Isolation of innate immune response genes, expression analysis, polymorphism identification and development of genetic markers for linkage analysis in common carp (Cyprinus carpio)
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Since the late 1990s, common carp and koi production enterprises around the world have suffered enormous losses due to a viral disease caused by cyprinid herpesvirus-3 (CyHV-3). Genetic variation in resistance to CyHV-3 infection was observed in different common carp strains, indicating that disease resistance can be improved by selective breeding. Marker-assisted selection is a breeding strategy that can accelerate genetic gain; however, this approach requires genetic markers and a genetic linkage map. To develop molecular tools for breeding CyHV-3-resistant aquaculture stock, several candidate genes for antiviral innate immune response from common carp were isolated, and single nucleotide polymorphisms (SNPs) were identified. SNP markers for common carp immune response genes were developed for testing their linkage to disease resistance and for generating a genetic linkage map. Common carp immune response genes were isolated using degenerate primers developed from conserved peptide regions among other fish species for polymerase chain reaction (PCR) amplification. The amplified products were cloned and sequenced. Gene-specific primers were designed based on the isolated carp gene sequences to amplify gene fragments from genomic DNA of three carp strains and koi. The amplified products were cloned and sequenced to identify SNPs. For the genes that are duplicated, locus-specific primers were used for PCR amplification. SNPs were identified in several genes, including TLR2, TLR3a, TLR3b, TLR4a, TLR4b, TLR7a, TLR7b, TLR9, TLR21, TLR22, MyD88a, MyD88b, TRAF6a, TRAF6b, type I IFN, IL-1β, IL10a and IL10b. Putative SNPs were genotyped in a SNP discovery panel consisting of different common carp strains and koi to evaluate their allele frequencies and in a full-sib family to validate their segregation patterns using the SNaPshot method. Validated SNPs were used to genotype a mapping family. Twenty-three SNPs (19 exonic and 4 intronic SNPs) were informative in a mapping family. Among these genes, polymorphisms in IL10a suggested a possible association with resistant and susceptible phenotypes of CyHV-3-challenged fish. These SNPs will be analyzed with a set of approximately 300 microsatellites to generate a second-generation genetic map and to identify quantitative trait loci (QTLs) affecting resistance to CyHV-3. Among the common carp genes that were isolated and sequenced, TLR9 is known for its ability to detect viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. Therefore TLR9, MyD88 and TRAF6 may be important candidate genes for mediating host antiviral response to CyHV-3. To elucidate possible functions of these genes, full-length cDNAs of common carp TLR9, MyD88 and TRAF6 were isolated and tissue-specific mRNA expression was determined. cDNA sequences of MyD88 and TRAF6 revealed that these genes are duplicated. These findings were the first report of MyD88 and TRAF6 duplications in a vertebrate. Protein domain characterization demonstrated that structural characteristics of these genes are conserved and resemble those of other vertebrates, indicating that common carp TLR9, MyD88 and TRAF6 genes may have identical functions with their mammalian orthologs. The mRNA expression of TLR9, MyD88a and b, and TRAF6a and b varied among tissues. Differential expression of the MyD88 and TRAF6 paralogous transcripts were observed in muscle tissues, suggesting that one paralog has evolved and attained a non-immune function. This genomic information will facilitate further research to better understand the ligand specificity of TLR9 and the role of TLR9, MyD88 and TRAF6 in the common carp immune response.
- Doctoral Dissertations