.2.3DNA的纯化回收………………………………………………………………10

2.2.4序列分析………………………………………………………………11

3结果分析和讨论………………………………………………………………12

3.1结果分析………………………………………………………………12

3.1.1序列特征分析………………………………………………………………12

3.1.2遗传多样性分析……………………………………………12

3.1.3种群遗传分化分析………………………………………………………………13

3.1.4单倍型系统进化分析………………………………………………………………13

3.2小结………………………………………………………………14

3.2.1圆尾斗鱼的遗传多样性………………………………………………………14

3.2.2圆尾斗鱼的种群遗传结构及分化……………………………………………………15

4结论………………………………………………………………16

致谢……………………………………………………………17

参考文献………………………………………………………18

1 前言

1.1圆尾斗鱼介绍

1.1.1圆尾斗鱼的生物学特征

圆尾斗鱼（Macropodus ocellatus）属斗鱼属Macropodus，斗鱼属鱼类是具有很高观赏价值的小型淡水鱼类,隶属于鲈形目Percifomes,攀鲈亚目Anabantoidei,斗鱼科Belontidae,斗鱼亚科Macropodinal^[1]。圆尾斗鱼广泛分布于中国长江流域北部的池塘，沟渠，稻田，Wang等认为圆尾斗鱼最初由西朝鲜半岛开始扩散到长江以北^{[1, 2]}。

而鱼类的鱼鳍、鱼鳞等生物学特征在鱼类分类上有极其重要的意义，是使该鱼种与其他相似鱼种区分的重要指标。圆尾斗鱼鳍有：臀鳍X V-X X-7-15, 背鳍XI-XIX-5-9，胸鳍鳍条9-12。而鱼鳞有：侧线鳞26到31,尾柄周围鳞片15到17。而脊椎骨27到29, 第一鳃弓上的角鳃骨上有6到13个鳃耙。鱼鳍有：背鳍后部有4至5根分支鳍条延伸至尾鳍后部。肛门鳍与背鳍的起始点相对并具有相似的形状。而鰓盖的后角靠后有尾部呈弧形的长条形胸鳍。胸部位置的腹鳍是倾斜的，第一鳍是硬鳍，第二和第三鳍是细长的节段性软鳍。尾鳍为扇形，红色且散布亮蓝色小点。体色棕褐色，有一横带由纵贯眼部直至鳃盖上的斑块前，争斗和繁殖期身体前部出现暗纹后部近黑色，奇鳍外缘呈亮蓝色，雄鱼尤其明显。圆尾斗鱼的染色体组型为2n=46,核型为4m 4sm 38t。圆尾斗鱼以尾鳍近圆形、头部被圆鳞和鳃杷数少等形态特征与斗鱼属其他种类截然区分^[1]。