Phosphine resistance molecular diagnostics David Schlipalius Research Scientist Department of Agriculture Fisheries and Forestry
biosecurity built on science Cooperative Research Centre for National Plant Biosecurity
Project aim  To develop DNA markers for phosphine resistance in two species: - Rhyzopertha dominica- no previous genome sequence - Tribolium castaneum- genome sequenced
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Background  Importance of phosphine: - Major fumigant worldwide - Future effectiveness threatened by high-level resistance evolving in pest insects  Need for rapid diagnostics for evaluation of management strategies
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Classical genetics  High-level resistance is conferred by two genes (rph1 and rph2) that act in synergy: Resistance factors compared to sensitive strains Gene
R. dominica
T. castaneum
rph1
~25X (rph1Rd)
~4X (rph1Tc)
rph2
~12.5X
~12-20X
rph1 + rph2
>250X
>400X biosecurity built on science
Complementation analysis  Crossing of resistant strains from Australia and India  The same two genes are responsible internationally in both species T. castaneum
R. dominica 99.999
99.999
99.995 99.99
99.995 99.99
99.95 99.9 99.8
99.95 99.9 99.8 99.5 99 98
Mortality (%)
99.5 99 98
95
95
90
90
80 70 60 50 40 30 20
80 70 60 50 40 30
10 5
20 10
2 1 0.5 0.2 0.1 0.05
QRD569 F2 F1 IRD01
5 2 1 0.5
QTC 931 F1 MADURAI MADURAI
0.01 0.1
0.1
1 Phosphine (mg/L)
1
10
100
10
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Results- T.castaneum Whole genome sequencing & Single NP analysis rph1 on Chromosome 8 rph2 on Chromosome 9
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Results- T. castaneum  Fine scale linkage mapping defines the genomic regions
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Molecular genetics – R. dominica Strategy: de novo genome and transcriptome sequencing Candidate gene list - Compare to T. castaneum mapping - Gene homolog (rph2) is the same between species - Mapped in F90 advanced intercross
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C. elegans also rph2 mutant  The rph2 homolog was also found to be the mutant pre-33 in the nematode Caenorhabditis elegans
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C. elegans -genetic proof Using C. elegans we were able to demonstrate that the rph2 gene is responsible for resistance ‘Gene rescue’ assay: insert wild type copy of gene into mutant- makes them sensitive ‘Gene knockout’: RNAi gene suppression causes wild-type animals to be resistant
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C. elegans -genetic proof Gene rescue (insert wild-type copy)
Gene knockout (RNAi)
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rph2  Highly conserved metabolic gene
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rph2 has geographical variation
 Mutations can differ between strains - but always the same gene
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rph2- 3D structure  Mutations are clustered around an active site
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rph2 - metabolism  Metabolite profiling in C. elegans shows how phosphine affects metabolism pre-33
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Field study – rph2 Developed marker for rph2 from Millmerran strain of R. dominica (QRD569) Tested against field samples from organic farms in SE Qld collected in 2006 and 2011
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Field study – rph2 Measured increase in allele frequency over 5 years
Increase even on organic farmslocal area movement
Overall, in 2006 most farms had no resistance alleles
In 2011, most farms that were measured had resistance alleles at an average ~5-9% frequency
0%, 9.2%
Resistance present Resistance absent
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Outcomes  DNA markers for resistance now a reality  Advantages: - Small sample size required - Can genotype dead insects (post fumigation, trap catches etc. ) - Large numbers of samples can be processed biosecurity built on science
Outcomes Mechanisms are the same globally Found specific exploitable metabolic weakness of rph2 Can now extend what is known about rph2 to other species and countries biosecurity built on science
Thank you For more information, please email David.Schlipalius@daff.qld.gov.au
QDAFF Dr. Pat Collins Dr. Manoj Nayak Andrew Tuck Linda Bond Hervoika Pavic Rajeswaran Jagadeesan Lawrence Smith
UQ Prof. Paul Ebert Ramandeep Kaur Yosep Mau Amelia Fotheringham Horst Schirra Massimo Hilliard Nick Valmas
Murdoch University Prof. Matthew Bellgard Paula Moolhuijzen Roberto Barrero
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