The Australian brushtail possum (Trichosurus vulpecula) is a pervasive
marsupial pest of New Zealand. Impacting on the native
flora and fauna
and the nation's livestock industry by its vectoring of bovine turberculosis,
T. vulpecula is a priority for control and eventual eradication. Current pest
control initiatives involve aerial deployment of chemical poisons, baiting
and trapping. To establish the success of such control operations, estimates
of possum population size pre- and post culling are required. Currently
several monitoring methodologies - requiring the detection and trapping
of individuals - are available to estimate indices of abundance (e.g. the
residual trap-catch index). But these monitoring protocols are constrained
by logistical and analytical considerations. The necessity to overcome the
limitations of traditional monitoring schemes presents the opportunity to
develop and evaluate the implementation of non-invasive genetic monitoring
systems for possums. This thesis aimed to optimise an efficient amplification
system for a panel of eight microsatellite loci that allow the identification of
individual possums, characterise the occurrence of genotyping error across a range of conditions, and evaluate the use of salivary DNA retrieved from
interference devices as template for amplification.
Optimisation of amplification conditions for all loci in the panel was
evaluated with DNA extracted from possum tissue collected at three localities
in Canterbury region. Allele polymorphism was analysed by capillary
electrophoresis and
uorescence based detection of fragments. After optimisation,
locus Tv16 was discarded from the panel due to its linkage with
locus Tv27 and amplfication of unspecific fragments. Microsatellite diversity
patterns of the seven remaining loci revealed moderate to high polymorphism
and heterozygosity, no evidence of genetic structuring between
localities across Canterbury (Fst = 0:03), and a sufficiently low overall
probability of identity adjusted for siblings (PIsib) (3 x 10β»Β³) to ensure a
robust identification of individual possums based on their multi-locus genotype.
Further exclusion of locus Tv54 was recommended based on its high
PIsib (0.63-1.00) and incidence of genotyping error.
Amplification of template DNA extracted from tissue was not exempt
from genotyping error (mean error rate per locus (el) = 4,8% and observed
error rate per multi-locus genotype (eobs) = 33.3%), these errors being associated
in equal measure with stochastic causes (e.g. allele drop-out and
false alleles) and systematic causes (e.g. scoring errors, sample swapping or
contamination). No evidence of null alleles was detected.
Six loci were successfully assembled into a multiplex PCR assay. The
implementation of mutliplex PCR had no significant effects on the incidence
of genotyping error or the consistency of allele size estimation compared
to standard PCR, and represented a substantial reduction in labour and
resources needed to obtain a genotype (92% cost reduction relative to singleplex). While 1:6 dilution of DNA extracted from tissue did not show significant
effects on the amplification success and the mean genotyping error rate per
locus, the use of template DNA retrieved from saliva decreased the performance
of the microsatellite amplification system significantly. Only 18 of 24
samples were able to generate positive or partially positive genotypes, loci
with amplicons > 200 bp being the most affected, while the mean error rate
per locus increased to 45%.
Altogether, these results indicate that locus characteristics (i.e. amplicon
size) and quality of template DNA are crucial factors affecting the sensitivity
and reliability of the protocol developed. Potential ways to improve the
remote collection of DNA from saliva are recommended