Genotying tools for Pacific oyster (Magallana gigas) have not advanced as rapidly as other aquaculture and agricultural species. To facilitate genome-enabled selection strategies a genotyping panel that is inexpensive, scalable and effective for confidently assigning kinship among samples is needed. In this study we describe the development of a microhaplotype panel from oyster populations used by the USDA ARS Pacific Oyster Genomic Selection (POGS) project.
Initially a haphazard sample from three populations of adult Pacific oysters were taken and sequenced using Whole Genome Resequencing methods to obtain a robust library of variation within and among breeding populations. Using the WGS data a greedy-algorithm was implemented to choose 288 loci that maximized heterozygosity and even distribution along the 10 chromosomes of M. gigas.
To develop and refine the panel a data set of 48 full-sibling families was used with 4 offspring per family. The increased number of offspring was used to identify loci with significant null allele frequencies. DNA was extracted, quantified and then standardized for library preparation. A GT-seq laboratory protocol was used to produce individual libraries that were then pooled by plate and submitted for Illumina sequencing.
Reads were demultiplexed, mapped to the amplicon sequences and processed using Microtyper. Run statistics were evaluated to quantify evenness in reads per locus, per sample and per plate. The microhaplotype panel was refined using multiple rounds of library preparation and sequencing where primer concentrations were primarily modified to achieve even coverage among loci.
The panel will be used to assign parentage to samples from the POGS project that have been part of Ostreid-Herpes Virus 1 experiments using a microvariant from San Diego Bay, California. Results from these studies will be presented to demonstrate the utility of the microhaplotype panel for enabling Pacific oyster breeding for increasing survival to exposure of a highly pathogenic virus that has the potential to significantly impact the Pacific Coast shellfish industry if the virus expands its range beyond San Diego Bay.