Genotyping and final genetic dataset.
Single nucleotide polymorphism (SNP) genotyping data were generated from
blood or tissue samples, collected during routine veterinary
examinations from 452 individual koalas. Blood samples were stored at
-20 °C, and tissue samples were stored in 70% ethanol. DNA was
extracted using the DNeasy Blood and Tissue Kit (QIAGEN), following the
manufacturer’s protocol, and DNA extracts were stored at -80° C. SNP
genotyping was conducted as in Kjeldsen et al. (Kjeldsen et al.2019) by Diversity Arrays Technology, Canberra, using their proprietary
DArTseqTM technology. DArTseqTMutilises a combination of next-generation sequencing platforms and DArT
complexity-reduction methods (Kilian et al. 2012; Courtoiset al. 2013; Cruz et al. 2013; Raman et al. 2014).
The protocol is also optimised for organism and application by selecting
the most appropriate complexity reduction method. This is assessed based
on minimising skewed size ranges, non-ideal numbers of fragments, and
percentages of repetitive elements. Samples were then processed as per
Kilian et al. (2012). SNP genotyping produced a total of 8649 SNPs. We
then filtered those SNPs for minor allele frequency ≥ 0.05, 90%
individual call-rate, ≥ 99% technical replication average which
resulted in 3655 SNPs from 367 koalas in total.
The successfully genotyped koalas
were organised into three genetic datasets to isolate the effect of the
linear infrastructure from those linked to ‘natural’ processes on the
genetic composition of the impacted koala population post-construction
of the linear transport infrastructure (i.e., rail line). As our point
of reference, we created Dataset 1 (n = 270) which contains all the
genotypes from the monitored koalas collected during the 4 years. To
account for ‘natural’ processes, we created Dataset 2 (n = 114) which
contains all genotyped koalas from dataset 1 minus those that died of
‘natural’ processes, these included death by predation, disease, trauma
and unknown. Comparing changes in the genetic composition of dataset 1
and 2 allows us to understand the impact of population decline due to
‘natural’ processes. To then estimate the immediate and longer-term
effect of the linear infrastructure on our studied koala population, we
created Dataset 3 which contains all genotyped koalas from dataset 2
minus those that were translocated because their core home range sat in
the linear transport infrastructure footprint and any new koalas born
after the linear transport infrastructure was built. Koalas in dataset 3
were then divided into two to represent their locations above (n = 27)
and below (n =75) the linear transport infrastructure. Comparing changes
in the genetic composition of dataset 2 and 3 (above and below) allowed
us to isolate the immediate and predict the longer-term genetic
consequences of the linear transport infrastructure project (i.e.
population size reduction by translocation and population subdivision).