新型红鳙生物学特征及其遗传多样性研究
摘要
鳙鱼(Aristichthys nobilis)属鲤形目,鲤科,鲢亚科,鳙属。俗称大头鱼(bighead carp),是我国最主要的养殖鱼类之一。广泛分布于亚洲东部,我国各大水系均有此鱼。由于多年的人工繁殖,鳙鱼的种质资源大幅衰退,遗传多样性显著降低,对鳙鱼种质资源进行遗传改良和提纯复壮是一项紧迫的任务。新型红鳙是本实验室通过近十年的选育工作,从野生的鳙鱼种群中选育出一种体色鲜红,肉质鲜嫩,生长速度快的新类型鳙鱼,这是一种优良的养殖鱼种和很有的价值实验材料。为了保持其优良性状,通过异源雌核发育等生物技术手段对湘云红鳙群体进行种质纯化。本文对普通鳙鱼(BC)、红鳙(RBC)和雌核发育红鳙(GRBC)三个种群的生物学特征和遗传多样性进行了研究,主要内容如下：
1.三个鳙鱼群体的可数性状和可量性状均处于种质异动标准之内。通过测量代表鳙鱼生物学特征的13个度量性状,处理9项比值并进行T值检验。实验结果表明,红鳙和雌核发育的红鳙的各项性状比值基本一致,这表明了雌核发育红鳙稳定地遗传了红鳙的优良性状。普通鳙鱼和两种红鳙在头长／吻长、体长／体高、尾柄长／尾柄高等性状比值上有着显著差异,尤其以体长／体高最为显著,红鳙具有体宽、尾柄短等优良的性状。
2.对红鳙进行了遗传多样性的RAPD和微卫星分析(来自湘江流域的普通鳙鱼作为对照)。在优化RAPD检测条件基础上,从230个随机引物中筛选出来的45个扩增较好且多态性强的引物对这两个群体的DNA多态性及分子标记进行了分析。结果显示在45个随机引物的扩增谱带中找到了2个引物(S20、S46)的特异扩增谱带,可以作为这两个种群间的分子遗传标记。普通鳙鱼群体内个体之间的遗传距离范围从0.04-0.13,群体内的平均遗传距离为0.075,而红鳙群体内个体之间的遗传距离范围从0.02-0.35,群体内的平均遗传距离为0.139。这表明红鳙群体遗传多样性明显强于普通鳙鱼群体。从32对微卫星标记中筛选9对微卫星标记用于两个群体24个个体的扩增。平均每个微卫星位点在红鳙群体和普通鳙鱼群体中检测到的平均有效等位基因数分别为2.44和1.89。普通鳙鱼群体的平均杂合度期望值(0.34)和平均杂合度观测值(0.39)要比红鳙群体的平均杂合度期望值(0.51)和平均杂合度观测值(0.54)的小得多。由此可见,RAPD和微卫星这两种方法得出的结果是一致。
3.对红鳙和雌核红鳙的mtDNA基因组全序列进行测序,并对其基因组结构进行了分析。两种红鳙的mtDNA基因组序列完全相同,全长均为16619 bp,其碱基比例如下：%A=31.64,%G=15.94,%T=25.32,%C=27.10,%A+T=56.96。这也表明了线粒体严格的母性遗传。本文得到的红鳙的线粒体基因组全序列结果可以为红金鳙及其近缘种的演化、生物地理学研究,为检验鱼类形态学、生理学和进化学等特征提供分子依据。
After the years been artificial breeding, the bighead carp (Aristichthys nobilis) had substantial decline in germplasm resources and significant reduction of genetic diversity. Purification and rejuvenation of the bighead-carp germplasm is an urgent task. Red bighead carp is a new type of special bighead crap be selected and bred by our laboratory form wild bighead crap in recent years and those red bighead carp been named XiangYun Golden Bighead-carp. That bighead crap in red color has fast growth, is good breeding species and valuable experimental material. At the same time we had gotten the offspring of those red bighead crap be bred by parthenogenesis. Three kinds of bighead crap——the normal bighead crap in black color (BC), XiangYun Golden Bighead-carp(RBC) and the offspring of those red bighead crap be bred by parthenogenesis(GRBC) were collected for use. The study is focus on the difference between three kings of bighead crap on morphological features and molecular biology.
The ordinary regulations and T value test were chose to study their morphological features. The results showed notable difference on the lateral-line scale, number of vertebra of the three stocks. The RBC and the gRBC almost had same morphological features. The difference of the ratio of head- length to snout- length, body-length to body-highness and caudal- peduncle- length to caudal- peduncle- highness between BC and RBC with gRBC was obvious.
Genetic variation was comparatively analyzed between the Red bighead carp (RBC) population and the bighead carp (BC) population of aquaculture strains originating from xiangjiang river used as the control, using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. The specific fragments for RBC and BC population was detected by S20 and S46 respectively. The average genetic distances of BC ranged from 0.04 to 0.13 with a mean of 0.075. And the average genetic distances of RBC ranged from 0.02 to 0.35 with a mean of 0.139. The average genetic distances estimated by Lynch's index of BC population were significantly lower than those of RBC population. Where as the average genetic distances between BC and RBC ranged from 0.06 to 0.37 with a mean of 0.123. The mean number of alleles per microsatellite locus (1.89) in BC population was considerably lower than that (2.44) in RBC population. The average observed (0.34) and expected heterozygosis (0.39) in BC population were lower than those (0.54 and 0.51, respectively) in RBC population, indicating that the RBC presented higher genetic diversity than the BC. The results from RAPD assay and microsatellite analysis were in agreement with each other, that is to say, the BC presented lower level of polymorphism than RBC. Furthermore, as expected, microsatellite analysis revealed more detailed information on genetic diversity than RAPD assay.
The complete mitochondrial DNA sequences from two kinds of of bighead carp (RBD and *RBD) were determined. The result showed that the complete mitochondrial DNA sequences of two kinds of bighead carp (RBD and *RBD) are resemble. The genome is a circular molecule with 16618 bp in length, and the overall base composition included 31.64% of Adenine (A),27.10% of cytosine (C),15.94% of Guanine (G), 25.32%, of thymine (T), and with 56.96% the A+T content.
1.三个鳙鱼群体的可数性状和可量性状均处于种质异动标准之内。通过测量代表鳙鱼生物学特征的13个度量性状,处理9项比值并进行T值检验。实验结果表明,红鳙和雌核发育的红鳙的各项性状比值基本一致,这表明了雌核发育红鳙稳定地遗传了红鳙的优良性状。普通鳙鱼和两种红鳙在头长／吻长、体长／体高、尾柄长／尾柄高等性状比值上有着显著差异,尤其以体长／体高最为显著,红鳙具有体宽、尾柄短等优良的性状。
2.对红鳙进行了遗传多样性的RAPD和微卫星分析(来自湘江流域的普通鳙鱼作为对照)。在优化RAPD检测条件基础上,从230个随机引物中筛选出来的45个扩增较好且多态性强的引物对这两个群体的DNA多态性及分子标记进行了分析。结果显示在45个随机引物的扩增谱带中找到了2个引物(S20、S46)的特异扩增谱带,可以作为这两个种群间的分子遗传标记。普通鳙鱼群体内个体之间的遗传距离范围从0.04-0.13,群体内的平均遗传距离为0.075,而红鳙群体内个体之间的遗传距离范围从0.02-0.35,群体内的平均遗传距离为0.139。这表明红鳙群体遗传多样性明显强于普通鳙鱼群体。从32对微卫星标记中筛选9对微卫星标记用于两个群体24个个体的扩增。平均每个微卫星位点在红鳙群体和普通鳙鱼群体中检测到的平均有效等位基因数分别为2.44和1.89。普通鳙鱼群体的平均杂合度期望值(0.34)和平均杂合度观测值(0.39)要比红鳙群体的平均杂合度期望值(0.51)和平均杂合度观测值(0.54)的小得多。由此可见,RAPD和微卫星这两种方法得出的结果是一致。
3.对红鳙和雌核红鳙的mtDNA基因组全序列进行测序,并对其基因组结构进行了分析。两种红鳙的mtDNA基因组序列完全相同,全长均为16619 bp,其碱基比例如下：%A=31.64,%G=15.94,%T=25.32,%C=27.10,%A+T=56.96。这也表明了线粒体严格的母性遗传。本文得到的红鳙的线粒体基因组全序列结果可以为红金鳙及其近缘种的演化、生物地理学研究,为检验鱼类形态学、生理学和进化学等特征提供分子依据。
After the years been artificial breeding, the bighead carp (Aristichthys nobilis) had substantial decline in germplasm resources and significant reduction of genetic diversity. Purification and rejuvenation of the bighead-carp germplasm is an urgent task. Red bighead carp is a new type of special bighead crap be selected and bred by our laboratory form wild bighead crap in recent years and those red bighead carp been named XiangYun Golden Bighead-carp. That bighead crap in red color has fast growth, is good breeding species and valuable experimental material. At the same time we had gotten the offspring of those red bighead crap be bred by parthenogenesis. Three kinds of bighead crap——the normal bighead crap in black color (BC), XiangYun Golden Bighead-carp(RBC) and the offspring of those red bighead crap be bred by parthenogenesis(GRBC) were collected for use. The study is focus on the difference between three kings of bighead crap on morphological features and molecular biology.
The ordinary regulations and T value test were chose to study their morphological features. The results showed notable difference on the lateral-line scale, number of vertebra of the three stocks. The RBC and the gRBC almost had same morphological features. The difference of the ratio of head- length to snout- length, body-length to body-highness and caudal- peduncle- length to caudal- peduncle- highness between BC and RBC with gRBC was obvious.
Genetic variation was comparatively analyzed between the Red bighead carp (RBC) population and the bighead carp (BC) population of aquaculture strains originating from xiangjiang river used as the control, using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. The specific fragments for RBC and BC population was detected by S20 and S46 respectively. The average genetic distances of BC ranged from 0.04 to 0.13 with a mean of 0.075. And the average genetic distances of RBC ranged from 0.02 to 0.35 with a mean of 0.139. The average genetic distances estimated by Lynch's index of BC population were significantly lower than those of RBC population. Where as the average genetic distances between BC and RBC ranged from 0.06 to 0.37 with a mean of 0.123. The mean number of alleles per microsatellite locus (1.89) in BC population was considerably lower than that (2.44) in RBC population. The average observed (0.34) and expected heterozygosis (0.39) in BC population were lower than those (0.54 and 0.51, respectively) in RBC population, indicating that the RBC presented higher genetic diversity than the BC. The results from RAPD assay and microsatellite analysis were in agreement with each other, that is to say, the BC presented lower level of polymorphism than RBC. Furthermore, as expected, microsatellite analysis revealed more detailed information on genetic diversity than RAPD assay.
The complete mitochondrial DNA sequences from two kinds of of bighead carp (RBD and *RBD) were determined. The result showed that the complete mitochondrial DNA sequences of two kinds of bighead carp (RBD and *RBD) are resemble. The genome is a circular molecule with 16618 bp in length, and the overall base composition included 31.64% of Adenine (A),27.10% of cytosine (C),15.94% of Guanine (G), 25.32%, of thymine (T), and with 56.96% the A+T content.
引文
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