Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

BMC Genomics

Research Article

Amelia Fotheringham¹  Paul R Ebert¹  Rajeswaran Jagadeesan²  David I Schlipalius³ 

[1]School of Biological Sciences, University of Queensland, St. Lucia, 4072, QLD, Australia

[2]School of Biological Sciences, University of Queensland, St. Lucia, 4072, QLD, Australia

[3]Agri-Science Queensland, Department of Agriculture, Fisheries and Forestry (DAFF), Ecosciences Precinct, GPO Box 267, Level 3C-West, QLD 4001, Brisbane, Australia

[4]School of Biological Sciences, University of Queensland, St. Lucia, 4072, QLD, Australia

[5]Agri-Science Queensland, Department of Agriculture, Fisheries and Forestry (DAFF), Ecosciences Precinct, GPO Box 267, Level 3C-West, QLD 4001, Brisbane, Australia

[6]Plant Biosecurity Cooperative Research Centre (PBCRC), LPO Box 5012, 2617, Bruce, ACT, Australia

关键词: Insecticide resistance;    Rhyzopertha dominica;    Linkage disequilibrium;    Fitness cost;    Gene interactions;   

DOI  :  10.1186/1471-2164-14-650

 received in 2013-05-28, accepted in 2013-09-17,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundNext-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically.ResultsProgeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations.ConclusionWe have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

【 授权许可】

Unknown   
© Jagadeesan et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.