Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae

Abstract

BackgroundThe genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density.MethodsWe designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine.ResultsIn the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection.ConclusionsWe present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2009-z) contains supplementary material, which is available to authorized users.

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii

“…Candidate enhancers were amplified from DNA of mosquitoes from the following A. coluzzii colonies: Ngousso, initiated in Cameroon in 2006 47 , Fd03, Mali, 2008, Fd05, Mali 2008, Fd09, Burkina Faso, 2008, and Fd33, Burkina Faso, 2014. Fd colonies were previously described 48 . Primers are listed in Supplementary Table S2.…”

Identifying coevolving loci using interspecific genetic correlations

“…As a consequence, established approaches for identifying genes influencing single species phenotypes, such as genetic association tests, can identify genes important in each species, but never which genes in one species interact with which genes in the other. A result of this difficulty is that we have a substantial collection of studies identifying candidate genes for resistance in hosts (e.g., Atlija, Arranz, Martinez-Valladares, & Gutierrez-Gil, 2016;Benavides et al, 2015;Kim, Sonstegard, da Silva, Gasbarre, & Van Tassell, 2015;Kover & Caicedo, 2001;Kover, Wolf, Kunkel, & Cheverud, 2005;Magwire et al, 2012;Redmond et al, 2015;Wilfert & Schmid-Hempel, 2008) and infectivity in parasites (e.g., Molina-Cruz et al, 2013;Scanlan, Hall, Lopez-Pascua, & Buckling, 2011) , but virtually no studies that identify evolutionarily coupled genes in host and parasite.…”

Rapidly Evolving Toll-3/4 Genes Encode Male-Specific Toll-Like Receptors in Drosophila

“…Some of these lineage-specific Tolls are important for pathogen recognition and host fitness. For instance, Toll-11 paralogs found only in mosquitos can protect the host from Plasmodium falciparum infection ( Redmond et al 2015 ). Nevertheless, most lineage-restricted Tolls remain poorly studied.…”