DNA fingerprinting procedure for ultra high‐throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non‐existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA finge...

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Bibliographic Details
Published in:Insect molecular biology 2001-12, Vol.10 (6), p.579-585
Main Authors: Schlipalius, D. I, Waldron, J, Carroll, B. J, Collins, P. J, Ebert, P. R
Format: Article
Language:English
Subjects:
Animals
chromosome mapping
cluster analysis
Coleoptera - classification
Coleoptera - genetics
DNA
DNA fingerprinting
DNA Fingerprinting - methods
DNA primers
genetic mapping
genetic markers
genetic variation
insects
molecular markers
Multigene Family
pests
phosphine
Random Amplified Polymorphic DNA Technique
resistance
Rhyzopertha dominica
stored grain
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Summary:Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non‐existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced ~50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty‐nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 ± 6.0% between clades and of 21.0 ± 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.
ISSN:0962-1075
1365-2583
DOI:10.1046/j.0962-1075.2001.00297.x