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Before addressing point by point the aforementioned topics in
Our recent QTL mapping study revealed that the adaptor genes, including the NCK genes and the ABI genes, are located within the genomic regions harboring the most significant QTL (Zhou et al., 2017), suggesting that these genes could be involved in the determination of resistance, and possibly through their roles as adaptors assisting the entry of the pathogen into the host. As a first step for the understanding of the roles of the adaptor genes in ESC pathogenesis, in this study, we identified the adaptor genes and determined their guanidine hydrochloride after ESC infection. Here we report the identification, annotation, and expression analysis of three NCK and six ABI genes.

Materials and methods


Adaptor genes NCK and ABI are important regulators for disease response. ABI1 was reported as a positional candidate gene for ESC disease resistance in catfish (Zhou et al., 2017), and bacterial cold water disease (BCWD) resistance in rainbow trout (Palti et al., 2015). NCK1 was reported to be a candidate gene for ESC disease resistance of catfish by using genome wide association analysis (Zhou et al., 2017). Blue catfish is more resistant to ESC than channel catfish (William and Terrence, 2004). Identification of NCK and ABI genes in channel and blue catfish and the expression analysis will provide insight for the research of molecular mechanism of ESC resistance.
Three NCK and six ABI genes were identified in both channel and blue catfish. The annotations of the NCK and ABI genes were explicit based on the phylogenetic, syntenic and protein domain structure analysis. NCK and ABI genes from channel and blue catfish showed closest phylogenetic relationship. Domain structures were well conserved for all the NCK and ABI genes in channel catfish. The domain of channel and blue catfish ABI3b protein was similar to zebrafish ABI3b protein, which contains only ABI_HHR domain but lacks the T_SNARE and SH3 domains. However, some domains were not observed in blue catfish NCK and ABI proteins. Future studies with more tissue source of RNA samples and increased sequence depth are needed to reassembly and validate the NCK and ABI genes in blue catfish. The predicted amino acid sequences of NCK1 from channel and blue catfish were exactly the same with each other. Three base variations were found in the shared 3′ untranslated region (UTR) between channel and blue catfish NCK1 mRNA. Genetic variants in 3’ UTR have been implicated in disease susceptibility because of the change in RNA structure and protein translation (Lu et al., 2015). Future studies of NCK protein expression level in channel and blue catfish are needed to figure out the molecular mechanism of ESC disease resistance difference between channel and blue catfish.
With the publication of channel catfish genome (Liu et al., 2016), the annotation is available in NCBI genome database. The three NCK genes were annotated by NCBI (gi 108272450, gi 108266513, gi 108273233), however, only five ABI genes (gi 108272842, gi 108267643, gi 108266499, gi 108272690, gi 108276098) were identified in NCBI annotation, with absence of ABI3b. The genomic region encode ABI3b gene in channel catfish was predicted to be “formin-like protein 5” (gi 108273397) in NCBI annotation. However, phylogenetic (Supplementary Fig. 2) and syntenic analysis (Supplementary Fig. 3) supported our annotation that this region encodes ABI3b.
The copy numbers of NCK and ABI genes in teleost fish and tetrapods should provide insights to the evolution of NCK and ABI genes. Mammals and amphibian genomes contain single copy of NCK and ABI genes, while teleost fish genomes contain one to three copies of NCK and ABI genes. Most of the teleost fish contain one or two copies of NCK and ABI genes. The duplicated catfish NCK and ABI genes were likely derived from the teleost-specific whole genome duplication as well as lineage-specific tandem duplications because they were found on separate chromosomes (Hoegg et al., 2004).
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