Abstract
Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.
Highlights
Understanding the genetic regulation of traits of importance to farmed animal production is key to guiding optimal use of genomic in formation in selective breeding programmes [1,2]
Families where both parents were heterozygous for the quantitative trait locus (QTL) were identified (n = in 2007, and n = in 2008), and from each of those families two ho mozygous resistant (RR) fish and two homozygous susceptible (SS) fish were selected for pooling of genomic DNA at equimolar concentrations and sequencing (2 x pools of RR fish and 2 x pools of SS fish, sequence reads available at NCBI Short Read Archive PRJNA614520) Following alignment of sequence reads to the Atlantic salmon reference genome (GenBank accession GCA_000233375.4), variants were called and the allele frequency dif ferences between the RR and SS pools were calculated (Fig. 1A)
Fine mapping of the major infectious pancreatic necrosis virus (IPNV) resistance QTL using whole genome sequencing combined with differential expression between homozygous resistant and homozygous susceptible fish both pointed to nae1 as a strong candidate causative gene
Summary
Understanding the genetic regulation of traits of importance to farmed animal production is key to guiding optimal use of genomic in formation in selective breeding programmes [1,2] Such production traits are typically underpinned by a polygenic architecture, with many loci of minor effect contributing to their heritability [1,2]. There are exceptions where major effect loci segregate within farmed animal populations, and a single genomic region underlies the majority of genetic variation in a trait of interest One such example is the case of host resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon, a species with a global aquaculture production of >2.4 million tonnes, worth >$17.1 billion USD in 2018 [3]. Identification of functional mecha nisms and variants leads to new opportunities for disease control [8,9,10,11], including genome editing to introduce resistance to salmonid strains or species which do not carry the major resistance allele for the QTL
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