Study On The Time Of The Fourth Round Of Genome Duplication And CC Chemokine Gene In Common Carp (Cyprinus Carpio)

Background:Common carp(Cyprinus carpio) is thought to have undergone one extra round of genome duplication compared to zebrafish. Transcriptome analysis has been used to study the existence and timing of genome duplication in species for which genome sequences are incomplete. Large-scale transcriptome data for the common carp genome should help reveal the timing of the additional duplication event. Chemokines belong to a family of chemotactic cytokines and are involved in activation and migration of leukocytes. They play important roles in the innate immune system of fish species. CC chemokines are one of the biggest superfamilies of chemokines. With the launch of more and more fish genome sequencing and transcriptome sequencing projects, rapid accumulation of the data set make comparative genomics more accessible to identify fish CC chemokines. However, there are few reports on Cyprinus carpio CC chemokine so far. Only one Cyprinus carpio CC chemokine (GenBank accession:AB010469) was reported by Kazuhiro et al. The existing transcriptome sequence data of Cyprinus carpio from public database can help reveal Cyprinus carpio CC chemokine.Results:We have sequenced the transcriptome of common carp using454pyrosequencing. After assembling the454contigs and the published common carp sequences together, we obtained49,669contigs and identified genes using homology searches and an ab initio method. We identified4,651orthologous pairs between common carp and zebrafish and found129,984paralogous pairs within the common carp. An estimation of the synonymous substitution rate in the orthologous pairs indicated that common carp and zebrafish diverged120million years ago (MYA). We identified one round of genome duplication in common carp and estimated that it had occurred5.6to11.3MYA. In zebrafish, no genome duplication event after speciation was observed, suggesting that, compared to zebrafish, common carp had undergone an additional genome duplication event. We annotated the common carp contigs with Gene Ontology terms and KEGG pathways. Compared with zebrafish gene annotations, we found that a set of biological processes and pathways were enriched in common carp. Besides, a full-length cDNA of CC chemokine CCL-C5a-like gene was amplified in Cyprinus carpio spleen by RACE method (GenBank accession No. JQ026408). The full-length cDNA was830bp in size, with174bp5’UTR,296bp3’UTR and360bp ORF. The ORF encodes a protein of119amino acids. Functional domain analysis showed that this protein contained signal peptide and conserved chemokine structure. Gene Ontology annotation indicated that this protein was involved in the biological process of immune cell migration. CCL-CSa was widely expressed in brain, skin, muscle, head kidney, body kidney, intestine, gill and heart and mainly in spleen and liver. After lipopolysaccharide stimulation, the expression of CCL-C5a showed significantly increase in peripheral blood leukocytes and head kidney leukocytes. The deduced amino acid sequence of Cyprimts carpio CCZ-CSa shared high identity with other seven species, which was from37%to61%, and the highest was61%with Danio rerio. Adaptive evolution analysis revealed that the Ka/Ks of CCL-C5a bewteen Cyprinus carpio and Danio rerio was less than1, indicating that CCZ-CSa was under negative selection pressure in the evolutionary process of fish species.Conclusions:The assembled contigs helped us to estimate the time of the fourth-round of genome duplication in common carp. The resource that we have built as part of this study will help advance functional genomics and genome annotation studies in the future. Besides, a full-length cDNA of CC chemokine CCZ-C5a-like gene was amplified in Cyprinus carpio spleen, and tissue expressions and evolutional analysis of this gene were conducted. The results showed that Cyprinus carpio CCL-CSa are principally expressed in immune organs and up-regulated after lipopolysaccharide stimulation and that this gene is under negative selection pressure in fish species. These results indicate that Cyprinus carpio CCZ-CSa might be a immune-relevant gene.