Modi K, P., Sonalismita M., Narsingh K., Kalpita T. and Deepak Agarwal*
Institute of Fisheries Postgraduate Studies, TNJFU, OMR-campus, Chennai, India-603103,
Aquaculture is the fastest growing sector that provides highly nutritious food to mitigate global challenges such as hunger, malnutrition, medicinal therapeutics and human health. In the past few decades, the fisheries sector possesses potential threats of overfishing, anthropogenic activities, germplasm degradation, climate change and disease (viral, bacterial and parasitic). Advance genomics technology such as Next generation sequencing (NGS), Single molecule real-time sequencing (SMRT), Nanopore sequencing, Cas9-assisted targeting chromosome segments (CATCH) and quantum dot and signal amplification by exchange reaction (QD-SABER) were used to assess & prevent these challenges. In the last two decades, 594 fish genomes have been sequenced, which is 1.85% of the total reported fish species (34,000).
Genome mapping technology allows the identification of novel and causal genetic variation, sex determination, disease, biomass prediction (abundance and spawning stock biomass), andgenetic basis performance and trait determination for selective breeding programs.
What is genome mapping?
A genome contains information on all functional and non-functional DNA sequences of an organism.Genome mapping is a technique used to locate genes on a chromosome and measure the distances between them.It includes short DNA sequences regulatory sites that turn genes on and off or the genes themselves. There are two types of the genome mapping
1. Genetic mapping: it shows the location or arrangement of genes and genetic markers along the chromosomes based on how frequently they are inherited together.These maps rely on recombination and crossing over. Numerous DNA-based genetic markers such as restrictionfragment length polymorphism(RFLP), random amplifiedpolymorphic DNA (RAPD), amplified fragment length polymorphism(AFLP), sequence-tagged site (STS), microsatellitesor simple sequence repeats (SSRs), and single nucleotide polymorphism(SNP) were developed to detect polymorphism between two parental lines.
2.Physical mapping: Physical mapping represents chromosomes and the physical distance between known DNA sequences (including genes) by measuring the number of base pairs (A-T, C-G) between them.Chemically staining andviewing whole chromosomes using techniques such as in situhybridization (ISH), C-banding and fiber-fluorescence in situhybridization (FISH) were used for physical mapping. These are further subdivided into three groups: (a.) Cytogenic maps (chromosomal maps) (b.) Radiation hybrid maps and (c.) Sequence maps.
Based on the application and drawbacks leads to the development of the different generation of DNA sequencing platform mentioned below:
Advance Genome mapping techniques
1. Next generation sequencing (NGS):
NGS works on sequencing by synthesis method and reversible dye-terminators allow for the detection of single bases as they are inserted into DNA strands. It directly uses genomic DNA /complementary DNA libraries for sequencing.It has numerous applications, such as transcription analysis, whole genome sequencing, methylation profiling, small RNA discovery, metagenomics, targeted region sequencing and genome wide analysis of protein-nucleic acid interactions. The basic workflow of the NGS is illustrated below (Fig. 1).
2. Single Molecule Real-Time sequencing (SMRT)
Single molecule real time sequencing allows sequencing from individual DNA molecules without amplifying and fragmenting the target sequence. It works on the sequencing while synthesizing principle. SMRT uses 4-color fluorescence labeled dNTPs and zero-mode waveguides (ZMW) to sequence single DNA molecules whereas the DNA template will be captured by DNA polymerase. Different bases will emit different fluorescent lights during the base pairing phase and the wavelength and peak value of fluorescent light can be used to determine the type of base entering. The phosphate group of dNTPis the place where the fluorescence signal is attached,and when the subsequent dNTP is synthesized, the phosphate group is automatically detached, ensuring detection consistency, accelerating detection, and working with the high-resolution optical detection system for real-time detection. The steps of the SMRT sequencing are presented below (Fig. 2).
Fluorescent phospholinked labeled nucleotides are introduced into the zero-mode waveguides.
The base being incorporated is held in the detection volumefor tens of milliseconds, producing a bright flash of light.
The phosphate chain is cleaved, releasing the attached dye molecule. The process repeats.
3. Nanopore sequencing
It’s a fourth-generation DNA sequencing technology developed by Oxford Nanopore Technologies Ltd. and is the most powerful method for the rapid generation of long-read sequences.Without PCR amplification or chemical labeling of the samplesingle molecule of DNA or RNA can be sequenced using nanopore sequencing.Single strands of DNA or RNA molecules are passed through a tiny protein channel (nanopore)embeddedin an electrically resistant membrane.The ion current changes as the DNA molecule moves through the nanopore, which provides information on the sequence and base modifications (Fig. 3).
4. Cas9-Assisted Targeting Chromosome segments (CATCH)
For genetic analysis,enrichment of large genomic fragments was used.Intact moleculesof the target DNA are extracted, enabling for multiscale examination of both short- and long-range data.A Cas9 nuclease is guided to the site of cleavage by the sequence homology of an RNA subunit. This method relies on capturing cells in gel plugs for lysis and their high-molecular weight chromosomal DNA extracted. Then the DNA is digested invitro at two sites flanking the locus of interest using the Cas9 enzyme.Gibson assembly can be used by CATCH to efficiently clone large genomic segments. The workflow of the CATCH technique is illustrated below (Fig. 4).
5. Quantum Dot and Signal Amplification By Exchange Reaction (QD-SABER)
Recruitment of fluorescently labeled detectionoligos to the specific nucleic acid or protein targets, the regulated synthesis of long DNA concatemers from a short primer serves as an effective substrate for multiplexed and amplified signal detection in cells.So, this technology utilizes quantum dots (higher levelof brightness, photostability, and multiplexing) and the adaptability and programmability of DNA nanotechnology (such as signal amplification, and reduced antibody incubation cycles) due to unique optical properties (Fig. 5).
Application of advanced Genome mapping techniques
Assessment and understanding of the molecular mechanism of abundant non-coding element divergence, phenotypic diversity, excessive gene duplications, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulationby novel microRNAs using genome analysis.
– Accurate identification of fish stocks for capture fish management.
– Conservation of fish genetic resources.
– Development of therapeutics based on analysis and comparison of the genome using the immune-related molecular understanding and production of disease-free/resistant fish stock.
– Genomic data is used forhow genetic and environmental factors control the formation and development of morphological traits such as growth and development.
– Whole genome sequencing data is used for the identification of sex determination and sex differentiation of potential aquaculture species.
– Transcriptomic data were utilized for the selective breeding program.
– To understand the biochemical response of fishes to climate change.
– Understanding the interaction between genotype and nutritional profile.
Advance genome sequencing technology have modernized fisheries and aquaculture sciences and practices. Genome sequenced data can be utilized to mitigate upcoming challenges such as disease resistance and climate change, increasing production using selective breeding programs, sexual determination and fisheries resource management. Further, this advance techniques such QD-SABER, CATCH and SMRT could be used for the genome sequencing of non-model fish species.
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