Genome evolution trend of common carp （Cyprinus carpio L.） as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.     

鲤鱼微卫星突变速率和模式(英文)

利用150个微卫星分子标记在F1代家系的基因型分析过程中,共有27600个等位基因从亲本向子代传递,其中在5个微卫星座位上检测到6个突变的等位基因。对突变的等位基因数目进行统计分析后得出:鲤鱼平均每个世代每个微卫星座位的突变速率为2.53×10-4。在发现突变的5个位点中,经测序发现,突变序列中插入1个以上的重复单元就导致了突变的发生。这些突变表明,鲤鱼的微卫星突变没有遵循严格的渐变突变模型(stepwise mutation model,SMM)。该文关于鲤鱼微卫星突变速率和模式的研究将会对统计鲤鱼有效群体的统计提供有效参数。     

Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.     

Development of 10 tri- and tetranucleotide microsatellite loci for population studies in the common carp (Cyprinus carpio L.)

Ten tri- and tetranucleotide microsatellite DNA markers were isolated and characterized from common carp (Cyprinus carpio L.) to estimate genetic potential. These markers were tested in the samples from two closely related carp populations (Cyprinus carpio var. xingguonensis and Cyprinus carpio var. wananensis). The number of the alleles ranged from three to nine, and observed and expected hererozygosities varied from 0.207 to 1.000 and from 0.499 to 0.900 in each population, respectively. No evidence for linkage disequilibrium was found, indicating that these markers will be useful for population studies.     

微卫星DNA标记分析德国镜鲤的遗传潜力

结合体重、体长、体高等数量性状, 用30个微卫星分子标记, 评估了3个德国镜鲤群体的遗传潜力, 共检测到287个等位基因, 559种基因型, 片段长度109~400 bp, 有效等位基因数1.1014~6.4665, 观察杂合度0.0968~0.9892, 期望杂合度0.0926~0.8554, 位点多态信息含量0.08787~0.8559, 其中中度多态(0.25≤PIC≤0.5)13个, 高度多态(PIC≥0.5)13个。统计结果显示: 3个群体的遗传潜力处于中度水平, 双来养殖群体的遗传潜力比换新和松浦繁殖群体低。同时, 用30个基因座的不同等位基因和基因型与双来群体的体重、体长、体高进行了连锁分析, 得到2个与镜鲤体长相关的微卫星分子标记(HLJ319, HLJ693)和1个与体高相关的位点(HLJ677), 与体长相关的1个位点(HLJ693)还与体重连锁。将此遗传标记在鲤鱼重组自交系中验证, 结果显示: 主要经济性状相连锁的3个遗传标记中HLJ319与鲤鱼体长性状的QTL定位结果基本一致。     

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.     

Salt stress and resistance to hypoxic challenges in the common carp (Cyprinus carpio L.)

Long term exposure to brackish water (171 mm NaCl) affected the capacity of common carp Cyprinus carpio to deal with hypoxic conditions and the critical oxygen concentrations for oxygen consumption increased. In addition, regulation of ammonia excretion was lost. The cytosolic phosphorylation potential (the index of the energy status of a cell in terms of potential transferable phosphate groups) in the lateral muscle on the other hand remained relatively unaffected, indicating that oxygen transport to the tissues was not severely compromised. It appears that exposure to brackish water reduces the capacity of common carp to cope with hypoxic conditions mainly because of the high energetic cost of hyperventilation under conditions where energy stores are depleted, and not because of any impeded oxygen transport mechanisms.     

鲤鱼体长性状的QTL定位及其遗传效应分析

以大头鲤/荷包红鲤抗寒品系的重组自交系群体及其遗传连锁图谱, 利用Windows Map Manager 2.0的标记回归法进行QTL单标记定位分析和复合区间作图法进行QTL区间检测, 通过置换实验(1 000次重复)确定连锁群显著性水平阈值。在体长性状的标记回归研究中, 共7个标记达到显著水平(P<0.01), 对性状的贡献率为14.00%～27.00%, 其中3个标记达到极显著水平(P<0.001)。HLJ534, HLJ319, HLJ370座位可能与影响鲤鱼体长性状的主效基因连锁。在体长性状的QTL区间定位研究中, 共6个QTL达到连锁群显著水平(P=0.05), 对性状的贡献率为11.33.%～23.12%, 其中2个达到连锁群极显著性水平(P=0.01), 它们的加性效应方向并不一致。HLJ190-HLJ497区间和HLJ479-HLJ483区间是影响鲤鱼体长性状的主效QTL区间。     

The ingestion of bacteria in suspension by the common carp Cyprinus carpio L.

Groups of large (65 -75 g) and small (8-17 g) common carp ( Cyprinus carpio L.) fingerlings were exposed to the bacterium Chromobacterium violaceurn in order to establish whether they could detect and ingest unattached bacteria. Small fish exposed to both bacteria and to cell-free bacterial extracts showed a significant increase in opercular beat rates, thus demonstrating that they are able to detect the presence of unattached bacteria in suspension. Examination of carp gut contents showed that the proportion of small fish ingesting bacteria increased with exposure time although no significant relationship was observed among larger fish. Significant, positive correlations between numbers of viable bacteria isolated from the intestinal tracts and concentration in the environment were observed. Possible mechanisms of bacterial ingestion are discussed.     

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.     

Divergent selection for antibody production in common carp (Cyprinus carpio L.) using gynogenesis

A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH. to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring do indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 ± 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.     

Polymorphism in ornamental and common carp strains (Cyprinus carpio L.) as revealed by AFLP analysis and a new set of microsatellite markers

Forty-seven new microsatellite markers were generated and applied, together with the AFLP (Amplified Fragment Length Polymorphism) technique using two different enzyme combinations, to the genetic analysis of two carp species, Cyprinus carpio L. and Ctenopharyngodon idella. The extent of polymorphism and the genetic relationships between nine carp populations were studied. The incidence of microsatellites containing CA and CT motifs was estimated to be one every 17.4 and one every 126.3 kb, respectively, and their average allele numbers were four and five, respectively. Across populations, the average proportion of individuals that were heterozygous for microsatellite markers was 44.2% and the average allele number was 4.02. The EcoRI/TaqI combination generated more analyzable AFLP bands than the EcoRI/MseI pair, making the former preferable for the analysis of carp populations. The proportion of polymorphic AFLP bands within populations ranged from 6.7% in grass carp to 59.9% in Kohaku strain (Koi) of the ornamental carp. The fixation index (FST) for microsatellites in these populations was estimated to be 0.37, and for AFLP markers the value was 0.39. Genetic distance matrices derived from microsatellites and from two AFLP analyses were positively correlated. Grass carp showed fewer AFLP bands than other populations and was genotyped by only half of the microsatellite markers. These findings agree with genetic distance estimates in suggesting that the grass carp is phylogenetically quite remote from all the other populations examined.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Monoclonal antibodies against spermatozoa of the common carp (Cyprinus carpio L.)

Summary Eleven monoclonal antibodies that recognize membrane determinants on spermatozoa of the carp Cyprinus carpio L. have been produced. Indirect immunofluorescence revealed that these determinants are uniformly distributed on the surface of head and midpiece. Most of them are also present on the outer membrane of precursor sperm cells. Although none of the monoclonal antibodies reacted with carp somatic tissue, five monoclonal antibodies were positive for surface membrane determinants of oogonia and early prophase oocytes in carp ovary. Preliminary analysis of the testis and ovary of three other species of fish showed that some carp determinants are shared with germ cells from Barbus conchonius, Clarias lazera, or Salmo gairdneri.Abbreviation WCS Wageningen Carp Sperm antibody     

When everything changes at once: finding a new normal after genome duplication

Whole-genome duplication (WGD), which leads to polyploidy, is implicated in adaptation and speciation. But what are the immediate effects of WGD and how do newly polyploid lineages adapt to them? With many studies of new and evolved polyploids now available, along with studies of genes under selection in polyploids, we are in an increasingly good position to understand how polyploidy generates novelty. Here, I will review consistent effects of WGD on the biology of plants, such as an increase in cell size, increased stress tolerance and more. I will discuss how a change in something as fundamental as cell size can challenge the function of some cell types in particular. I will also discuss what we have learned about the short- to medium-term evolutionary response to WGD. It is now clear that some of this evolutionary response may ‘lock in’ traits that happen to be beneficial, while in other cases, it might be more of an ‘emergency response’ to work around physiological changes that are either deleterious, or cannot be undone in the polyploid context. Yet, other traits may return rapidly to a diploid-like state. Polyploids may, by re-jigging many inter-related processes, find a new, conditionally adaptive, normal.     

Growth and survival of carp (Cyprinus carpio L.) larvae in alkaline water

Growth of carp larvae was significantly retarded in water with pH between 9.4 and 10.3, as compared with control conditions of pH 7.8–8.2. The survival rate was lower the higher the pH.     

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.)

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.     

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.)

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5′-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3′,5′-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5′-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1 mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5′-deiodination in liver is potently stimulated by 1 mM DTT. The marked biochemical differences between 5′-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

A linkage map of common carp (Cyprinus carpio) based on AFLP and microsatellite markers

Common carp (Cyprinus carpio) is an important fish for aquaculture, but genomics of this species is still in its infancy. In this study, a linkage map of common carp based on Amplified Fragment Length Polymorphism (AFLP) and microsatellite (SSR) markers has been generated using gynogenetic haploids. Of 926 markers genotyped, 151 (149 AFLPs, two SSRs) were distorted and eliminated from the linkage analyses. A total of 699 AFLP and 20 microsatellite (SSR) markers were assigned to the map, which comprised 64 linkage groups and covered 5506.9 cM Kosambi, with an average interval distance of 7.66 cM Kosambi. The normality tests on interval map distances showed a non‐normal marker distribution. Visual inspection of the map distance distribution histogram showed a cluster of interval map distances on the left side of the chart, which suggested the occurrence of AFLP marker clusters. On the other hand, the lack of an obvious cluster on the right side showed that there were a few big gaps which need more markers to bridge. The correlation analysis showed a highly significant relatedness between the length of linkage group and the number of markers, indicating that the AFLP markers in this map were randomly distributed among different linkage groups. This study is helpful for research into the common carp genome and for further studies of genetics and marker‐assisted breeding in this species.