High-throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Abstract

Aquatic genetic resources (AqGR) include DNA, tissues, gametes, embryos, and other early life stages, wild and farmed individuals, and communities of organisms of actual or potential value for food and aquaculture. Monitoring the AqGR at national, regional, and global levels would not only help to improve production traits, enhance disease resistance, and ensure the long-term sustainability of aquatic species but also provide valuable information on the state of rare or endangered aquatic species. While the importance of monitoring and reporting of AqGR is becoming more and more apparent among different stakeholders, efforts to date are still insufficient (FAO, 2022).

A common method to estimate the AqGR is genotyping, which is a process of determining the genotype at positions within the genome of an individual and comparing it to other individuals' sequences. It is often used to understand association between genotype and phenotype. Sequence variations like single-nucleotide polymorphisms (SNPs) or microsatellite loci are applied as markers in linkage and association studies to determine genes relevant to specific traits. SNPs are the most common sequence variant widely used in genome-wide association studies (GWAS). With more and more SNPs being discovered, SNP genotyping technologies have been greatly promoted and include low-throughput and high-throughput methods. Nowadays, demands of high-throughput SNP genotyping are increasing, especially for hybridization-based SNP arrays and various next-generation sequencing (NGS)-enabled genotyping methods, such as genotyping-by-sequencing (GBS).

Besides genotyping AqGR, related molecular methods have also been wildly used in pathogen diagnosis in aquaculture. For farmers, early detection of pathogens can help to prevent spread of disease and minimize economic losses due to disease outbreaks. Rapid, pond-side methods allow for quick and efficient diagnosis of diseases, which can lead to more timely and effective treatment options. Furthermore, effective disease management can help to minimize the use of antibiotics and other treatments, thus reducing the risk of antimicrobial resistance and promoting more sustainable aquaculture practices.

Both good management of AqGR and disease control are key elements of sustainable aquaculture. Here, we summarize the methods used for genotyping AqGR and detecting disease by molecular diagnosis with an emphasis on high-throughput and onsite solutions.

The common laboratory procedure for genotyping involves sample collection, DNA extraction, PCR, and subsequent detection of genetic variation. For example, tissue samples are collected from fish, usually by taking a small piece of tissue such as a fin clip. DNA is extracted from the tissue sample using standard laboratory techniques, such as phenol-chloroform extraction or commercial DNA extraction kits. Then, specific molecular markers may need to be designed for detecting any genetic variation; an example is the mitochondrial cytochrome oxidase subunit 1 (COI) gene. Microsatellite markers can be used for genotyping, but SNP markers are now more commonly used for genotyping since more loci and greater information can be extracted from genome-wise SNP data. SNP genotyping can be performed using various methods, such as restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), or sequencing. RFLP and SSCP are based on differences in the size and/or shape of the DNA fragments resulting from SNP variation, while sequencing provides direct information on the nucleotide sequences.

Compared with high-throughput genotyping, traditional methods are generally less efficient and time consuming, and may require a larger amount of starting material. Traditional genotyping methods may also have a higher error rate and be less sensitive to low-frequency variants. Additionally, traditional methods may not be able to genotype as many loci simultaneously as high-throughput methods, and this can limit their utility in certain applications such as genome-wide association studies.

With the rapid development of next-generation sequencing (NGS), many high-throughput genotyping methods have been developed and are primarily divided into SNP arrays and genotyping by sequencing (GBS) methods (Scheben et al., 2017). The latter has various related methods, such as whole genome resequencing (WGR) and reduced representation sequencing (RRS), including sequence capture, restriction-site-associated DNA sequencing (RAD-seq), genotyping-in-thousands by sequencing (GT-seq), etc.

Intensive aquaculture with high stocking density can lead to outbreaks of disease. The best way to control aquatic disease is by prevention, but detection of a problem and timely treatment can be equally important. A rapid, pond-side pathogen detection method would be essential and aid in the control of disease in aquaculture.