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

Abstract

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.

Prevalence and potential fitness cost of weak phosphine resistance in Tribolium castaneum (Herbst) in eastern Australia

“…Second, there were two replicate populations in each case. The finding of no evidence of a fitness cost associated with weak resistance in T. castaneum is similar to that obtained in a recently published study on T. castaneum using the population cage approach and phenotype testing of a single population derived by a susceptible  weakly resistant cross (Jagadeesan et al, 2012), although subsequent molecular screening of sub-samples from that study showed a slight increase in the frequency of the rph1 resistance allele over time (Jagadeesan et al, 2013). The finding of no apparent fitness cost is also similar to a recently published study on S. oryzae using the population cage approach and phenotype testing of replicated populations derived from a susceptible and weakly resistant cross (Daglish et al, 2014).…”

“…The study used a single segregating population each from the following crosses: susceptible  weakly resistant, weakly  strongly resistant cross and susceptible  strongly resistant. Subsequently, Jagadeesan et al (2013) reported that molecular analysis of the susceptible  strongly resistant population showed an increase in the frequency of the rph1 allele and a decrease in the frequency of the rph2 resistance allele over time indicating a fitness cost associated with that allele.…”

“…Therefore, knowing if there are fitness costs associated with phosphine resistance could help in resistance management. Fitness has been investigated in a range of insect pests of stored grain using different experimental approaches (Pimentel et al, 2007;Schlipalius et al, 2008;Sousa et al, 2009;Pimentel et al, 2011;Jagadeesan et al, 2012Jagadeesan et al, , 2013Daglish et al, 2014). Some studies have used the population cage approach in which susceptible and resistant strains are hybridized and the resulting populations are reared in the absence of phosphine selection.…”

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

“…As with other phosphine resistant pest species, the very high level of resistance exhibited by the Strong-R (505×) appears to be the result of strong synergistic interactions between two major genes. These results are consistent with inheritance of weak and strong resistance to phosphine in R. dominica, T. castaneum and S. oryzae, where the weak resistance phenotype was predominantly controlled by a single major gene, rph1, and strong resistance was governed by the synergistic interaction of two major genes, rph1 and rph2 [6][7][8]37].…”

Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera : Tenebrionidae) populations from Turkey

“…This would allow survival under strong selection pressures and maintenance in insect populations that breed outside of storages and are not exposed to phosphine. It has been noted that some rph2 alleles reported in T. castaneum do carry significant fitness costs (Jagadeesan et al, 2013), however this has yet to be studied in detail for the P45S allele.…”