长薄鳅（Leptobotia elongata）野生资源分布及其种群遗传多样性研究
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摘要
长薄鳅(Leptobotia elongate Bleeker)是中国特有鱼种,著名的观赏淡水鱼,主要分布在长江上游的干流及其支流。采用渔民调访、拖网调查和产卵江段的规模估算等方法,调查了长薄鳅在长江干流上游及其支流的分布和数量,结果表明我国长薄鳅资源在长江干流及其支流正在快速衰竭,走向濒危。
为了分析长薄鳅野生种群结构并评估其遗传多样性,本研究测定了5个长薄鳅种群共110个样本的线粒体控制区(D-loop)序列(835bp),并进行比较分析,这些序列中,总共有49个变异位点,45个单倍型,单倍型多样性(h)和核苷酸多样性(π)分别是0.952和0.00454；样本间的平均遗传距离是0.0046±0.0010,群内的遗传距离和群间的遗传距离的变化分别是0.0033±0.0011至0.0050±0.0012和0.0037±0.0.0011至0.0050±0.001。Tajima's D中性检验(-1.86383,P<0.01)和Fu'sFs检验(-25.92536,P<0.01)以及错配分布分析都表明,长薄鳅在最近历史上经历过种群扩张或瓶颈效应。分子变异分析(AMOVA)发现,绝大多数的变异来自群内个体间(98.3%),群间的变异很少(1.7%)。分化系数和基因流分析结果表明,种群间的分化系数较低(Fst=0.01702,P>0.05)而有高的基因流水平(Nm=28.88),提示长薄鳅种群分化水平较低,没有显著的地理种群结构。这些结果与长薄鳅属产漂流性卵的鱼类,以及其个体频繁地溯江上游相一致。同时,长薄鳅的种群遗传结构表明,分布在长江上游的这些种群应该被当作一个遗传单元进行管理和保护。
本研究通过设计保守引物、使用高保真PCR反应和引物步移测序法测序了长薄鳅全线粒体基因组序列。长薄鳅线粒体基因组全序列有16591个碱基,其中包含13个蛋白编码基因、两个核糖体RNA (ribosomal RNA, rRNA)基因、22个转运RNA (transfer RNA, tRNA)基因和一个控制区,其基因组成与大部分脊椎动物线粒体完全一样。轻链复制起点(origin of L-strand replication, OL)有30个碱基,位于一个由5个tRNA基因串联组成区(WANCY区)。控制区有925个碱基,位于tRNA-Pro和tRNA-Phe基因之间。基于长薄鳅线粒体重链上12个蛋白编码基因的序列,连同12个其它鳅科鱼类线粒体重链上12个蛋白编码基因的序列,使用贝叶斯(Bayesian)和最大简约法(maximum parsimony, MP)分析了长薄鳅的进化关系,结果表明,整个进化树分成两枝,即Acantopsis、Pangio、Cobitis、Niwaella、Koreocobitis、Misgurnus属聚为一枝,共同构成鳅亚科(Cobitinae); Chromobotia和(?)Leptobotia属聚为另一枝,共同构成沙鳅亚科(Botiinae)。
长薄鳅绝对怀卵量为6160-47239粒,平均绝对怀卵量为27511粒；相对生殖力为每克体重26.6-48.8粒,平均为37.7粒。采用LRH-A2和脑垂体混合使用能有效促使长薄鳅的排卵,催产率可达75%-81.8%,受精率和孵化率分别高达82%-83%和87%-89%。
Elongate loach (Leptobotia elongate Bleeker), an endemic fish species to China, is a famous ornamental freshwater fish, which disturbuted in the upper reaches of the Yangtze River of China. The resource of Elongate loach in Yangtze River was investigated by visiting fishermen, trawling, and so on. The results showed that the Elongate loach resources in Yangtze River has rapidly decreased and at the brink of extinction.
To analyze wild population structure and evaluate genetic diversity of the famous ornamental fresh water fish, elongate loach (Leptobotia elongata (Bleeker)), a comparative analysis using partial mtDNA control region (D-loop)(835bp) sequences was performed for110individuals from five localities in upper reaches of the Yangtze River, China. A total of49polymorphic sites and45haplotypes were detected, and the haplotype diversity (h) and nucleotide diversity (π) were0.952and0.00454, respectively. The overall mean genetic distance among individuals was0.0046±0.0010. The genetic distance of intra-group ranged from0.0033±.0011to0.0050±0.0012, and that between pairwise groups ranged from0.0037±0.0.0011to0.0050±0.0012. The neutral test of Tajima's D (-1.86383, P<0.01) and Fu's Fs (-25.92536, P<0.01), together with uni-modal mismatch distribution, indicated a recent genetic bottleneck or population expansion of the species in history. Analysis of molecular variance (AMOVA) revealed that small variances occurred among groups (1.7%), while most variances occurred within groups (98.3%). Also, there were no significant population structure (Fst=0.01702, P>0.05), and extremely high gene flows among groups (Nm=28.88), suggesting low genetic divergence in the species. It should be attribute to that the fish spawn drift eggs and the individuals migrate downstream or upstream in its distribution ranges frequently. Thus, the gene flows could frequently occur among these groups. These groups of Elongate loach distributed in upper reaches of the Yangtze River should be considered as a whole unit for management.
The complete mitochondrial genome of Elongate loach was determined using pairs of conservation primers, high-fidelity PCR reaction and primers walking sequencing, which was16591bp in length. It contained13protein-coding genes, two ribosomal RNA (rRNA) genes,22transfer RNA (tRNA) genes and one control region, with an identical sequence order to that of most vertebrates.The origin of L-strand replication (OL) in Elongate loach mitochondrion was located in a cluster of five tRNA genes (WANCY region) with30nucleotides in length. The control region was925nucleotides in length,and was located between tRNA-Pro and tRNA-Phe genes. Based on the12protein-coding genes on the heavy strand of Elongate loach and other cobitidae species, the phylogenetic relationship analysis among13cobitidae species was performed by Bayesian and maximum parsimony methods. The result suggested that phylogenetic tree was divided into two branchs. Acantopsis, Pangio, Cobitis, Niwaella, Koreocobitis and Misgurnus clustered into a sub-family (cobitinae), Chromobotia and Leptobotia clustered into another sub-family (botiinae).
The absolute fecundity for Elongate loach was between6160and47239, with the average of27511. The relative fecundity was between26.6and48.8particles per gram of body weight, with the average of37.7. The mixture of LRH-A2and pituitary were used to promote Elongate loach spawning, and spawing rate was75%-81.8%. The fertilization rate and hatching rate were up to82%-83%and87%-98%, respectively.
为了分析长薄鳅野生种群结构并评估其遗传多样性,本研究测定了5个长薄鳅种群共110个样本的线粒体控制区(D-loop)序列(835bp),并进行比较分析,这些序列中,总共有49个变异位点,45个单倍型,单倍型多样性(h)和核苷酸多样性(π)分别是0.952和0.00454；样本间的平均遗传距离是0.0046±0.0010,群内的遗传距离和群间的遗传距离的变化分别是0.0033±0.0011至0.0050±0.0012和0.0037±0.0.0011至0.0050±0.001。Tajima's D中性检验(-1.86383,P<0.01)和Fu'sFs检验(-25.92536,P<0.01)以及错配分布分析都表明,长薄鳅在最近历史上经历过种群扩张或瓶颈效应。分子变异分析(AMOVA)发现,绝大多数的变异来自群内个体间(98.3%),群间的变异很少(1.7%)。分化系数和基因流分析结果表明,种群间的分化系数较低(Fst=0.01702,P>0.05)而有高的基因流水平(Nm=28.88),提示长薄鳅种群分化水平较低,没有显著的地理种群结构。这些结果与长薄鳅属产漂流性卵的鱼类,以及其个体频繁地溯江上游相一致。同时,长薄鳅的种群遗传结构表明,分布在长江上游的这些种群应该被当作一个遗传单元进行管理和保护。
本研究通过设计保守引物、使用高保真PCR反应和引物步移测序法测序了长薄鳅全线粒体基因组序列。长薄鳅线粒体基因组全序列有16591个碱基,其中包含13个蛋白编码基因、两个核糖体RNA (ribosomal RNA, rRNA)基因、22个转运RNA (transfer RNA, tRNA)基因和一个控制区,其基因组成与大部分脊椎动物线粒体完全一样。轻链复制起点(origin of L-strand replication, OL)有30个碱基,位于一个由5个tRNA基因串联组成区(WANCY区)。控制区有925个碱基,位于tRNA-Pro和tRNA-Phe基因之间。基于长薄鳅线粒体重链上12个蛋白编码基因的序列,连同12个其它鳅科鱼类线粒体重链上12个蛋白编码基因的序列,使用贝叶斯(Bayesian)和最大简约法(maximum parsimony, MP)分析了长薄鳅的进化关系,结果表明,整个进化树分成两枝,即Acantopsis、Pangio、Cobitis、Niwaella、Koreocobitis、Misgurnus属聚为一枝,共同构成鳅亚科(Cobitinae); Chromobotia和(?)Leptobotia属聚为另一枝,共同构成沙鳅亚科(Botiinae)。
长薄鳅绝对怀卵量为6160-47239粒,平均绝对怀卵量为27511粒；相对生殖力为每克体重26.6-48.8粒,平均为37.7粒。采用LRH-A2和脑垂体混合使用能有效促使长薄鳅的排卵,催产率可达75%-81.8%,受精率和孵化率分别高达82%-83%和87%-89%。
Elongate loach (Leptobotia elongate Bleeker), an endemic fish species to China, is a famous ornamental freshwater fish, which disturbuted in the upper reaches of the Yangtze River of China. The resource of Elongate loach in Yangtze River was investigated by visiting fishermen, trawling, and so on. The results showed that the Elongate loach resources in Yangtze River has rapidly decreased and at the brink of extinction.
To analyze wild population structure and evaluate genetic diversity of the famous ornamental fresh water fish, elongate loach (Leptobotia elongata (Bleeker)), a comparative analysis using partial mtDNA control region (D-loop)(835bp) sequences was performed for110individuals from five localities in upper reaches of the Yangtze River, China. A total of49polymorphic sites and45haplotypes were detected, and the haplotype diversity (h) and nucleotide diversity (π) were0.952and0.00454, respectively. The overall mean genetic distance among individuals was0.0046±0.0010. The genetic distance of intra-group ranged from0.0033±.0011to0.0050±0.0012, and that between pairwise groups ranged from0.0037±0.0.0011to0.0050±0.0012. The neutral test of Tajima's D (-1.86383, P<0.01) and Fu's Fs (-25.92536, P<0.01), together with uni-modal mismatch distribution, indicated a recent genetic bottleneck or population expansion of the species in history. Analysis of molecular variance (AMOVA) revealed that small variances occurred among groups (1.7%), while most variances occurred within groups (98.3%). Also, there were no significant population structure (Fst=0.01702, P>0.05), and extremely high gene flows among groups (Nm=28.88), suggesting low genetic divergence in the species. It should be attribute to that the fish spawn drift eggs and the individuals migrate downstream or upstream in its distribution ranges frequently. Thus, the gene flows could frequently occur among these groups. These groups of Elongate loach distributed in upper reaches of the Yangtze River should be considered as a whole unit for management.
The complete mitochondrial genome of Elongate loach was determined using pairs of conservation primers, high-fidelity PCR reaction and primers walking sequencing, which was16591bp in length. It contained13protein-coding genes, two ribosomal RNA (rRNA) genes,22transfer RNA (tRNA) genes and one control region, with an identical sequence order to that of most vertebrates.The origin of L-strand replication (OL) in Elongate loach mitochondrion was located in a cluster of five tRNA genes (WANCY region) with30nucleotides in length. The control region was925nucleotides in length,and was located between tRNA-Pro and tRNA-Phe genes. Based on the12protein-coding genes on the heavy strand of Elongate loach and other cobitidae species, the phylogenetic relationship analysis among13cobitidae species was performed by Bayesian and maximum parsimony methods. The result suggested that phylogenetic tree was divided into two branchs. Acantopsis, Pangio, Cobitis, Niwaella, Koreocobitis and Misgurnus clustered into a sub-family (cobitinae), Chromobotia and Leptobotia clustered into another sub-family (botiinae).
The absolute fecundity for Elongate loach was between6160and47239, with the average of27511. The relative fecundity was between26.6and48.8particles per gram of body weight, with the average of37.7. The mixture of LRH-A2and pituitary were used to promote Elongate loach spawning, and spawing rate was75%-81.8%. The fertilization rate and hatching rate were up to82%-83%and87%-98%, respectively.
引文
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