Aplicação do sequenciamento de leituras curtas no estudo da variabilidade genômica de organismos relevantes para a carcinicultura

Abstract

Over the last two decades, short-read sequencing has become a central tool in genomic studies allowing the rapid and accurate discovery of high-quantity DNA sequences. This made it possible to apply sequencing in activities of economic interest such as shrimp farming, allowing, for example, the identification of pathogens and genotyping of hundreds to thousands of shrimps simultaneously and the detection of genetic variants of these pathogens. This sequencing can also help in the discovery of genetic markers, such as microsatellites and SNPs, which can be brought together in a genotyping panel, making it scalable and reducing the cost per sample of its application. Specifically in shrimp farming, this technology has proven to be extremely valuable, especially for studying the shrimp genome. Nuclear and mitochondrial genome analyzes provide crucial information about origin, adaptability and other evolutionary aspects that are vital for optimizing shrimp farming. Due to the high depth of coverage of these sequences, it is possible to capture genetic diversity in the samples, allowing the discovery of genetic variations in mitochondrial populations (heteroplasmy) occurring in shrimp and in pathogens, such as viruses that affect them. This presents a unique opportunity to study the impacts of this genetic diversity on the use of mitochondrial markers and the discovery of viral variants and quasispecies. This translates into more informed management practices, minimizing outbreaks and optimizing shrimp health. The implementation of panels based on SNPs is a clear example of the impact of genotyping, demonstrating how the technology can be used to improve genetic selection practices in shrimp farming. In this work, the impact of mitochondrial genetic diversity was evaluated in a virus that affects shrimp farming and shrimp using bioinformatics approaches. First, the occurrence of heteroplasmy was assessed by analyzing muscle sequencing data from a single shrimp, detecting patterns of variability and conservation in the mitogenomes of that individual, in addition to comparing this internal variability with that observed among other mitogenomes and observing the impact on markers in the control region, widely used in population studies. Second, the genetic variability of the shrimp infectious myonecrosis virus obtained from tanks in a viral outbreak situation was evaluated, obtaining the genotype of the most prevalent variant for phylogenetic analyses, revealing its possible origin and relationship with other existing lineages, and secondary variants, evaluating the occurrence of viral quasispecies. Finally, based on previously existing data, a panel of 25 SNP markers (15 genomic and 10 mitochondrial) was developed aiming at low-cost genotyping of the shrimp Penaeus vannamei by multiplex PCR amplification followed by silico parentage analysis. This last project is currently ongoing, and the primers have already been evaluated, and now it remains to continue with sequencing and bioinformatics analyses. In short, this work showed that short-read sequencing is capable of capturing genetic diversity, bringing new insights to shrimp farming by exposing the impact on markers, viral variability and, in the future, the impact of this variability on genotyping panels.