Genotype-by sequencing (GBS) is commonly used in aquaculture selective breeding programs to acquire SNP genotypes for various purposes including pedigree reconstruction, GWAS, and in the estimation of genetic parameters, genomic relationships, and breeding values. GBS if based on de-novo sequencing approaches (e.g., shallow resequencing, ddRADseq) may generate raw sequence data not only from the targeted species, but also from any type of microorganism (such as bacteria, viruses, and fungi) infecting the sampled tissue of the host at the time of collection. Thus, such GBS approaches may unintentionally generate important information on the presence and relative load of pathogens affecting the hosts, which could be a useful epidemiological tool and inform farmers about the health status of their stock.
In barramundi (Lates calcarifer ), scale drop disease virus (SDDV) is a pathogen responsible for mortality losses of 40-90%. Presently, the detection of SDDV involves qPCR testing of immune competent tissues, such as spleen or kidney, which are sacrificial, labour intensive to obtain and impractical for high-valued broodstock. In this study, we used ddRADseq GBS data from 4,484 barramundi (2,239 clinically sick and 2,249 clinically healthy) from four commercial cohorts genotyped for a breeding program to also identify SDDV DNA in the fish fin clip samples. Here we blasted ddRADseq raw sequences against the SDDV genome and standardized SDDV read counts by the ratio of viral sequences per 1 million sequences (Reads Per Million, RPM) present in each sample (viral load) . Results showed a high association between SDDV prevalence and load and fish health status. Sick fish had 88.9% SDDV prevalence with 21.8 ± 0.56 RPM, whereas healthy fish had 0.2% SDDV prevalence with 0.002 ± 0.001 RPM. To validate these findings, qPCR for SDDV copy number was performed on the fin and spleen samples from 172 barramundi (81 sick and 91 healthy) and compared against the ddRADseq GBS sequence read data from the same fish . A strong correlation was found between ddRADseq GBS RPM and the viral load from qPCR of fin and spleen (Fin-ddRADseq Spearman’s ρ = 0.84; Spleen-ddRADseq ρ = 0.76). Higher viral loads were detected in the fin (278 copies/ng DNA), compared to the spleen (185 copies/ng DNA) of sick fish. Furthermore, comparable prevalence was found across the four cohorts, demonstrating that fin samples can be used to reliably test for SDDV. The results demonstrate the ability to use genomic data generated via dd-RADseq GBS as an epidemiological tool to assess SDDV presence and load in barramundi breeding programs.
These findings suggest that breeding programs generating large ddRADseq GBS datasets may also be used for pathogen surveillance purposes in aquaculture breeding programmes where the target pathogen infects the host genotyped tissue.