IDENTIFICATION OF DISEASE-RESISTANCE MARKERS BY NEXT-GENERATION SEQUENCING IN THE EASTERN OYSTER  

The eastern oyster (Crassostrea virginica Gmelin) is a major aquaculture species in the United States. It faces two serious diseases: MSX (caused by Haplosporidium nelsoni) and Dermo (caused by Perkinsus marinus). The identification of disease-resistance markers may facilitate marker-assisted selection and improve selection efficiency. Next-generation sequencing has opened new avenues for marker development. Genome-wide candidate-gene association studies (GCAS) with next-generation sequencing may provide rapid and cost-effective identification of disease-resistance markers. In this study, we used GCAS and next-generation sequencing of pooled samples to identify disease-resistance markers in the eastern oyster.

We assembled a set of 1002 candidate genes of the eastern oyster that are involved in immune and stress responses based on gene function and transcriptome profile. Primers were designed with Ion AmpliSeq^TM Designer to amplify fragments of all candidate genes in a single-tube multiplex PCR. Oysters were collected from three populations before and after significant mortalities that were primarily caused by Dermo and MSX. DNAs from 480 oysters were pooled in equal amounts producing 6 pooled samples: before and after-mortality in three populations. The pooled DNA samples were used as template for amplification of 1002 candidate genes with the AmpliSeq^TM primer panel. Amplified products were sequenced to about 1200x per gene with the Ion Torrent PGM 400 bp module. Of the 1002 genes targeted, 616 genes were successfully amplified and sequenced, with 455 genes being recovered in all six samples. Single-nucleotide polymorphisms (SNPs) and indels were identified and analyzed for post-mortality frequency shifts. Variations in 21 genes showed significant (>20%) frequency shift in all three populations, suggesting they are likely associated with disease resistance. They include some well-known immune response genes encoding c-type lectins, tissue necrosis factor receptor-associated factor and interferon induced protein, as well as proteins whose involvement in immune response is less clear such as epidermal growth factor, G-protein coupled receptor, hemicentin, L-ascorbate oxidase and glutamate decarboxylase. These genes and markers provide good candidates for further studies and marker-assisted selection.