*2.2.13 DNA vaccines*

*Synthetic Biology - New Interdisciplinary Science*

*2.2.11 Genomic marker development*

to detect the association.

*2.2.12 Metagenomics*

output of NGS technologies. Original RAD-Seq was first described by Baird et al. [20] and several variants of this methodology have been described since then [21]. But, only the original RAD-Seq [20], 2b-RAD and ddRAD are extensively used in aquaculture research. In aquaculture, RAD-Seq has been used in genetic mapping [22], reference genome assembly sex determination loci mapping [23–26], etc. Some of the main reasons for its instant success is that RAD-Seq does not require any prior genomic knowledge, it allows generation of population-specific genotype data (i.e., no ascertainment bias) and it offers flexibility in terms of desired marker density across the genome. The use of different REs or innovative modifications to the base technique allows a high level of control over the number of markers obtained for a specific study. RAD-Seq and similar techniques are also amenable tools for aquaculture breeding, where genetic markers have typically been used in family assignment and pedigree reconstruction [27]. Mass spawning species are common in aquaculture, where mixed rearing and unknown parental contribution necessitate the use of genotyping for family-based breeding. RAD-Seq potentially facilitates a single experiment whereby pedigrees are reconstructed, genetic diversity is quantified, QTL are mapped, and genomic breeding values calculated [28].

Most of the genetic improvement in fish and shellfish species to date has been made through the use of traditional selective breeding of Atlantic salmon, Rainbow trout, tilapia and many other fish [29]. Notably, spontaneous mutations in the genome create genetic variability (or polymorphism) and this variability can be an effective means to analyze fish trait and geological pedigree. Boom in whole genome sequencing technology, though still costly, encourage fish researchers to investigate genomic marker's potential in selective breeding and aquaculture production. There are several available markers for fish research: AFLP, RAPD, etc., but most prevalent ones are microsatellite and SNPs. Microsatellite markers, identified using microsatellite sequence enriched genomic library or Expressed tagged sequence library, are simple tandem sequence repeats scattered across the genome and used increasingly in aquaculture species [29]. SNPs are generally identified using in depth genome sequencing and require huge financial and bioinformatical investment. MAS (marker assisted selection) is useful for traits that are difficult to measure on breeding candidates, particularly when they are largely linked to QTL (quantitative trait loci). With the help of MAS and GS (genomic selection), several studies have demonstrated increased accuracy of breeding value predictions in growth and disease resistance in yellowtail and Atlantic salmon [30–33]. Nevertheless, this approach requires a great amount of detailed information in order to choose which gene explains the greatest effect and to have sufficient power

There are two main methods for studying the microbiome using highthroughput sequencing: marker-gene studies and whole-genome-shotgun (WGS) metagenomics. While marker-gene studies, amplify a particular gene (16S rRNA for bacteria/archaea, 18S for fungi), metagenomics refer to the sequencing of DNA from the entire genome of samples obtained directly from the environment (water, soil) or tissues. Advances in metagenomics have themselves been driven by advances in second- and third-generation sequencing technologies, which are now capable of producing hundreds of gigabases of DNA sequenced data at a very low cost [34]. Unlike bacteria that use the 16S ribosomal RNA as a common gene

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DNA vaccines are composed of bacterial plasmids which has two units-antigen expressing unit that comprises of promoter/enhancer sequences, antigen coding and polyadenylation sequences; and the production unit comprising of sequences necessary for plasmid amplification and selection [39]. The vaccine inserts are constructed by molecular cloning and transformed into bacterial cells, and the purified plasmid DNA is injected into fish. Hansen et al. [40] first introduced vaccination in fish by injecting plasmid constructs encoding viral glycoprotein directly into skeletal muscle of common carp that resulted in efficient protection of the fish against rhabdoviruses. More than 20 different virus DNA vaccines have been developed experimentally for prophylactic use in fish targeting viruses such as rhabdoviridae, orthomyxoviridae, togaviridae and nodaviridae [41, 42]. However, despite this huge prospect, DNA vaccines for farmed animals remain at the moment experimental. DNA vaccines seem to be more harmless and more stable than ordinary vaccines [42]. Plasmids are non-viable and do not multiply, and therefore have a low risk of developing secondary disease and infection. The main concern about the potential DNA vaccines is that they might integrate into the host genome and generate immune responses. However, extensive surveys have found little evidence of integration, and the merger risk appears to be less than normal mutation. Significant advantages of these vaccines include cheapness, simplicity of


#### **Table 1.**

*Prevalent examples of established disease diagnostics in aquaculture.*

production and consumption, transport and higher resistance. The other important feature of these vaccines is the ability to put several antigens in the plasmid, resulting in immunization against all agents [43]. In 2005, APEX-IHN (Novartis/Elanco) became the first DNA vaccine licensed for commercial use in aquaculture for protection of Atlantic salmon against Infectious Hematopoietic Necrosis Virus (IHNV) in British Colombia. In 2017, the European Commission through the European Medicines Agency (EMA) granted marketing authorization of CLYNAV (Elanco), a polyprotein-encoding DNA vaccine against Salmon Pancreas Disease Virus (SPDV) infection in Atlantic salmon (*Salmo salar*) for use within the EU. However, administration of vaccines typically requires individual handling and treatment of all production fish, which can be expensive and impractical in a large-scale production environment.
