| Topmouth culter (Culter alburnus) (order Cypriniformes, family Cyprinidae) is widely distributed throughout large rivers, reservoirs and lakes in China. In the process of fishery resources survey, reproductive difference of C. alburnus was detected that it lays pelagic eggs in Danjiangkou reservoir while lays demersal eggs in other regions in Yangtze River basin (unpublished). The phenomenon of laying pelagic eggs was also found in Xingkaihu lake, Heilongjiang province. The demersal egg has thick and complex structural membrane while the pelagic egg has thin and simple structural membrane. Does this mean that the topmouth culter populations of Danjiangkou Reservoir and Xingkaihu lake have a larger degree of differentiation with the species in other waters? What is the main reason causing egg-type variation? Everything is unknown. Many morphological structure of the egg membrane can be used as distinguishing traits of fish family or genus, deeply the egg fertilized hole features can be used to distinguish some fish species. Therefore the genetic relationships of these C. alburnus populations, could provide valuable genetic information for fishery resource protection, should be urgently determined. Additionally, as an important economic farmed fish, the germplasm degradation risk is gradually faced for the fish species in recent years. Thence genetic diversity needs to be investigated in favor of rational fishery resource exploitation and utilization. Based on the above, in present study, we employed mitochondrial DNA and microsatellite markers to assess the C. alburnus genetic diversity and population structure of various mild populations, analyzed the genetic variability between wild and breeding populations. Additionally, the full-length cDNA of C. alburnus ZP3 gene was cloned, characterized, analyzed for expression and inspected for single nucleotide polymorphism (SNP). Following the relationship between SNPs and egg shape variation was analysied.Important results are as follows:1. Development of new polymorphic microsatellite markers in topmouth culter (Culter alburnus) and determination of their applicability in Culter mongolicusFifteen new polymorphic microsatellite markers were developed for Culter alburnus. In 32 individuals representing a wild population of the Danjiangkou Reservoir, Hubei, China, the number of alleles at these loci varied between 2 and 10, with an average of 5.5. The average observed and expected heterozygosities were 0.664 and 0.681, respectively. The polymorphism information content of 11 loci was more than 0.5 whereas that of the other 4 loci was less than 0.5 but more than 0.25. In addition, the genomes of 30 C. mongolicus individuals were successfully amplified with these primer pairs. Amonst,12 sties showed polymorphic and the polymorphism information content of 11 loci was more than 0.5, while the rest one loci was less tha 0.5 but more than 0.25. As indicated, these novel loci had extensive university across the closely related species.2. Assessing the geneti c diversity and population structure of Culter alburnus in China based on mitochond rial 16S rRNA and COI gene sequencesThe genetic diversity and population genetic structure of Culter alburnus were investigated using mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal 16S subunit (16SrRNA) gene sequences. A total of 89 individuals from four local ities were in cluded in the analysis. Overall,12 polymorphic sites were observed and 10 haplotypes were defined. The C. alburnus populations were characterized by high haplotype diversity (0.587±0.047) and low nucleotide diver sity (0.00197±0.00073). Pairwise fixation index (FST) analysis indicated significant genetic differentiation among different populations. The hierarchic alanalysis of molecular variance (AMOVA) analysis also showed significant genetic divergence (FST=0.3792, P< 0.01) among these populations. The present results suggest that subdivisions exist among four C. alburnus populations, and should be considered as different management unit for effective conservation and management purposes. This study provides new in formation for genetic assessment and will be crucial for establishing fisheries management and strategies for this species.3. Genetic diversity of wild and cultured topmouth culter(Culter alburnus) inhabiting China as determined with mtDNA and microsatellitesVery limited information is available regarding the genetic diversity and population structure of the topmouth culter(Culter alburnus), one of the most commercially important freshwater fish species inhabiting China. This is hindering the stock improvement of this fish species. Understanding of the level of genetic diversity of wild and cultured topmouth culter populations is highly relevant to successful hatchery management. With microsatellites and mitochondrial DNA, the genetic diversity and structure of five wild and two cultured populations were determined in this study. We found a significant decrease in the genetic diversity of wild C. alburnus, indicating that wild fishery resources should be urgently protected in order to prevent degeneration and extinction. It was interesting to find that the genetic diversity of wild populations was not significantly higher than that of the cultured populations, demonstrating that the cultured populations have relatively great breeding potential. Subdivisions among wild populations were found, indicating different units should be considered for conservation and hatchery management. These findings could broaden our understanding of fishery resources protection and development of topmouth culter.4. Conserved zona pellucid glycorprotein 3 in topmouth culter (Culter alburnus): molecuallar cloning, characterization, expression analysis and SNPs discoveryIn the present study, we cloned the full-length cDNA of zona pellucida glycoprotein 3 (ZP3) gene in topmouth culter Culter alburnus (the new gene named CaZP3) using reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced peptide sequence encoding 436 amino acid residues, showed considerable conservation across all species characterized with n-terminal signal peptide, ZP domain, internal hydrophobic patch, external hydrophobic patch, one conserved N-linked and two O-linked glycosylation sites,8 conserved cysteine residues in ZP domain. The ZP3 site cleaved during embryo hatching in fish was also identified in C.alburnus ZP3. We also cloned the ZP3 gene segment with the coverage of coding region, and detected eight exons and seven introns as found in other species. qRT-PCR indicated that the C.alburnus zp3 was highly expressed in the ovary and heart of females, and weakly expressed in female kidney, muscle and gill. Unexpectedly the zp3 mRNA was slightly detected in the testis and muscle of males. Certain fragments of CaZP3 are inspected for single nucleotide polymorphism (SNP) by direct sequencing of PCR production,15 novel SNP sites are detected. Amonst site Snp12 was detected distinct in the two populations, suggesting a possible function contributed to egg-type variation. |
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