Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Polymorphism of the cytochrome oxidase b gene (cyt b) in Russian populations of common carp and wild common carp (Cyprinus carpio L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (pi) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, pi = 0.67%, h = 0.7; and BB, pi = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (pi = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (pi = 0.035%, h = 0.4; and pi = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (pi = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

从江及周边地区稻田鲤的遗传结构及系统发育关系

为探究贵州从江及邻近的榕江、黎平和锦屏以及广西三江、湖南的通道和靖州等地稻田鲤Cyprinus carpio Linnaeus的遗传结构及系统关系, 研究基于mtDNA Cyt b基因测序技术对6个地理群体进行了分析。447尾稻田鲤中共界定出29种Cyt b基因序列单倍型, 结合NCBI中鲤同源序列的系统发育和序列比对分析表明, 从江及邻近苗侗族地区稻田鲤是一个含欧亚2大谱系、3个鲤亚种和多个母系血统成分的混合群体, 主体类群是华南鲤C. carpio rubrofuscus, 同时混有欧洲鲤C. carpio carpio和远东鲤C. carpio haematopterus, 遭受国内外鲤养殖品种的遗传渗析。遗传多样性分析显示, 欧洲鲤亚种群体为低H_d低π, 遗传多样性贫乏, 远东鲤和华南鲤2个亚种群体呈高H_d低π特点。华南鲤6个稻田群体除锦屏为低H_d低π外, 其余都为高H_d低π, 从江的H_d最高, 黎平的π最高。遗传分化和分子方差(AMOVA)分析显示, 谱系间和亚种间存在显著的遗传分化(P<0.001), 锦屏和三江与其他群体存在中等-高度的分化等级, 其余群体间分化较弱, 群体间变异贡献率为16.69%, 群体内为81.14%。综合分析表明, 从江及邻近地区稻田鲤群体间存在有限的遗传交流, 群体内和群体间有不同的遗传结构。研究丰富了该地区稻田鲤遗传背景资料, 可为湘黔桂交界苗侗族地区稻田鲤的种质资源鉴定与保护提供理论依据。     

应用微卫星多态分析四个鲤鱼群体的遗传多样性

选择12个斑马鱼功能基因的微卫星标记和12个鲤鱼微卫星标记,检测黑龙江鲤(Cyprinus carpiohaematopterusTemminck et Schlegel)、散鳞镜鲤(C.carpio)、荷包红鲤抗寒品系(C.carpiovar.wuyuanensis)、松浦鲤(C.carpioSongpu carp)的群体遗传多样性。共检测到3882个扩增片段,长度为126～489bp,在群体间扩增出1～5个等位基因不等,共计59个等位基因,平均等位基因2.46个。数据经PHYLIP V3.6软件估算和MEGA3软件作图,确立4个群体间的亲缘关系。并应用Bootstrap检验估计系统树中节点的自引导值,并进行了系统发生分析。结果表明①4个群体检测的有效等位基因数都在55个以上,平均观测杂合度为0.36～0.43,平均期望杂合度为0.49～0.53,平均多态信息含量为0.21～0.25,说明这几个群体多态性属于中度偏高水平,遗传多样性较高;②群体间相似系数都在0.84以上,遗传距离较近,为0.067～0.170,与前人研究结果一致。聚类分析显示,松浦鲤与散鳞镜鲤亲缘关系最近,荷包红鲤抗寒品系与它们的亲缘关系较黑龙江鲤更近;③在3个斑马鱼功能基因相关的微卫星位点上,黑龙江鲤缺失特异扩增条带,这可能与几种鱼类的育成史有关。     

一个锂品种线粒体基因片段序列保守性

在红锂、镜锂和野锂中进行线粒体１６rRNA和Ｃytb基因片段的序列测定。结果发现,３个鲤品种在多达８５９个碱基长度上无变异,但是与已报道的鲤全序列中的同源序列则有约１．５％的趋异。这个结果提示３个鲤品种可能在起源中是独立的一支。并且分化极低。     

Genetic Variation Analysis within and among Six Varieties of Common Carp (Cyprinus carpio L.) in China Using Microsatellite Markers

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles ranging from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (P < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F _st distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.     

Mitochondrial genetic diversity and gene flow of common carp from main river drainages in China

1. The common carp (Cyprinus carpio L.) is one of the most widely distributed and important freshwater fishes in the world. In China, the common carp has been recognised as three subspecies: C. carpio haematopterus, C. carpio rubrofuscus and C. carpio carpio. The Nanling Mountains have been suggested as providing the dividing line between C. carpio haematopterus and C. carpio rubrofuscus. However, the demographic history and gene flow of the common carp in China is not clear. 2. We collected mitochondrial COII and D‐loop sequences (1494 bp) from 241 individuals distributed in eight major river drainages across China. The objective was to provide the first investigation into population genetic structure, demographic history and migration patterns of the common carp from these river drainages, and to assess the validity of the three subspecies. 3. Phylogenetic analysis did not result in three major monophyletic lineages corresponding to the three subspecies. The Nanling Mountains do not form a border separating C. carpio haematopterus and C. carpio rubrofuscus. amova showed low population differentiation, with 11.60% of the molecular variance found among river drainages. Pairwise F_ST values between river drainages were moderate (0.0331–0.2617). Substantial gene flow detected in coalescent analysis between drainages showed that the Yangtze drainage was the centre from which migrants moved northward, southward and north‐westward. Human‐mediated translocation has confounded our ability to identify subspecies of common carp in China.     

Discovery of an ancient lineage of Cyprinus carpio from Lake Biwa, central Japan, based on mtDNA sequence data, with reference to possible multiple origins of koi

MtDNA sequence data ( c . 2100 bp of complete D‐loop + cytochrome b gene regions) showed a principal phylogenetic split within common carp Cyprinus carpio between the Lake Biwa wild form ( n  = 3) and 'Eurasian' wild ( n  = 18) and domesticated ( n  = 35) forms. This supports the idea of the ancient origin of the Lake Biwa wild form, as well as the East Asian origin of wild common carp, previously proposed on the basis of fossil data. In addition, a possible multiple origin of koi carp was indicated by the polyphyletic distribution of five mtDNA haplotypes of koi carp within the 'Eurasian' clade.     

Genetic variation analysis within and among six varieties of common carp (Cyprinus carpio L.) in China using microsatellite markers

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (p < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F(st) distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.     

mtDNA sequence data supports an Asian ancestry and single introduction of the common carp into the Danube Basin

Common carp Cyprinus carpio ( n =24) from the upper Danube (wild or feral forms, scaleless varieties) as well as Japanese koi shared the identical mtDNA haplotype (565 bp). In contrast, Amur River carp ( n =5) exhibited unique haplotypes, 1–12 bp divergent from the Danubian one. These results support an Asian origin and single introduction or domestication event leading to the present diversity of common carp in Europe, as well as the ornamental Japanese koi.     

转基因红鲤体细胞的核移植

以F4代转hGH基因红鲤体细胞（肾脏和尾鳍）及培养18代的F4代转hGH基因红鲤尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植,以探讨外源F4代转基因鱼体外源基因的分布与存在形式,稳定性和克隆转基因鱼的可能性。F4代红鲁肾脏细胞核与泥鳅卵配合的核移植胚胎有12．4％发育到囊胚,0．33％发育到神经胚;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚,神经胚、肌节期和肌肉效应期的胚胎分别为24．5％、0．3％、0．2％和0．1％;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有50．53％发育到囊胚,5．69％发育到原肠胚,0．53％发育到神经胚,0．4％发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性,早期发育率高;而由于培养细胞的异倍化,后期的发育率降低。用PCR技术对供体鱼不同个体及同一体不同组织外源基因检测,结果100％个体为阳性鱼,而且不同组织的阳性率也是100％,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体,核移植胚胎中hGH基因的检出率为100％。说明F4代转基因红鲤个体不同细胞都存在hGH基因,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基因已基本稳定,体细胞核移植可以作为获得同质化转基因鱼的有效手段,但核移植效率还很低。另外还讨论了核质的相容性、细胞周期的协调、染色体的变异等因素对核移植的影响。     

人工诱导荷包红鲤雌核发育子代与亲本的染色体组型及几种酶的组织化学比较

作者使用元江鲤失活的精子,诱导荷包红鲤卵子进行雌核发育所获得的子代,同亲本作了染色体组型和几种酶的组织化学比较。雌核发育子代的染色体组型与母本荷包红鲤完全相同,与父本元江鲤有不同。元江鲤的体色黑灰色,是由于肌肉中有酪氨酸酶存在,能将酪氨酸氧化成黑色素的缘故;荷包红鲤肌肉中无此酶,不能形成黑色素,所以体色呈桔红色;雌核发育子代肌肉中,也无酪氨酸酶,其体色同母本一样为桔红色;乳酸脱氢酶、非特异性酯酶的分布和含量也都与母本一致,与父本有差异。     

New species of myxosporidians in the genus Myxobolus (Myxosporidia, Myxobolidae) from cyprinid fishes of the Amur River

Five new species are described as follows: M. haematopterus from gills, fins and skin of Cyprinus carpio haematopterus; M. gibelio from connective tissue of gill filaments, fin rays and kidneys of Carassius auratus gibelio; Myxobolus waleckii from gills of Leuciscus waleckii; Myxobolus junchisi from gills, muscles, kidneys and spleen of Cyprinus carpio haematopterus; M. alacaudatus of Cyprinus and Carassius Myxobolus divergens carassii from gills, muscles, fins, and cartilaginous tissue of operculim of Carassius auratus gibelio and Cyprinus carpio haematopterus.     

鲤鱼不同品种（系）红血球抗原特异性的初步研究

用血球凝集和血清吸收试验的方法,对若干个鲤鱼品种及鲤鱼人工雌核发育系的红血球中的某些同种异型抗原纯合性进行了初步研究。①以抗雌核发育红鲤抗血清(abs／R)为试剂,三个雌核发育系(R8305,R8413,RMGF1)的RBC反应值分别为1521—2360,1521-2028,853—1521,普通红鲤、镜鲤、白鲢的反应值在80—782。②abs／8305,与abs／8413经靶细胞相互吸收后的残留活性显著降低(均为320),而以白鲢RBC吸收后抗血清活性不变。作者认为这部分抗原在鲤鱼种以下的品种和品系中具有特异性,可考虑将其作为鱼类品系鉴定的一种手段。     

Genetic Diversity in Crucian Carp (Carassius auratus)

A survey of restriction fragment polymorphism in mitochondrial DNA of three subspecies of Carassius auratus throughout four provinces in China was undertaken using 17 restriction enzymes. Two carp, Cyprinus carpio rubbrofuscus and Cyprinus carpio carpio, were included as the outgroup. A total of 16 haplotypes was observed: 5 in tetraploids of C. auratus auratus; 8 in hexaploids of C. auratus auratus; and 2 in C. auratus gibelio and C. auratus cuvieri, respectively. The tetraploids and hexaploids share three common haplotypes as I, V, and VI. C. a. Cuvieri may have diverged first among the three subspecies. Interestingly, C. a. auratus and C. a. cuvieri did not form monophyletic clades, which indicated that the classification of Carassius auratus required further studies. The current hypothesis, that hexaploids originated from tetraploids by a polyploidy event, is less favorable, based on the distribution of haplotypes and the lower diversity in tetraploids than in hexaploids. Our data also indicate that divergence of hexaploids and tetraploids might be recent and mtDNA polymorphism existed before the divergence. Meanwhile, genetic isolation exists between the hexaploids and the tetraploids.     

Genetic evolution and diversity of common carp Cyprinus carpio L.

Knowledge of genetic variation and population structure of existing strains of both farmed and wild common carp Cyprinus carpio L. is absolutely necessary for any efficient fish management and/or conservation program. To assess genetic diversity in common carp populations, a variety of molecular markers were analyzed. Of those, microsatellites and mitochondrial DNA were most frequently used in the analysis of genetic diversity and genome evolution of common carp. Using microsatellites showed that the genome evolution in common carp exhibited two waves of rearrangements: one whole-genome duplication (12–16 million years ago) and a more recent wave of segmental duplications occurring between 2.3 and 6.8 million years ago. The genome duplication event has resulted in tetraploidy since the common carp currently harbors a substantial portion of duplicated loci in its genome and twice the number of chromosomes (n = 100–104) of most other cyprinid fishes. The variation in domesticated carp populations is significantly less than that in wild populations, which probably arises from the loss of variation due to founder effects and genetic drift. Genetic differentiation between the European carp C.c. carpio and Asian carp C.c. haematopterus is clearly evident. In Asia, two carp subspecies, C.c. haematopterus and C.c. varidivlaceus, seem to be also genetically distinct.     

Polymorphism of microsatellite markers in Russian common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.     

Polymorphism of the cytochrome oxidase b gene (<Emphasis Type="Italic">cyt b</Emphasis>) in Russian populations of common carp and wild common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (π) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, π = 0.67%, h = 0.7; and BB, π = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (π = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (π = 0.035%, h = 0.4; and π = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (π = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

杞麓湖鲤鱼鳞片表面结构的扫描电镜观察

采用扫描电镜技术对鲤科鱼类鳞片表面结构进行观察研究,目前国内尚未见报道。本工作主要以杞麓湖的鲤鱼为研究对象,同时观察了鲤亚科其他属鱼类的鳞片表面结构,发现鳞纹上的齿状粒突可能是从无到有,同时探察此结构有无可能作为分类的一项指标。鉴于杞麓鲤的鳞纹上具齿状粒突,而华南鲤则无,再综合考虑二者骨骼性状的差异,作者认为,这些性状可作为将杞麓鲤由普通鲤的一个亚种提升为一个独立种级地位的证据。从鳞片辐射沟的亚显微结构推测,辐射沟具有两方面的功能,一方面可对鳞片的热胀冷缩起缓冲作用:另一方面可增强鳞片的柔软弯曲性。