*2.1.2 Maintenance of the environment and biodiversity*

Since farmed fish are selected and bred for certain genetic criteria like size, quick growth and hardiness, escaped species can become invasive and pose a massive threat to global biodiversity. The ever-growing aquaculture industries also have to bear the public concern in regard to pollution and other environmental effects and thus maintaining and sustaining the environment is of paramount importance. Attention to genetic variability and biodiversity in aquaculture development, proper stock maintenance and aquatic resource management are therefore crucial elements for sustainable environment. In this sense, traceability tools are essential to assess the impact of aquaculture escapees in natural populations or distinguish the farmed and wild specimens.

## *2.1.3 Reproductive medley*

Reproduction is crucial for steady and quality seed supply and hence of utmost importance for aquaculture sustainability. Fish gonadal development is influenced by intrinsic (genetics, growth, behavior, etc.), and extrinsic (temperature, hormone, environmental pollution, etc.) factors. Though, large diversity of aqua animals has its own advantages, each species has distinct reproductive and embryonic development biology that hinders the timely breeding and smooth progression of commercial aquaculture. For instance, some gonochoristic fish harbors sex chromosome while others do not, and several commercially lucrative fish sequentially changes their sex. Moreover, some hybrids tend to grow bigger with the expense of reproductive unfitness (e.g., hybrids of Atlantic and pacific salmon).

### *2.1.4 Improper growth*

Fish growth largely depends on feeding, environment and genetic background. For example, farmed Atlantic salmon tend to grow faster than wild ones, and genetically modified (GM) farmed salmons are even better. Though FDA recently approved GM salmon, till date it is not ethically preferable to use GM fish for commercial aquaculture. There are few more success stories of using myostatin knockout to improve growth of tilapia, red sea bream and common carp; however, yellow catfish [12] did not display similar results, suggesting functional variation among species.

#### **2.2 Application of molecular cloning in aquaculture**

#### *2.2.1 Restriction enzyme/endonuclease digestion*

Restriction enzymes (or restriction endonucleases, RE) are enzymes or better known as "molecular scissors" that recognize and cleave the DNA into fragments at or near specific "recognition" sites. The DNA fragments are observed by gel electrophoresis and the pattern of bands are used to generate the "fingerprint" of a particular DNA molecule. The cut DNA can be observed by gel electrophoresis and the pattern of bands compiled to create a restriction enzyme map [13]. This map is useful to identify and characterize a particular DNA region and analyze genetic variation. Restriction enzymes are used to manipulate DNA and are vital tools in molecular cloning. They form the basis for several diagnostic tools like RFLP, AFLP, Southern blotting, etc. For instance, RFLP recognizes size variations, and in combination with PCR can be used to reduce the labor-intensive DNA isolation for RFLP analysis [14]. SNPs (single-nucleotide polymorphism) or INDELs change the restriction endonuclease recognition sites that cause differences in restriction fragment lengths. AFLP technique is based on cutting with two Res (one average (e.g., EcoRI), and another rare (e.g., MseI) cutter), ligation of adapters to these restriction fragments and followed by a PCR-based selective amplification with adapter-specific radioactive or fluorescent-labeled primers.

#### *2.2.2 Random amplified polymorphic DNA (RAPD)*

RAPDs are DNA fragments that are amplified using short random primers (~10 bp) and are used to detect polymorphisms. RAPDs are randomly distributed throughout the genome and have high abundance. This technique is quick and easy and requires low quantity of DNA. Fish pathogens have been studied using RAPD, but problems with reproducibility and risks of contamination render the method unsuitable as a stand-alone method of diagnosis. However, RAPD can be a useful technique as a first step in the development of specific primers or probes and has been used in such a way in the study of bacteria.

#### *2.2.3 Polymerase chain reaction (PCR)*

The polymerase chain reaction is a robust technique used to produce large copies of the target DNA sequence by amplifying the specific region of interest. The reaction includes template DNA, primers, polymerase enzyme to catalyze creation of new copies of DNA, and nucleotides to form the new copies. The template DNA can be collected from sample tissue, blood, serum, fluid, mucus or can be a purified DNA. The principle of PCR is based on the repetitive cycling of denaturation, annealing and extension. Each copy of the DNA then serves as another template for further amplification and copy number of PCR products then doubles in each cycle. After "n" rounds of replication, 2n copies of the target sequence are theoretically produced. After thirty cycles, PCR can produce 230 or more than ten billion copies of a single target DNA sequence. The PCR product can be detected by gel electrophoresis. The whole process just needs 2–5 h depending on the number and types of nucleotide. PCR has distinct advantages over conventional microbiological diagnostic methods as it can detect slow growing and unculturable pathogens. PCR is faster, extremely efficient and sensitive and can be used to amplify sequences from wide variety of samples even if they only have a small amount of DNA. Some of the shortcomings of PCR are the false positive results from DNA contamination, limited detection platform for simultaneous identification of multiple samples, etc.

**55**

*Applied Molecular Cloning: Present and Future for Aquaculture*

single PCR is not sufficient to identify pathogen.

be free of genomic DNA to avoid false positive results.

*2.2.4 Loop-mediated isothermal amplification (LAMP)*

*2.2.5 Fluorescence in situ hybridization (FISH)*

In most cases, the target DNA sequence is the rRNA operon and in bacteria, the most frequently used is the variable region of the evolutionary conserved 16S rRNA gene. Nevertheless, other types of genes or sequences of unknown sequences can

To overcome the shortcoming and to increase the diagnostic capacity of conventional PCR, multiplex PCR was developed to simultaneously amplify several target sequences by using more than one pair of primers. It can detect multiple pathogens, which save time and cost without compromising test utility, but might require further analysis such as DNA sequencing to confirm the identity of the species. Nested PCR, which uses two pairs of primers and two successive PCR run, was developed to increase specificity and sensitivity of conventional PCR. The first set of primers is used to amplify target sequence in first run and the PCR products are used as template for the second run and amplification is conducted with the second set of primers. Though, it is popular for unknown/homologous gene identification, due to the lengthy process and complexity, this type of PCR is limited to cases where

Though DNA is reliable, RNA is often a more accurate indicator of viable microorganism. Therefore, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was developed to first synthesize cDNA from RNA by reverse transcription (RT) and later amplify the cDNA by PCR. However, for effective detection, sufficient amount of detectable RNA concentrations is required, and the RNA sample should

Most recently, real time PCR is used to detect, confirm and quantify PCR products at "real time" during the amplification process using Fluorescent dyes. Two types of dyes are generally used; one is the use of non-sequence specific dyes like SYBR green I or ethidium bromide and the second is the use of fluorescently labeled internal probe like TaqMan, FRET (fluorescence resonance energy transfer), etc. The real time PCR has three novel features—temperature cycling occurs considerable faster than in standard PCR assays, hybridization of specific DNA probes occurs continuously during the amplification and the dye fluoresces only when hybridization takes place. This technique is quick and convenient, and with the recent introduction of multiplex real time PCR, detection of multiple targets in a single reaction can be achieved at cheaper cost, shorter time and faster diagnosis.

It is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers to recognize six distinct regions of the target DNA. Unlike PCR, LAMP is carried out in constant temperature (60–65°C) using an auto-cycling strand displacement DNA synthesis and does not require thermal cycler. The amplified product can be detected as white precipitate or yellow green color solution after addition of SYBR Green. It is cost effective and when combined with reverse transcription, this method can also amplify RNA sequences with high efficiency. It can be used to detect the identification of genus and species-specific parasites. However, this technique is not effective for detection of different pathogens simultaneously.

*In situ* hybridization refers to detection of DNA or RNA on actual tissues, cells, or any biological sample in their natural positions within a chromosome, by using a complementary probe. *ISH* correlates DNA localization and mRNA expression

*DOI: http://dx.doi.org/10.5772/intechopen.88197*

also be used.

*Synthetic Biology - New Interdisciplinary Science*

*2.2.1 Restriction enzyme/endonuclease digestion*

**2.2 Application of molecular cloning in aquaculture**

adapter-specific radioactive or fluorescent-labeled primers.

*2.2.2 Random amplified polymorphic DNA (RAPD)*

been used in such a way in the study of bacteria.

*2.2.3 Polymerase chain reaction (PCR)*

cycle. After "n" rounds of replication, 2n

Restriction enzymes (or restriction endonucleases, RE) are enzymes or better known as "molecular scissors" that recognize and cleave the DNA into fragments at or near specific "recognition" sites. The DNA fragments are observed by gel electrophoresis and the pattern of bands are used to generate the "fingerprint" of a particular DNA molecule. The cut DNA can be observed by gel electrophoresis and the pattern of bands compiled to create a restriction enzyme map [13]. This map is useful to identify and characterize a particular DNA region and analyze genetic variation. Restriction enzymes are used to manipulate DNA and are vital tools in molecular cloning. They form the basis for several diagnostic tools like RFLP, AFLP, Southern blotting, etc. For instance, RFLP recognizes size variations, and in combination with PCR can be used to reduce the labor-intensive DNA isolation for RFLP analysis [14]. SNPs (single-nucleotide polymorphism) or INDELs change the restriction endonuclease recognition sites that cause differences in restriction fragment lengths. AFLP technique is based on cutting with two Res (one average (e.g., EcoRI), and another rare (e.g., MseI) cutter), ligation of adapters to these restriction fragments and followed by a PCR-based selective amplification with

RAPDs are DNA fragments that are amplified using short random primers (~10 bp) and are used to detect polymorphisms. RAPDs are randomly distributed throughout the genome and have high abundance. This technique is quick and easy and requires low quantity of DNA. Fish pathogens have been studied using RAPD, but problems with reproducibility and risks of contamination render the method unsuitable as a stand-alone method of diagnosis. However, RAPD can be a useful technique as a first step in the development of specific primers or probes and has

The polymerase chain reaction is a robust technique used to produce large copies of the target DNA sequence by amplifying the specific region of interest. The reaction includes template DNA, primers, polymerase enzyme to catalyze creation of new copies of DNA, and nucleotides to form the new copies. The template DNA can be collected from sample tissue, blood, serum, fluid, mucus or can be a purified DNA. The principle of PCR is based on the repetitive cycling of denaturation, annealing and extension. Each copy of the DNA then serves as another template for further amplification and copy number of PCR products then doubles in each

cally produced. After thirty cycles, PCR can produce 230 or more than ten billion copies of a single target DNA sequence. The PCR product can be detected by gel electrophoresis. The whole process just needs 2–5 h depending on the number and types of nucleotide. PCR has distinct advantages over conventional microbiological diagnostic methods as it can detect slow growing and unculturable pathogens. PCR is faster, extremely efficient and sensitive and can be used to amplify sequences from wide variety of samples even if they only have a small amount of DNA. Some of the shortcomings of PCR are the false positive results from DNA contamination, limited detection platform for simultaneous identification of multiple samples, etc.

copies of the target sequence are theoreti-

**54**

In most cases, the target DNA sequence is the rRNA operon and in bacteria, the most frequently used is the variable region of the evolutionary conserved 16S rRNA gene. Nevertheless, other types of genes or sequences of unknown sequences can also be used.

To overcome the shortcoming and to increase the diagnostic capacity of conventional PCR, multiplex PCR was developed to simultaneously amplify several target sequences by using more than one pair of primers. It can detect multiple pathogens, which save time and cost without compromising test utility, but might require further analysis such as DNA sequencing to confirm the identity of the species.

Nested PCR, which uses two pairs of primers and two successive PCR run, was developed to increase specificity and sensitivity of conventional PCR. The first set of primers is used to amplify target sequence in first run and the PCR products are used as template for the second run and amplification is conducted with the second set of primers. Though, it is popular for unknown/homologous gene identification, due to the lengthy process and complexity, this type of PCR is limited to cases where single PCR is not sufficient to identify pathogen.

Though DNA is reliable, RNA is often a more accurate indicator of viable microorganism. Therefore, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was developed to first synthesize cDNA from RNA by reverse transcription (RT) and later amplify the cDNA by PCR. However, for effective detection, sufficient amount of detectable RNA concentrations is required, and the RNA sample should be free of genomic DNA to avoid false positive results.

Most recently, real time PCR is used to detect, confirm and quantify PCR products at "real time" during the amplification process using Fluorescent dyes. Two types of dyes are generally used; one is the use of non-sequence specific dyes like SYBR green I or ethidium bromide and the second is the use of fluorescently labeled internal probe like TaqMan, FRET (fluorescence resonance energy transfer), etc. The real time PCR has three novel features—temperature cycling occurs considerable faster than in standard PCR assays, hybridization of specific DNA probes occurs continuously during the amplification and the dye fluoresces only when hybridization takes place. This technique is quick and convenient, and with the recent introduction of multiplex real time PCR, detection of multiple targets in a single reaction can be achieved at cheaper cost, shorter time and faster diagnosis.

### *2.2.4 Loop-mediated isothermal amplification (LAMP)*

It is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers to recognize six distinct regions of the target DNA. Unlike PCR, LAMP is carried out in constant temperature (60–65°C) using an auto-cycling strand displacement DNA synthesis and does not require thermal cycler. The amplified product can be detected as white precipitate or yellow green color solution after addition of SYBR Green. It is cost effective and when combined with reverse transcription, this method can also amplify RNA sequences with high efficiency. It can be used to detect the identification of genus and species-specific parasites. However, this technique is not effective for detection of different pathogens simultaneously.

#### *2.2.5 Fluorescence in situ hybridization (FISH)*

*In situ* hybridization refers to detection of DNA or RNA on actual tissues, cells, or any biological sample in their natural positions within a chromosome, by using a complementary probe. *ISH* correlates DNA localization and mRNA expression

with morphological findings [15]. Most current *in situ* hybridization methods use *FISH* [16, 17] in which fluorescent labeled pieces of DNA or RNA (probe) hybridize to target nucleic acid in cells under appropriate conditions. These labeled cells can then be visualized by flow cytometry or fluorescence microscopy. *FISH* can be used on formalin fixed paraffin embedded tissues, frozen tissues, etc. The technique has also been used to detect bacterial and viral DNA in an infected cell. Since the probe has to reach the target inside the cells, only probes that are small (~300 bases) can be used for tissue penetration, hence sensitivity is limited to the accessibility of the target in the cell.

#### *2.2.6 Molecular padlock probes (MPP)*

Padlock probes (PLPs) are single stranded long oligonucleotides whose 5′ and 3′ ends are complementary to two immediately adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, effectively circularizing the probe with a nick. DNA ligase is added to convert this linear PLP into a covalently closed circular molecule. Single strand specific DNA exonucleases can be used to "chew up" the linear strands and only make available the intact circular molecules. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray. However, synthesis of long probes can be little expensive as compared to short primers for PCR. At present, the most common application for PLPs is the detection of single nucleotide polymorphisms (SNPs) and multiplex pathogen detection assays.

#### *2.2.7 Rolling circle amplification (RCA)*

RCA is an isothermal enzymatic process where short DNA/RNA primer amplified to form a long single stranded DNA/RNA using a circular DNA template and special DNA/RNA polymerases. The product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. By manipulating the circular template, RCA can be employed to generate complex DNA nanostructures such as DNA origami, nanotubes, nanoribbons and DNA based metamaterials which can be used for bio-detection, drug delivery, etc. Millard et al. [18] combined RCA, MPP and hyperbranching (Hbr) to develop a multiplex detection assay for IHNV and ISAV.

#### *2.2.8 Microarray*

This technology is used to assess expression rate of thousands of genes and identify wide range of pathogens from complex samples in one single reaction. This technique usually involves hybridization of DNA with large number of probes and can overcome the shortcomings of multiplex PCR, which can detect only a maximum of six pathogens at a time. There are two types of DNA microarrays that are widely used—cDNA microarrays and oligonucleotide/DNA chips. There are a number of ways of using DNA microarrays. One method is the use of fluorescent labeled DNA sequences that are hybridized to the microarray slide. The data is detected by fluorescent array detection and analyzed by computer programs. The second and more practical method is the use of fluorescent labeled competitor oligonucleotide. When target DNA does not hybridize to the tethered oligonucleotide in the microarray, fluorescent labeled competitor oligonucleotide will bind to the tethered oligonucleotide on the chip and displace the test DNA. Then the fluorescent microarray detector and computer program will analyze the fluorescent array for the presence or absence of the species/strain specific DNA sequence. Microarray does not require

**57**

*Applied Molecular Cloning: Present and Future for Aquaculture*

clear length differences between PCR products and therefore, PCR assays can be designed to generate smaller sized amplicons that can improve efficiency and probability of template recovery from degraded DNA and reduces PCR template biasedness. Compared to traditional nucleic acid hybridization with membranes, microarrays offer the additional advantages of high density, high sensitivity, rapid detection, lower cost, automation, and low background levels. Since most of the pathogens genetic sequences are known, oligonucleotide probes complementary to all pathogens can be used for microarray. Although the set-up cost for the use of DNA microarrays is high, once the equipment is available and microarrays are prepared, cost per unit of sample analyzed becomes low. In the post-genome sequencing era, microarrays have been developed from model and non-model fish and have the possibility of heterologous application. Though majority of them are publicly available, however, they vary in type, size, complexity, methodological development and motivation and degree of annotation, so it is advisable to carefully

DNA sequencing is used to determine the four chemical blocks—adenine, guanine, thymine and cytosine, that make up the DNA molecule. The sequence information can help determine changes in the gene that may cause disease. First generation sequencing techniques include the Sanger method and the Maxam-Gilbert techniques. Maxam-Gilbert are based on chemical modification of DNA and subsequent cleavage at specific bases while Sanger method requires that each read start be cloned for production of single-stranded DNA. Maxam–Gilbert sequencing is less popular due to its technical complexity. The chain-terminator method or Frederick Sanger method, which uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators, became a popular method of DNA sequencing due to its greater efficiency, use of fewer toxic chemicals and lower amounts of radioactivity than Maxam-Gilbert method. Second generation sequencing includes technologies such as Illumina and Ion Torrent that produce massive parallel sequencing of short read length of reads of DNA (150–400 bp), which require extensive assembly. Third generation sequencing method includes PacBio and ONT and involves sequencing through extended repetitive regions in the genome to produce much longer reads (6–20 kb) but far fewer reads per run (typically hundreds of thousands). The second and third generation sequencing methods, collectively known as the next generation sequencing (NGS) or high throughput sequencing allows the sequencing of DNA and RNA more quickly and cheaply. The goal of NGS is to investigate functional genome, epigenome and transcriptome elements in cells and tissues, and their temporal expression, which permits the definition of variation in gene expression among the different types of tissue, organs or life stages of the target organism. Over the past decade, the cost of NGS has decreased significantly, making it possible to use non-model fish species to investigate emerging environmental issues, understand the cell-cell interactions, and whole organismal physiology. To cope with it, bioinformatics is also rapidly evolving and new algorithms are being published. It is expected that NGS with bioinformatics is the way to revolutionize the field of fisheries and might also help clarify the previous findings and dogmas

Restriction-site associated DNA sequencing (RAD sequencing or RAD-Seq) combine the use of genome complexity reduction with REs and the high sequencing

*DOI: http://dx.doi.org/10.5772/intechopen.88197*

select the array beforehand [19].

prevalent in aquaculture and biology.

*2.2.10 RAD sequencing*

*2.2.9 DNA sequencing*

#### *Applied Molecular Cloning: Present and Future for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.88197*

clear length differences between PCR products and therefore, PCR assays can be designed to generate smaller sized amplicons that can improve efficiency and probability of template recovery from degraded DNA and reduces PCR template biasedness. Compared to traditional nucleic acid hybridization with membranes, microarrays offer the additional advantages of high density, high sensitivity, rapid detection, lower cost, automation, and low background levels. Since most of the pathogens genetic sequences are known, oligonucleotide probes complementary to all pathogens can be used for microarray. Although the set-up cost for the use of DNA microarrays is high, once the equipment is available and microarrays are prepared, cost per unit of sample analyzed becomes low. In the post-genome sequencing era, microarrays have been developed from model and non-model fish and have the possibility of heterologous application. Though majority of them are publicly available, however, they vary in type, size, complexity, methodological development and motivation and degree of annotation, so it is advisable to carefully select the array beforehand [19].

### *2.2.9 DNA sequencing*

*Synthetic Biology - New Interdisciplinary Science*

*2.2.6 Molecular padlock probes (MPP)*

(SNPs) and multiplex pathogen detection assays.

multiplex detection assay for IHNV and ISAV.

*2.2.8 Microarray*

*2.2.7 Rolling circle amplification (RCA)*

target in the cell.

with morphological findings [15]. Most current *in situ* hybridization methods use *FISH* [16, 17] in which fluorescent labeled pieces of DNA or RNA (probe) hybridize to target nucleic acid in cells under appropriate conditions. These labeled cells can then be visualized by flow cytometry or fluorescence microscopy. *FISH* can be used on formalin fixed paraffin embedded tissues, frozen tissues, etc. The technique has also been used to detect bacterial and viral DNA in an infected cell. Since the probe has to reach the target inside the cells, only probes that are small (~300 bases) can be used for tissue penetration, hence sensitivity is limited to the accessibility of the

Padlock probes (PLPs) are single stranded long oligonucleotides whose 5′ and 3′ ends are complementary to two immediately adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, effectively circularizing the probe with a nick. DNA ligase is added to convert this linear PLP into a covalently closed circular molecule. Single strand specific DNA exonucleases can be used to "chew up" the linear strands and only make available the intact circular molecules. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray. However, synthesis of long probes can be little expensive as compared to short primers for PCR. At present, the most common application for PLPs is the detection of single nucleotide polymorphisms

RCA is an isothermal enzymatic process where short DNA/RNA primer amplified to form a long single stranded DNA/RNA using a circular DNA template and special DNA/RNA polymerases. The product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. By manipulating the circular template, RCA can be employed to generate complex DNA nanostructures such as DNA origami, nanotubes, nanoribbons and DNA based metamaterials which can be used for bio-detection, drug delivery, etc. Millard et al. [18] combined RCA, MPP and hyperbranching (Hbr) to develop a

This technology is used to assess expression rate of thousands of genes and identify wide range of pathogens from complex samples in one single reaction. This technique usually involves hybridization of DNA with large number of probes and can overcome the shortcomings of multiplex PCR, which can detect only a maximum of six pathogens at a time. There are two types of DNA microarrays that are widely used—cDNA microarrays and oligonucleotide/DNA chips. There are a number of ways of using DNA microarrays. One method is the use of fluorescent labeled DNA sequences that are hybridized to the microarray slide. The data is detected by fluorescent array detection and analyzed by computer programs. The second and more practical method is the use of fluorescent labeled competitor oligonucleotide. When target DNA does not hybridize to the tethered oligonucleotide in the microarray, fluorescent labeled competitor oligonucleotide will bind to the tethered oligonucleotide on the chip and displace the test DNA. Then the fluorescent microarray detector and computer program will analyze the fluorescent array for the presence or absence of the species/strain specific DNA sequence. Microarray does not require

**56**

DNA sequencing is used to determine the four chemical blocks—adenine, guanine, thymine and cytosine, that make up the DNA molecule. The sequence information can help determine changes in the gene that may cause disease. First generation sequencing techniques include the Sanger method and the Maxam-Gilbert techniques. Maxam-Gilbert are based on chemical modification of DNA and subsequent cleavage at specific bases while Sanger method requires that each read start be cloned for production of single-stranded DNA. Maxam–Gilbert sequencing is less popular due to its technical complexity. The chain-terminator method or Frederick Sanger method, which uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators, became a popular method of DNA sequencing due to its greater efficiency, use of fewer toxic chemicals and lower amounts of radioactivity than Maxam-Gilbert method. Second generation sequencing includes technologies such as Illumina and Ion Torrent that produce massive parallel sequencing of short read length of reads of DNA (150–400 bp), which require extensive assembly. Third generation sequencing method includes PacBio and ONT and involves sequencing through extended repetitive regions in the genome to produce much longer reads (6–20 kb) but far fewer reads per run (typically hundreds of thousands). The second and third generation sequencing methods, collectively known as the next generation sequencing (NGS) or high throughput sequencing allows the sequencing of DNA and RNA more quickly and cheaply. The goal of NGS is to investigate functional genome, epigenome and transcriptome elements in cells and tissues, and their temporal expression, which permits the definition of variation in gene expression among the different types of tissue, organs or life stages of the target organism. Over the past decade, the cost of NGS has decreased significantly, making it possible to use non-model fish species to investigate emerging environmental issues, understand the cell-cell interactions, and whole organismal physiology. To cope with it, bioinformatics is also rapidly evolving and new algorithms are being published. It is expected that NGS with bioinformatics is the way to revolutionize the field of fisheries and might also help clarify the previous findings and dogmas prevalent in aquaculture and biology.

#### *2.2.10 RAD sequencing*

Restriction-site associated DNA sequencing (RAD sequencing or RAD-Seq) combine the use of genome complexity reduction with REs and the high sequencing 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].

### *2.2.11 Genomic marker development*

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 to detect the association.

#### *2.2.12 Metagenomics*

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

**59**

**Table 1.**

*Applied Molecular Cloning: Present and Future for Aquaculture*

and molecular characterization using PCR ([38], **Table 1**).

for their identification, viruses lack a single common gene for their identification which makes it difficult to monitor their population dynamics in different aquatic environments [35]. Metagenomics also holds the promise of revealing the genomes of the majority of microorganisms that cannot be readily obtained in pure culture [36]. Breitbart et al. [37] have shown that it is possible to sequence entire genomes of uncultured marine viruses using metagenomics. For metagenomic sequences linked to novel diseases, there is need to isolate the virus involved followed by verification using conventional diagnostic approaches such as cell culture to exhibit the cytopathic effect (CPE), morphological characterization using electron microscopy,

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

**Pathogens Detection method** *V. vulnificus*, *L. anguillarum*, *P. damselae*, *V. parahaemolytocus* Multiplex PCR, DNA

*Y. ruckeri*, *A. salmonicida*, *F. psychrophilum* Multiplex PCR Infectious salmon anemia virus (ISAV) RT PCR *Myxobolus cerebralis* Real time PCR *Edwardsiella tarda* LAMP

*Infectious hematopoietic necrosis virus (IHNV) & ISAV* Molecular padlock

*A. salmonicida*, *E. ictaluri* and *F. psychrophilum* PCR and DNA

Aeromonas (*A. hydrophila*, *A. sobria*, *A. caviae* and *A. veronii*) Multiplex PCR *P. salmonis* (Salmonid Rickettsial Septicaemia) PCR-RFLP

*R. salmoninarum*, *A. salmonicida*, *E. ictaluri*, *F. columnare*, *F. psychrophilum*,

*Prevalent examples of established disease diagnostics in aquaculture.*

*Y. ruckeri*, *P. salmonis*, *T. maritimum*

microarray

DNA microarray

microarrays

*DOI: http://dx.doi.org/10.5772/intechopen.88197*

*2.2.13 DNA vaccines*

#### *Applied Molecular Cloning: Present and Future for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.88197*

for their identification, viruses lack a single common gene for their identification which makes it difficult to monitor their population dynamics in different aquatic environments [35]. Metagenomics also holds the promise of revealing the genomes of the majority of microorganisms that cannot be readily obtained in pure culture [36]. Breitbart et al. [37] have shown that it is possible to sequence entire genomes of uncultured marine viruses using metagenomics. For metagenomic sequences linked to novel diseases, there is need to isolate the virus involved followed by verification using conventional diagnostic approaches such as cell culture to exhibit the cytopathic effect (CPE), morphological characterization using electron microscopy, and molecular characterization using PCR ([38], **Table 1**).
