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.