2.1 | Sample collection and DNA sequencing
Tissue biopsies were collected from a total of 343 individual H. rubra from 14 locations spanning the Victorian Western and Central Zone fisheries. Locations were selected based on their known virus exposure history according to confidential records held by the Victorian wild fishing sector and the Victorian Fisheries Authority (10 AVG affected and 4 AVG unaffected locations; Table 1, Figure 1). Sampling for 5 of the locations within the Western Zone fishery was coordinated in 2009 by the Department of Economic Development, Jobs, Transport and Resources (DEDJTR). It is expected that animals from these locations during this sampling period survived the virus event and provide a reliable snap shot of the post-virus allele frequency distributions in AVG affected and unaffected populations. Sampling of the remaining 9 locations was performed between 2015 and 2020. To avoid the potential swamping effects of inter-generational gene flow since the disease outbreak, sampling was biased towards fishing stocks expected to be largely self-recruiting based on biophysical connectivity models (Young et al. 2020), and toward large adult animals (expected to be either direct survivors or first generation post-virus survivors). This sampling was performed by contract divers, commercial fisherman, and our research team. At each location, individual abalone were collected within a 100 m2 area, with tissue biopsies consisting of 20 mg of muscle tissue from the abalone lip obtained using sterile dissection tools to avoid cross-contamination. Biopsied material was transferred to 2 ml microcentrifuge tubes containing 80-100% ethanol and stored at 4 °C until required for genomic analysis.
Total genomic DNA was extracted from 10 mg of tissue using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. Resulting DNA extracts were quantified using a Qubit version 2 fluorometer (Life Technologies, Carlsbad, CA, USA). To obtain population genomic data, we applied the Pool-Seq approach (Futschik & Schlotterer 2010), which involves pooling the DNA of a large number of individuals from the same population and then sequencing the ‘population variability genome’. This was achieved by pooling individual DNA extracts from each sample location equimolar, splitting the 25 individuals per location into 2 x pools per locations consisting of DNA from 12 and 13 individuals, respectively, to account for potential sequencing bias. The resulting 28 pooled libraries were prepared for sequencing using the NexteraTM DNA Sample Preparation kit, and sequenced using the Illumina NovaSeq platform (Illumina, San Diego, CA, USA), with the 150 base pair (bp) paired-end protocol. Sequencing was performed allowing for 3 x genome coverage per individual per pool, equating to 80 - 100 x genome coverage per population.